Highly selective and sensitive detection of glutamate by an electrochemical aptasensor.

An electrochemical aptamer-based sensor was developed for glutamate, the major excitatory neurotransmitter in the central nervous system. Determining glutamic acid release and glutamic acid levels is crucial for studying signal transmission and for diagnosing pathological conditions in the brain. Glutamic acid-selective oligonucleotides were isolated from an ssDNA library using the Capture-SELEX protocol in complex medium. The selection permitted the isolation of an aptamer 1d04 with a dissociation constant of 12 µM. The aptamer sequence was further used in the development of an electrochemical aptamer sensor. For this purpose, a truncated aptamer sequence named glu1 was labelled with a ferrocene redox tag at the 3'-end and immobilized on a gold electrode surface via Au-thiol bonds. Using 6-mercapto-1-hexanol as the backfill, the sensor performance was characterized by alternating current voltammetry. The glu1 aptasensor showed a limit of detection of 0.0013 pM, a wide detection range between 0.01 pM and 1 nM, and good selectivity for glutamate in tenfold diluted human serum. With this enzyme-free aptasensor, the highly selective and sensitive detection of glutamate was demonstrated, which possesses great potential for implementation in microelectrodes and for in vitro as well as in vivo monitoring of neurotransmitter release.

Electrophysiological and Behavioral Responses of an Ambrosia Beetle to Volatiles of its Nutritional Fungal Symbiont.

Ambrosia beetles (Coleoptera: Scolytinae) cultivate their fungal symbiont within host substrates as the sole source of nutrition on which the larvae and adults must feed. To investigate a possible role for semiochemicals in this interaction, we characterized electrophysiological and behavioral responses of Xylosandrus germanus to volatiles associated with its fungal symbiont Ambrosiella grosmanniae. During still-air walking bioassays, X. germanus exhibited an arrestment response to volatiles of A. grosmanniae, but not antagonistic fungi Beauveria bassiana, Metarhizium brunneum, Trichoderma harzianum, the plant pathogen Fusarium proliferatum, or malt extract agar. Solid phase microextraction-gas chromatography-mass spectrometry identified 2-ethyl-1-hexanol, 2-phenylethanol, methyl benzoate and 3-methyl-1-butanol in emissions from A. grosmanniae; the latter two compounds were also detected in emissions from B. bassiana. Concentration-responses using electroantennography documented weak depolarizations to A. grosmanniae fungal volatiles, unlike the comparatively strong response to ethanol. When tested singly in walking bioassays, volatiles identified from A. grosmanniae elicited relatively weak arrestment responses, unlike the responses to ethanol. Xylosandrus germanus also exhibited weak or no long-range attraction to the fungal volatiles when tested singly during field trials in 2016-2018. None of the fungal volatiles enhanced attraction of X. germanus to ethanol when tested singly; in contrast, 2-phenylethanol and 3-methyl-1-butanol consistently reduced attraction to ethanol. Volatiles emitted by A. grosmanniae may represent short-range olfactory cues that could aid in distinguishing their nutritional fungal symbiont from other fungi, but these compounds are not likely to be useful as long-range attractants for improving detection or mass trapping tactics.

A Complementary and Revised View on the N-Acylation of Chitosan with Hexanoyl Chloride.

The modification of the biobased polymer chitosan is a broad and widely studied field. Herein, an insight into the hydrophobization of low-molecular-weight chitosan by substitution of amino functionalities with hexanoyl chloride is reported. Thereby, the influence of the pH of the reaction media was investigated. Further, methods for the determination of the degree of substitution based on 1H-NMR, FTIR, and potentiometric titration were compared and discussed regarding their accuracy and precision. 1H-NMR was the most accurate method, while FTIR and the potentiometric titration, though precise and reproducible, underlie the influence of complete protonation and solubility issues. Additionally, the impact of the pH variation during the synthesis on the properties of the samples was investigated by Cd2+ sorption experiments. The adjusted pH values during the synthesis and, therefore, the obtained degrees of substitution possessed a strong impact on the adsorption properties of the final material.

