Direct multiplex recombinase polymerase amplification for rapid detection of Staphylococcus aureus and Pseudomonas aeruginosa in food

Foodborne illness undermines human health by causing fever, stomachache and even lethality. Among foodborne bacterial pathogens, Staphylococcus aureus and Pseudomonas aeruginosa are of extraordinary significance which drive reasons of food and beverage poisoning in numerous cases. Today, PCR has been widely used to examine the presence of different foodborne pathogens. However, PCR requires specialized equipment and skillful personnel which limit its application in the field. Recently, there is an emerging of isothermal PCR methods in which the reactions occur at low and constant temperatures, allowing their application in restricted-resource settings. In this work, multiplex Recombinase Polymerase Amplification (RPA) was used to simultaneously detect S. aureus and P. aeruginosa with high sensitivity and specificity. The limit detection of multiplex RPA was 10 and 30 fg/reaction of genomic DNAs of S. aureus and P. aeruginosa, respectively. Besides, the reaction time was reduced to only 25 minutes with a low incubation temperature of 39 °C. Markedly, multiplex RPA reactions succeeded to directly detect as low as 1 and 5 CFU/reaction of S. aureus and P. aeruginosa cells, respectively without the requirement of extracting DNA genome. Moreover, the multiplex RPA reliably detected the two foodborne bacteria in milk, fruit juice and bottled water samples. In general, the direct multiplex RPA described in this study is a rapid, simple, sensitive and efficient alternative tool that could be used to detect the presence of S. aureus and P. aeruginosa without the necessity of costly devices and high-trained staff.

Mercury Determination Using Stannous Chloride Reductant Followed by Atomic Absorption Spectrometric Measurement: Performance Characteristics, Uncertainty Estimation, and Compliance Assessment

Indonesian government has committed to reduce and eliminate mercury. Hence, the intensity of monitoring activities of mercury levels in various matrices would be increased and supported by qualified analytical data. Key characteristic performances, i.e., the limit of detection, linear range, precision, trueness, have been successfully carried out, and the method was shown to fit the purpose. The limit detection, LoD and LoQ, were found to be 0.26 and 0.86 µg/L, respectively, which were adequate to reach the tightest regulatory limit of mercury in surface water (1 µg/L). The examined linearity range of 1-20 µg/L has been found sufficient for its application since a high mercury concentration in the typical sample is seldomly expected. Precision and trueness aspects of the method were shown to have satisfaction performance, with CV of 1,24% and recovery of 104.54%. All the possible uncertainty sources have been identified in this study. Since no reference material was available, the uncertainty of bias was evaluated through the recovery of the spiked sample. Compliance assessment to six measurement results has been performed; one result was below LoQ, four were clearly below regulatory limit, and one was questionable. Hence a decision rule was applied.

Confirmatory Analysis of Per and Polyfluoroalkyl Substances in Milk and Infant Formula Using UHPLC–MS/MS

An ultra-high performance liquid chromatography tandem mass spectrometry method was developed and validated for the sensitive determination and unambiguous confirmation of residues of per and polyfluorinated alkyl substances (PFAS) in breastmilk, retail milk and infant formulas following two sample preparation methods. Sample pre-treatment was carried out by a simplified QuEChERS method without requiring dSPE or any further clean-up. The method was validated in accordance with the requirements of Commission Decision 657/2002/EC with slight modifications. The method displayed good linearity with R2 ranging from 0.9843–0.9998 for all target PFAS. The recovery and within-laboratory reproducibility of the method (n = 63) were in the range 60–121% and 5–28%, respectively. The decision limit, detection capability and limit of quantitation ranged from 30–60 ng kg−1 to 40–100 ng kg−1 and 5–50 ng kg−1, respectively. Acceptable matrix effect values in the range −45–29% were obtained with uncertainty of measurement lower than 25% for all target PFAS. The method displays its suitability for the sensitive and high-throughput confirmatory analysis of C4–C14 PFAS in breastmilk, dairy milk and infant formulas.

Hexaferrocenium tri[hexa(isothiocyanato)iron(III)] trihydroxonium complex as a new DNA intercalator for electrochemical DNA biosensor

Ferrocene or ferrocenium has been widely studied in the field of organometallic complexes because of its stable thermodynamic, kinetic and redox properties. Novel hexaferrocenium tri[hexa(isothiocyanato)iron(III)]trihydroxonium (HexaFc) complex was the product from the reaction of ferrocene, maleic acid and ammonium thiocyanate and was confirmed by elemental analysis CHNS, FTIR and single crystal X-ray crystallography. In this study, HexaFc was used for the first time as an electroactive indicator for porcine DNA biosensor. The UV–Vis DNA titrations with this compound showed hypochromism and redshift at 250 nm with increasing DNA concentrations. The binding constant (Kb) for HexaFc complex towards CT-DNA (calf-thymus DNA) was 3.1 × 104 M−1, indicated intercalator behaviour of the complex. To test the usefulness of this complex for DNA biosensor application, a porcine DNA biosensor was constructed. The recognition probes were covalently immobilised onto silica nanospheres (SiNSs) via glutaraldehyde linker on a screen-printed electrode (SPE). After intercalation with the HexaFc complex, the response of the biosensor to the complementary porcine DNA was measured using differential pulse voltammetry. The DNA biosensor demonstrated a linear response range to the complementary porcine DNA from 1 × 10−6 to 1 × 10−3 µM (R2 = 0.9642) with a limit detection of 4.83 × 10−8 µM and the response was stable up to 23 days of storage at 4 °C with 86% of its initial response. The results indicated that HexaFc complex is a feasible indicator for the DNA hybridisation without the use of a chemical label for the detection of porcine DNA.

