Ng μL Research Articles

Efficient extraction approach based on polydimethylsiloxane/ZIF-derived carbons sponge followed by GC–MS for the determination of volatile compounds in cumin

A sensitive, fast and simple approach based on polydimethylsiloxane/ZIF-derived carbons (PDMS/ZIF-C) sponge solid-phase extraction followed by gas chromatography-mass spectrometry was successfully developed to determine the volatile compounds in cumin. The PDMS/ZIF-C exhibits outstanding properties with high adsorption capacity and good reusability, and has shown its potentiality as an ideal adsorbent for extraction of volatile compounds. The effects of the experimental parameters were investigated. Excellent analytical performances were achieved, including wide linearity (10–920 μg mL−1) with high correlation coefficients (R2 ≥ 0.9989), satisfactory precision (RSDs ≤ 4.93 %), low limits of detection (1.20–2.65 ng μL−1) and quantification (3.40–6.01 ng μL−1). The optimal method was applied for analysis the volatile compounds in 17 cumin samples from four origins in Xinjiang, and 43 compounds were identified. Multivariate statistical analysis was used to establish the relationship between the origins and the volatile compounds. The method was effectively applied to determine the complex and trace volatile compounds in spices.

A SIMPLE AND VALIDATED HP-TLC METHOD FOR SIMULTANEOUS ANALYSIS OF ETHNO-MEDICINE GALLIC ACID AND EUGENOL

A new method has been developed and validated for the simultaneous estimation of gallic acid and eugenol for the poly-herbal formulation. The HP-TLC (High Performance Thin Layer Chromatography) method was developed using pre-coated silica gel 60 F254 on aluminum plate and the solvent system used was iso propyl alcohol : hexane: ethyl acetate: glacial acetic acid (50:30:30:1 V/V) and scanned at 254 nm wavelength. The Rf value for gallic acid was 0.608±0.041 and 0.752±0.035 for eugenol. The range of linearity detected for gallic acid and eugenol was 2-10 ng µL-1. The method was validated in accordance with ICH (International Council for Harmonization) guidelines for linearity, range, LOD, LOQ, precision, specificity and robustness. The developed method was found to be easy, simple, selective, specific and robust.

Fluorescent on-site detection of multiple pathogens using smartphone-based portable device with paper-based isothermal amplification chip.

The development of cost-effective, portable, and ease-of-use sensing system for on-site genetic diagnostics is highly desirable for pathogen screening and infectious disease diagnosis. This study develops (1) a paper-based biochip which is able to integrate the loop-mediated isothermal amplification (LAMP) protocols for simultaneous detection of Escherichia coli O157:H7, Salmonella spp., and Staphylococcus aureus, and (2) a stand-alone smartphone-based portable device which can control exactly 65 °C for isothermal amplification as well as collect and analyze the thus generated fluorescence signals. The reported sensing system has been successfully demonstrated for foodborne pathogen detection with a limit of detection of 2.8 × 10−5 ng μL−1. Spiked milk samples with concentration as low as 10 CFU mL−1 were successfully determined within 4 h, demonstrating the practicality of the reported sensing system in the fields. The reported sensing system featuring simplicity and reliability is ideally suited for genetic diagnostics in low resource settings.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00604-022-05419-x.

Visual Detection of Chicken Adulteration Based on a Lateral Flow Strip-PCR Strategy.

The aim of this study was to develop an accurate, easy-to-use, and cost-effective method for the detection of chicken adulteration based on polymerase chain reaction (PCR) and lateral flow strip (LFS). We compared six DNA extraction methods, namely the cetyltrimethylammonium bromide (CTAB) method, salt method, urea method, SDS method, guanidine isothiocyanate method, and commercial kit method. The chicken cytb gene was used as a target to design specific primers. The specificity and sensitivity of the PCR-LFS system were tested using a self-assembled lateral flow measurement sensor. The results showed that the DNA concentration obtained by salt methods is up to 533 ± 84 ng µL−1, is a suitable replacement for commercial kits. The PCR-LFS method exhibits high specificity at an annealing temperature of 62 °C and does not cross-react with other animal sources. This strategy is also highly sensitive, being able to detect 0.1% of chicken in artificial adulterated meat. The results of the test strips can be observed with the naked eye within 5 min, and this result is consistent with the electrophoresis result, demonstrating its high accuracy. Moreover, the detection system has already been successfully used to detect chicken in commercial samples. Hence, this PCR-LFS strategy provides a potential tool to verify the authenticity of chicken.