Host-Seeking Behavior of Aphidius gifuensis (Hymenoptera: Braconidae) Modulated by Chemical Cues Within a Tritrophic Context.

Aphidius gifuensis Ashmaed is a generalist endoparasitoid that parasitizes a variety of aphid species. In China, it is widely used as a biological control agent to protect vegetables and tobaccos in open fields; control efficiency is largely dependent on its host-seeking ability. In this study, a six-choice olfactometer was used to investigate the olfactory responses of A. gifuensis to tobacco plants that had suffered damage (either varying degrees of mechanical damage or from aphid-feeding at different time intervals) and tobacco volatiles with different dosages. Furthermore, the regularity of A. gifuensis females' response toward an aphid/tobacco complex was monitored using a Y-tube olfactometer. Our findings suggest that tobacco plants are significantly attractive to A. gifuensis after they have been punctured with 50 holes, or housed with Myzus persicae (Sulzer) at a density of 400 aphids, except at an infestation time of 12 h. Moreover, aphid density had a more significant effect on the response than the time interval since aphid application. Aphidius gifuensis was found to be active during the daytime and preferred to search for their aphid hosts at 14:00 h. Five EAG-active tobacco volatiles (trans-2-hexenal, methyl salicylate, benzaldehyde, cis-3-hexen-1-ol, and 1-hexanal) were found to significantly attract A. gifuensis females at different concentration ranges. The practical implications of these results are discussed in the framework of the sustainable biological control of pest aphids in agricultural production systems.

The Impact of an Anchoring Layer on the Formation of Tethered Bilayer Lipid Membranes on Silver Substrates.

Tethered bilayer lipid membranes (tBLMs) have been known as stable and versatile experimental platforms for protein-membrane interaction studies. In this work, the assembly of functional tBLMs on silver substrates and the effect of the molecular chain-length of backfiller molecules on their properties were investigated. The following backfillers 3-mercapto-1-propanol (3M1P), 4-mercapto-1-butanol (4M1B), 6-mercapto-1-hexanol (6M1H), and 9-mercapto-1-nonanol (9M1N) mixed with the molecular anchor WC14 (20-tetradecyloxy-3,6,9,12,15,18,22 heptaoxahexatricontane-1-thiol) were used to form self-assembled monolayers (SAMs) on silver, which influenced a fusion of multilamellar vesicles and the formation of tBLMs. Spectroscopic analysis by SERS and RAIRS has shown that by using different-length backfiller molecules, it is possible to control WC14 anchor molecules orientation on the surface. An introduction of increasingly longer surface backfillers in the mixed SAM may be related to the increasing SAMs molecular order and more vertical orientation of WC14 at both the hydrophilic ethylenoxide segment and the hydrophobic lipid bilayer anchoring alkane chains. Since no clustering of WC14 alkane chains, which is deleterious for tBLM integrity, was observed on dry samples, the suitability of mixed-component SAMs for subsequent tBLM formation was further interrogated by electrochemical impedance spectroscopy (EIS). EIS showed the arrangement of well-insulating tBLMs if 3M1P was used as a backfiller. An increase in the length of the backfiller led to increased defectiveness of tBLMs. Despite variable defectiveness, all tBLMs responded to the pore-forming cholesterol-dependent cytolysin, vaginolysin in a manner consistent with the functional reconstitution of the toxin into phospholipid bilayer. This experiment demonstrates the biological relevance of tBLMs assembled on silver surfaces and indicates their utility as biosensing elements for the detection of pore-forming toxins in liquid samples.

Attachment of Single-Stranded DNA to Certain SERS-Active Gold and Silver Substrates: Selected Practical Tips.