Rhodamine phenol-based fluorescent probe for the visual detection of GB and its simulant DCP

Rhodamine phenol-based fluorescent probes have been synthesized. The probe RBNP demonstrates a rapid response and extreme low limit detection to diethylchlorophosphate and can rapidly and visually detect a real nerve agent GB in vapor.

Detection of Single Molecules Using Stochastic Resonance of Bistable Oligomers

Ultra-sensitive elements for nanoscale devices capable of detecting single molecules are in demand for many important applications. It is generally accepted that the inevitable stochastic disturbance of a sensing element by its surroundings will limit detection at the molecular level. However, a phenomenon exists (stochastic resonance) in which the environmental noise acts abnormally: it amplifies, rather than distorts, a weak signal. Stochastic resonance is inherent in non-linear bistable systems with criticality at which the bistability emerges. Our computer simulations have shown that the large-scale conformational dynamics of a short oligomeric fragment of thermosrespective polymer, poly-N-isopropylmethacrylamid, resemble the mechanical movement of nonlinear bistable systems. The oligomers we have studied demonstrate spontaneous vibrations and stochastic resonance activated by conventional thermal noise. We have observed reasonable shifts of the spontaneous vibrations and stochastic resonance modes when attaching an analyte molecule to the oligomer. Our simulations have shown that spontaneous vibrations and stochastic resonance of the bistable thermoresponsive oligomers are sensitive to both the analyte molecular mass and the binding affinity. All these effects indicate that the oligomers with mechanic-like bistability may be utilized as ultrasensitive operational units capable of detecting single molecules.

Hexaferrocenium Tri [Hexa(Isothiocyanato)Iron(III)] trihydroxonium Complex as a New DNA Intercalator for Electrochemical DNA Biosensor

Ferrocene or ferrocenium has been widely studied in the field of organometallic complexes because of its stable thermodynamic, kinetic and redox properties. Novel Hexaferrocenium Tri[Hexa(Isothiocyanato)Iron (III)]Trihydroxonium (HexaFc) complex was the product from the reaction of ferrocene, maleic acid and ammonium thiocyanate and it was confirmed by elemental analysis CHNS, FTIR and single crystal x-ray crystallography. In this study, HexaFc was used for the first time as an electroactive indicator for DNA hybridisation biosensor. The UV-Vis DNA titrations with this compound showed hypochromism and redshift at 250 nm with increasing DNA concentrations. The binding constant (Kb) for HexaFc complex towards CT-DNA (calf-thymus DNA) was 3.1 × 104 M−1. These results indicated intercalator behaviour of the complex. To test the usefulness of this complex for DNA biosensor application, a porcine DNA biosensor was constructed. The recognition probes were covalently immobilised onto silica nanospheres (SiNS) via glutaraldehyde linker on a screen-printed electrode (SPE). After intercalation with the complex, the biosensor response to complementary porcine DNA was measured using differential pulse voltammetry. The DNA biosensor demonstrated a linear response range to the complementary porcine DNA from 1 × 10−6 to 1 × 10−3 µM (R2 = 0.9642) with a limit detection of 4.83 × 10−8 µM. The results indicated that HexaFc complex is a feasible indicator for DNA hybridisation evaluation without the need of chemical attachment as a label.

Multifunctional rhodamine B appended ROMP derived fluorescent probe detects Al3+ and selectively labels lysosomes in live cells

There a few reports of rhodamine-based fluorescent sensors for selective detection of only Al3+, due to the challenge of identifying a suitable ligand for binding Al3+ ion. The use of fluorophore moieties appended to a polymer backbone for sensing applications is far from mature. Here, we report a new fluorescent probe/monomer 4 and its ROMP derived polymer P for specific detection of Al3+ ions. Both monomer 4 and its polymer P exhibit high selectivity toward only Al3+ with no interference from other metal ions, having a limit detection of 0.5 and 2.1 µM, respectively. The reversible recognition of monomer 4 and P for Al3+ was also proved in presence of Na2EDTA by both UV–Vis and fluorometric titration. The experimental data indicates the behavior of 4 and P toward Al3+ is pH independent in medium conditions. In addition, the switch-on luminescence response of 4 at acidic pH (0 < 5.0), allowed us to specifically stain lysosomes (pH ~ 4.5–5.0) in live cells.

Fabrikasi dan Karakterisasi Sensor Elektrokimia untuk Mendeteksi Kadmium Berbasis Teknologi Film Tebal

Electrochemical sensor has been fabrication and characterization electrode to detect heavy metals of cadmium with thick film technology. Fabrication electrodes electrochemical sensors start with the design stage of electrodes, screen maker, screen printing, and substrate cutting. The fabrication process produces a ten-pieces of sensors to be used to sensors characterization of SEM, XRD, and Cyclic Voltammetry (CV). The process of characterization produces morphology and the structure of the composites N/IL/G, and linieritas 0,9623, precision ranges between 4,11 and 10,35%, sensitivity of 0,04112 µA.L-1/mg.mm2, limit detection 0,18 mg/L, and accuracy between 79,5 and 126,8%.