A mammalian messenger RNA sex determination method from humpback whale (Megaptera novaeangliae) blubber biopsies.

The large size of free-ranging mysticetes, such as humpback whales (Megaptera novaeangliae), make capture and release health assessments unfeasible for conservation research. However, individual energetic condition or reproductive health may be assessed from the gene expression of remotely biopsied tissue. To do this, researchers must reliably extract RNA and interpret gene expression measurements within the context of an individual's sex. Here, we outline an RNA extraction protocol from blubber tissue and describe a novel mammalian RNA sex determination method. Our method consists of a duplex reverse transcription-quantitative (real-time) polymerase chain reaction (RT-qPCR) with primer sets for a control gene (ACTB) and the X-chromosome inactivation gene (XIST). Products of each RT-qPCR had distinct melting temperature profiles based on the presence (female) or absence (male) of the XIST transcript. Using high-resolution melt analysis, reactions were sorted into one of two clusters (male/female) based on their melting profiles. We validated the XIST method by comparing results with a standard DNA-based method. With adequate quantities of RNA (minimum of approx. 9 ng µl−1), the XIST sex determination method shows 100% agreement with traditional DNA sex determination. Using the XIST method, future cetacean health studies can interpret gene expression within the context of an individual's sex, all from a single extraction.

A real time loop-mediated isothermal amplification (RealAmp) assay for rapid detection of Pleurostoma richardsiae in declining olive plants

Pleurostoma richardsiae is associated with host trunk diseases, known to cause dieback, cankers and wilting of woody trees, and human infections. This fungus was isolated from wood tissues of declining olive trees and grapevines showing esca disease symptoms, in the Apulia region of Italy. Fungus detection has been based on morphological and molecular features, which are time-consuming to identify and require well-trained personnel. Improvement of Pl. richardsiae detection in olive was achieved through development of real time loop-mediated isothermal amplification targeting the intergenic spacer (IGS) region of the fungus. Specificity of the assay was confirmed using ten Pl. richardsiae strains and 36 other fungus strains of species usually isolated from declining olive trees. The achieved limit of detection was 7.5 × 10-2 ng μL-1 of Pl. richardsiae genomic DNA. A preliminary validation of RealAmp was also performed using material from infected olive plants artificially inoculated in a greenhouse.

Knockdown of hexokinase in Diaphorina citri Kuwayama (Hemiptera: Liviidae) by RNAi inhibits chitin synthesis and leads to abnormal phenotypes.

Silencing specific genes in pests using RNA interference (RNAi) technology is a promising new pest-control strategy. The Asian citrus psyllid, Diaphorina citri Kuwayama, is the most important citrus pest because it transmits Candidatus Liberibacter asiaticus, which causes huanglongbing. Chitin is essential for insect development, and enzymes in this pathway are attractive targets for pest control. The hexokinase gene DcHK was characterized from D.citri to impair proper growth and chitin synthesis through RNAi. The transcription of DcHK was more highly developed in third-instar nymphs, adults and the Malpighian tube. The RNAi needed for D.citri is dose-dependent, with 600 ng μl-1 dsDcHK sufficient to knockdown endogenous DcHK expression. The messenger RNA (mRNA) level was lowest at 36 h after dosing, and there were significant effects on the relative levels of mRNA in the chitin synthesis pathway (DcTre, DcG6PI, DcGNAT, DcGFAT, DcPGM, DcUAP and DcCHS), leading to mortality, reduced body weight and abnormal or lethal phenotypes. RNAi can be triggered by orally delivered double-stranded RNA in D.citri. These results can provide support for HK genes as a new potential target for citrus psyllid control. © 2022 Society of Chemical Industry.