Layers formed from single-stranded DNA on nanostructured plasmonic metals can be applied as "working elements" in surface-enhanced Raman scattering (SERS) sensors used to sensitively and accurately identify specific DNA fragments in various biological samples (for example, in samples of blood). Therefore, the proper formation of the desired DNA layers on SERS substrates is of great practical importance, and many research groups are working to improve the process in forming such structures. In this work, we propose two modifications of a standard method used for depositing DNA with an attached linking thiol moiety on certain SERS-active structures; the modifications yield DNA layers that generate a stronger SERS signal. We propose: (i) freezing the sample when forming DNA layers on the nanoparticles, and (ii) when forming DNA layers on SERS-active macroscopic silver substrates, using ω-substituted alkanethiols with very short alkane chains (such as cysteamine or mercaptopropionic acid) to backfill the empty spaces on the metal surface unoccupied by DNA. When 6-mercapto-1-hexanol is used to fill the unoccupied places on a silver surface (as in experiments on standard gold substrates), a quick detachment of chemisorbed DNA from the silver surface is observed. Whereas, using ω-substituted alkanethiols with a shorter alkane chain makes it possible to easily form mixed DNA/backfilling thiol monolayers. Probably, the significantly lower desorption rate of the thiolated DNA induced by alkanethiols with shorter chains is due to the lower stabilization energy in monolayers formed from such compounds.

Subsecond-Resolved Molecular Measurements Using Electrochemical Phase Interrogation of Aptamer-Based Sensors.

Recent years have seen the development of a number of biosensor architectures that rely on target binding-induced changes in the rate of electron transfer from an electrode-bound receptor. Most often, the interrogation of these sensors has relied on voltammetric methods, such as square-wave voltammetry, which limit their time resolution to a few seconds. Here, we describe the use of an impedance-based approach, which we have termed electrochemical phase interrogation, as a means of collecting high time resolution measurements with sensors in this class. Specifically, using changes in the electrochemical phase to monitor target binding in an electrochemical-aptamer based (EAB) sensor, we achieve subsecond temporal resolution and multihour stability in measurements performed directly in undiluted whole blood. Electrochemical phase interrogation also offers improved insights into EAB sensors' signaling mechanism. By modeling the interfacial resistance and capacitance using equivalent circuits, we find that the only parameter that is altered by target binding is the charge-transfer resistance. This confirms previous claims that binding-induced changes in electron-transfer kinetics drive signaling in this class of sensors. Considering that a wide range of electrochemical biosensor architectures rely on this signaling mechanism, we believe that electrochemical phase interrogation may prove generalizable toward subsecond measurements of molecular targets.

Identification of a new class of non-electrophilic TRPA1 agonists by a structure-based virtual screening approach.

Recent work has gradually been clarifying the binding site of non-electrophilic agonists on the transient receptor potential A1 (TRPA1). This study searched for non-electrophilic TRPA1 agonists by means of in silico drug discovery techniques based on three-dimensional (3-D) protein structure. First, agonist-bound pocket structures were explored using an advanced molecular dynamics simulation starting from the cryo-electron microscopic structure of TRPA1, and several pocket structures suitable for virtual screening were extracted by structure evaluation using known non-electrophilic TRPA1 agonists. Next, 49 compounds were selected as new non-electrophilic agonist candidates from a library of natural products comprising 10,555 compounds by molecular docking toward these pocket structures. Measurement of the TRPA1 agonist activity of these compounds showed notable TRPA1 activation with three compounds (decanol, 2-ethyl-1-hexanol, phenethyl butanoate). Decanol and 2-ethyl-1-hexanol, which are categorized as fatty alcohols, in particular have a novel chemical scaffold for TRPA1 activation. The results of this study are expected to be of considerable use in understanding the molecular mechanism of TRPA1 recognition by non-electrophilic agonists.

Label-Free Detection of Zeptomol miRNA via Peptide Nucleic Acid Hybridization Using Novel Cyclic Voltammetry Method.