Non-faradaic electrochemical impedance spectroscopy analysis of C-MEMS derived bio-modified glassy carbon electrode

In this work, we have developed a carbon-microelectromechanical systems derived glassy carbon electrode (GCE) for non-faradaic electrochemical impedance spectroscopy (nf-EIS) measurement to detect electrode interfacial changes upon biomodification. This is carried out using a three-electrode system configuration. The fabricated electrode was electrochemically characterized in phosphate-buffered solution, and then changes in impedance were observed upon bio-modification of the electrode surface. The absence of any labelling molecule and no redox indicators makes the measurement system more straightforward and precise. The investigations were done using a standard deoxyribonucleic acid (DNA) immobilization on the GCE. The surface modification was done using a two-step assembly protocol linking the probe to the carbon electrode and blocking the unwanted sites using a spacer chemical-mercaptohexanol. The results obtained help us to understand the electrical signatures upon bio-modification of electrodes in the presence of a probe and its complementary 50 ng μl−1 DNA target. nf-EIS relies on various microscopic interactions, which occur at the electrode–electrolyte interface system. We are currently working on extending this study to develop a precise, accurate and sensitive sensor to detect bio-molecular interactions occurring on the GCE to detect disease-causing microorganisms in contaminated water samples.

Quantification of Global DNA Hydroxymethylation Level Using UHRF2 SRA-Luciferase Based on Bioluminescence Resonance Energy Transfer.

5-Methylcytosine (5mC) plays an important role in the regulation of gene expression. Ten-eleven translocation (TET) continuously oxidizes 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). High levels of 5hmC are found in the brain and embryonic stem cells, while global hydroxymethylation levels are reduced in several cancer cells. Moreover, alterations in hydroxymethylation levels occur in neurological diseases, such as Alzheimer's disease and Parkinson's disease. In this study, a convenient sensing method for the determination of global hydroxymethylation levels was developed. A bioluminescence resonance energy transfer (BRET) assay for global methylation level determination has been previously reported. In the assay, BOBO-3 DNA intercalating dye is excited by the bioluminescence of methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc); that is, the BRET signal depends on the content of methylated CpG on genomic DNA. To develop a hydroxymethylation level sensing method, SET- and RING-associated (SRA) domain of ubiquitin-like with PHD and RING finger domains 2 (UHRF2)-fused Fluc (UHRF2 SRA-Fluc) was prepared. UHRF2 SRA is known to bind to both hydroxymethylated and methylated CpG sites; thus, MBD was utilized to mask the methylated CpG on genomic DNA. We demonstrated that the BRET signal between UHRF2 SRA-Fluc and BOBO-3 depends on the global hydroxymethylation level in the presence of MBD (R2 = 0.99, and relative standard deviation < 2.3%). The limit of detection for hydroxymethylated genomic DNA was 0.75 ng μL-1. In this assay, the global hydroxymethylation level was quantified within 40 min in a single tube, indicating that the assay would be utilized not only for clinical diagnostics but also for the elucidation of 5hmC functions.

Draft Genome Sequence of Ralstonia syzygii subsp. celebesensis from Indonesia, the Causal Agent of Blood Disease of Banana.

Draft Genome Sequence of Ralstonia syzygii subsp. celebesensis from Indonesia, the Causal Agent of Blood Disease of Banana.

Novel polymorphisms in GPX5 gene and their association with reproductive and productive traits in Lumsniang (Hampshire x Niang Megha) pigs under subtropical hilly region of Meghalaya

Aim: To detect genetic variants of GPX5 gene and their associations with reproductive and productive traits in the Lumsniang (Hampshire x Niang Megha) pigs. Methodology: In this study, a total of 87 Lumsniang pigs were selected for collecting blood samples (3-5 ml) from Nucleus Pig Breeding Farm of ICAR Research Complex for North Eastern Hilly region, Umiam, Meghalaya. Genomic DNA was extracted from the blood leukocytes using the Qiagen DNeasy Blood and tissue kits and diluted to working concentration (50 ng μl-1) and stored at -20°C, which were used as templates for association of GPX5 variants with polymerase chain reaction. The data for reproductive and productive traits were recorded from all the animals taken in this study. The reproductive and productive traits were analyzed using a general linear model (GLM) procedure of SPSS Version 16.0. Results: Polymorphisms at Intron 1 of the GPX5 gene was determined by PCR-RFLP with HinfI restriction enzyme and DNA sequencing analyses. Two alleles and three genotypes were identified by HinfI digestion of the GPX5 gene. In the SNP-HinfI (g.1896A&gt;G) locus, significant associations were found between GPX5 genotypes and litter size at birth. The genotype AA demonstrated significantly (P&lt;0.05) superior litter size at birth as compared to AG and GG genotypes at the SNP-HinfI locus in Lumsniang pig. Interpretation: The study reported probable associations of GPX5 variants with reproduction and production traits in Lumsniang pig. The genotype AA at locus SNP-HinfI improved the litter size at birth in the studied population. The GPX5 gene may be considered as a DNA marker for marker-assisted selection after validation in a large pig population.

Visualization of copper nanoclusters for SARS-CoV-2 Delta variant detection based on rational primers design.

Here, based on the design of rational primers and copper nanoclusters (CuNCs), we present a method for the accurate detection of the SARS-CoV-2 Delta variant, which is capable of distinguishing the Delta variant with its single nucleotide polymorphism from the ‘wild type’ coronavirus (NC_045512.2), and realizing visualization signal out. Specifically, we show that dual priming oligonucleotide (DPO) primers and AT primers can be used to distinguish between wild types and mutations of this virus by polymerase chain reaction (PCR) analysis and that visualization can be achieved via the red fluorescence of CuNCs in ultraviolet radiation. Among the results, it was found that the R-1-down (DPO)-6I and F-1-30 AT, with the single nucleotide deletion site designed at the 3′ end of the downstream primer, showed the best specificity towards the Delta variant. Moreover, the use of AT primers increased the AT contents of the PCR products, thus meeting the template requirements generated by the CuNCs. It was also found that the AT primers could assist with improving detection specificity. Finally, we demonstrate that the visualization of the CuNCs-based detection assay exhibited a linear relationship in 0.5 pg μL−1–50 ng μL−1, with a limit of quantitation (LOQ) of 0.5 pg μL−1.

Rapid detection of Chattonella marina by PCR combined with dot lateral flow strip.

In this study a novel technique referred to as PCR combined with dot lateral flow strip (PCDS) is proposed and its application to the detection of harmful microalgae was explored. For this purpose, using Chattonella marina as a test algal species, PCR targeting the D1–D2 region of large subunit ribosomal gene of this alga was performed with the tagged specific primers. The amplicons were then analyzed with the manually prepared dot lateral flow strip, and the strip could produce a test dot and a control dot that are naked-eye detectable, indicating the successful establishment of PCDS. The established PCDS assay does not require expensive instruments for the detection, and the results can be observed visually after adding 7.5 μL of PCR amplicons in combination with 92.5 μL of chromatography buffer to the sample pad of the strip for about 10 min. The PCR conditions were optimized to enhance the effectiveness of detection. The cross-reactivity test with 23 microalgae species, including Chattonella marina, showed good specificity of the PCDS. The detection limit of PCDS was 1.25 × 10−2 ng µL−1 for genomic DNA and 101 cells mL−1 for crude cell extracts, which can meet the detection needs. In summary, the PCDS proposed in this study has low cost, clear, and intuitive detection results and good specificity and sensitivity, providing a novel detection method for C. marina.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10811-021-02667-x.

Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids.

An athermal approach to mRNA enrichment from total RNA using a self-immolative thioester linked nucleic acids (TENA) is described. Oligo(thymine) (oT) TENA has a six-atom spacing between bases which allowed TENA to selectively base-pair with polyadenine RNA. As a result of the neutral backbone of TENA and the hydrophobicity of the octanethiol end group, oT TENA is water insoluble and efficiently pulled down 93±2 % of EGFP mRNA at a concentration of 10 ng μL-1 . Self-immolative degradation of TENA upon ambient temperature exposure to nucleophilic buffer components (Tris, DTT) allowed recovery of 55±27 ng of mRNA from 3.1 μg of total RNA, which was not statistically different from the amount recovered using Dynabeads® mRNA DIRECT Kit (89±24 ng). Gene expression as measured by RT-qPCR was comparable for both enrichment methods, suggesting that the mild conditions required for enrichment of mRNA using oT TENA are compatible with RT-qPCR and other downstream molecular biology applications.

Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids

AbstractAn athermal approach to mRNA enrichment from total RNA using a self‐immolative thioester linked nucleic acids (TENA) is described. Oligo(thymine) (oT) TENA has a six‐atom spacing between bases which allowed TENA to selectively base‐pair with polyadenine RNA. As a result of the neutral backbone of TENA and the hydrophobicity of the octanethiol end group, oT TENA is water insoluble and efficiently pulled down 93±2 % of EGFP mRNA at a concentration of 10 ng μL−1. Self‐immolative degradation of TENA upon ambient temperature exposure to nucleophilic buffer components (Tris, DTT) allowed recovery of 55±27 ng of mRNA from 3.1 μg of total RNA, which was not statistically different from the amount recovered using Dynabeads® mRNA DIRECT Kit (89±24 ng). Gene expression as measured by RT‐qPCR was comparable for both enrichment methods, suggesting that the mild conditions required for enrichment of mRNA using oT TENA are compatible with RT‐qPCR and other downstream molecular biology applications.

Electrical conductivity measurement of λ DNA molecules by conductive atomic force microscopy

Conductive atomic force microscopy (C-AFM) is a powerful tool used in the microelectronics analysis by applying a certain bias voltage between the conducting probe and the sample and obtaining the electrical information of sample. In this work, the surface morphological information and current images of the lambda DNA (λ DNA) molecules with different distributions were obtained by C-AFM. The 1 and 10 ng μl−1 DNA solutions were dripped onto mica sheets for making randomly distributed DNA and DNA network samples, and another 1 ng μl−1 DNA sample was placed in a DC electric field with a voltage of 2 V before being dried for stretching the DNA sample. The results show that the current flowing through DNA networks was significantly higher than the stretched and random distribution of DNA in the experiment. The I–V curve of DNA networks was obtained by changing the bias voltage of C-AFM from −9 to 9 V. The currents flowing through stretched DNA at different pH values were studied. When the pH was 7, the current was the smallest, and the current was gradually increased as the solution became acidic or alkaline.

Detection of four foodborne pathogens based on magnetic separation multiplex PCR and capillary electrophoresis.

Foodborne pathogen contamination is a major safety issue for many foods and is causing concern worldwide. In this study, a detection system based on magnetic separation, multiplex PCR (MPCR) and capillary electrophoresis (CE) technologies was developed for the simultaneous detection of four foodborne pathogens. Magnetic separation technology is used to rapidly capture pathogenic bacteria in food samples, and then a combination of MPCR and CE can be used to greatly increase detection sensitivity. The detection limit for bacterial DNA reached 10-5 -10-7 ngμL-1 and in the analysis of mocked food samples, the assay showed good sensitivity for bacterial detection ranging from 101 to 105 CFU mL-1 with excellent specificity. Compared to similar detection methodologies, this technique avoids the need for time-consuming enrichment cultures, is more sensitive, and can be used to assay simultaneously four foodborne pathogens.

Pulse purge thermal desorption ion mobility spectrometer for rapid and sensitive determination of intravenous anesthetic etomidate in blood

Rapid and sensitive determination of etomidate in blood is of great significance for anesthesiologists to adjust the depth of anesthesia, ensure the safety of operation and reduce the incidence of adverse reactions in patients, especially those with unstable hemodynamics. Herein, a positive photoionization ion mobility spectrometry method with acetone doped into the drift gas was developed, increasing the reaction efficiency of ion-molecule reactions and improving the detection sensitivity of etomidate in blood. Furthermore, the proposed injection method of pulse purge thermal desorption in bidirectional flow mode not only successfully improved the analysis performance of etomidate by increasing the effective injection amount of gaseous etomidate molecules, but also significantly reduced the impact of pressure fluctuation caused by the pulse on the quantitative accuracy. In view of the effective blood concentration range of etomidate from 1.25 to 3.75 ng μL−1, the proposed method with the quantitative range of 0.1–10 ng μL−1 was suitable for etomidate determination throughout the perioperative period. As a demonstration, the proposed method has been applied clinically to determine the concentration of etomidate in blood of patients undergoing tonsillectomy or nasal endoscopic functional sinus surgery.

The development of genus-specific and species-specific real-time PCR assays for the authentication of Patagonian toothfish and Antarctic toothfish in commercial seafood products.

The substitution or mislabeling of toothfish is an issue of significant concern for seafood authorities; it also reduces the effectiveness of marine conservation and management programs for its over-exploitation and illegal trafficking, boosting the need for identification methods. Two species-specific real-time polymerase chain reaction (PCR) assays for the identification of Patagonian toothfish (Dissostichus eleginoides) and Antarctic toothfish (Dissostichus mawsoni) and a genus-specific real-time PCR assay for Dissostichus spp. identification were developed based on fragments of the 16S rRNA and COI (cytochrome c oxidase subunit I) genes. These methods were confirmed to be rapid, simple, and sensitive (absolute sensitivity of 0.0002 ng μL-1 and relative sensitivity of 0.1g kg-1 with good specificity). These methods can be applied to processed and commercial fish products. These approaches can be beneficial for protecting both consumers and producers from economic fraud and might also help protect toothfish from over-exploitation as well as combat illegal, unreported, and unregulated (IUU) fisheries. © 2021 Society of Chemical Industry.

Absolute quantification of senescence mediators in cells using multiple reaction monitoring liquid chromatography-Tandem mass spectrometry

BackgroundThe identification of unique senescence markers remains challenging. Current hallmarks of senescent cells, including increased senescence-associated β-galactosidase activity, increased levels of cell cycle regulators such as p16INK4a, p27, and p53, and altered levels of sirtuins and lamins, are detected commonly by Western blot and immunohistochemistry methods. Mass spectrometry outperforms these conventional quantification methods in terms of high throughput, specificity, and reproducibility. ObjectivesTo develop multiple reaction monitoring-based tandem mass spectrometric senescence assay for simultaneous measuring of p16INK4a, p27, p53, p53-β, the seven proteins of the sirtuins family and the four transcript variants of lamins proteins in aging cell model and cancerous cell lines. MethodologyMultiple reaction monitoring-tandem mass transitions per protein were developed for each signature peptide(s) and stable isotope-labeled internal standard. The developed assay was validated in a matrix using breast cancer MCF7 cell lines according to the US-FDA guidelines for bioanalytical assays. ResultsThe analytes chromatographic peaks were baseline separated and showed linear behavior in a wide dynamic range with r2 ≥ 0.98. The method for all proteins has passed the inter/intra-day precision and accuracy validation using three levels of quality control samples. The accuracy and the precision for most analytes were 80–120% and ≤20%, respectively. The method's sensitivity for the panels' signature peptides ranged from 1 ng μL−1 to 1 μg mL−1. Extraction recovery assessed in two quality control levels was >60% for most analytes. This LC-MS-MS validated senescence assay showed reduced lamin A, lamin A△10, lamin A△50, SIRT1, SIRT3, SIRT5, p53, and p16INK4a, as well as p53-β induction, are implicated in replicative senescence. Meanwhile, increased lamin C: lamin A ratio was evident and can diagnose breast carcinogenesis. Moreover, in breast cancer metastasis, reduced SIRT2 and p27 and elevated levels of lamin A△50, SIRT5, SIRT7, and p53-β are evident. ConclusionLC-MS/MS is a potent alternative tool to the currently available assays. The high throughput method established can study senescence's role in different pathophysiological processes.