To harness the applicability of microribonucleic acid (miRNA) as a cancer biomarker, the detection sensitivity of serum miRNA needs to be improved. This study evaluated the detection sensitivity of miRNA hybridization using cyclic voltammograms (CVs) and microelectrode array chips modified with peptide nucleic acid (PNA) probes and 6-hydroxy-1-hexanethiol. We investigated the PNA probe modification pattern on array chips using fluorescently labeled cDNA. The pattern was not uniformly spread over the working electrode (WE) and had a one-dimensional swirl-like pattern. Accordingly, we established a new ion-channel sensor model wherein the WE is negatively biased through the conductive π-π stacks of the PNA/DNA duplexes. This paper discusses the mechanism underlying the voltage shift in the CV curves based on the electric double-layer capacitance. Additionally, the novel hybridization evaluation parameter ΔE is introduced. Compared to conventional evaluation using oxidation current changes, ΔE was more sensitive. Using ΔE and a new hybridization system for ultrasmall amounts of aqueous solutions (as low as 35 pL), 140 zeptomol label-free miRNA were detected without polymerase chain reaction (PCR) amplification at an adequate sensitivity. Herein, the differences in the target molar amount and molar concentration are elucidated from the viewpoint of hybridization sensitivity.

Identification of the Volatile Components of Galium verum L. and Cruciata leavipes Opiz from the Western Italian Alps.

The chemical composition of the volatile fraction from Galium verum L. (leaves and flowers) and Cruciata laevipes Opiz (whole plant), Rubiaceae, was investigated. Samples from these two plant species were collected at full bloom in Val di Susa (Western Alps, Turin, Italy), distilled in a Clevenger-type apparatus, and analyzed by GC/FID and GC/MS. A total of more than 70 compounds were identified, making up 92%-98% of the total oil. Chemical investigation of their essential oils indicated a quite different composition between G. verum and C. laevipes, both in terms of the major constituents and the dominant chemical classes of the specialized metabolites. The most abundant compounds identified in the essential oils from G. verum were 2-methylbenzaldheyde (26.27%, corresponding to 11.59 µg/g of fresh plant material) in the leaves and germacrene D (27.70%; 61.63 µg/g) in the flowers. C. laevipes essential oils were instead characterized by two sesquiterpenes, namely ß-caryophyllene (19.90%; 15.68 µg/g) and trans-muurola-4(15),5-diene (7.60%; 5.99 µg/g); two phenylpropanoids, benzyl alcohol (8.30%; 6.71 µg/g), and phenylacetaldehyde (7.74%; 6.26 µg/g); and the green-leaf alcohol cis-3-hexen-1-ol (9.69%; 7.84 µg/g). The ecological significance of the presence of such compounds is discussed.

Structural Changes of Mercaptohexanol Self-Assembled Monolayers on Gold and Their Influence on Impedimetric Aptamer Sensors.

Despite a large number of publications describing biosensors based on electrochemical impedance spectroscopy (EIS), little attention has been paid to the stability and reproducibility issues of the sensor interfaces. In this work, the stability and reproducibility of faradaic EIS analyses on the aptamer/mercaptohexanol (MCH) self-assembled monolayer (SAM)-functionalized gold surfaces in ferri- and ferrocyanide solution were systematically evaluated prior to and after the aptamer-probe DNA hybridization. It is shown that the EIS data exhibited significant drift, and this significantly affected the reproducibility of the EIS signal of the hybridization. As a result, no significant difference between the charge transfer resistance (RCT) changes induced by the aptamer-target DNA hybridization and that caused by the drift could be identified. A conditioning of the electrode in the measurement solution for more than 12 h was required to reach a stable RCT baseline prior to the aptamer-probe DNA hybridization. The monitored drift in RCT and double layer capacitance during the conditioning suggests that the MCH SAM on the gold surface reorganized to a thinner but more closely packed layer. We also observed that the hot binding buffer used in the following aptamer-probe DNA hybridization process could induce additional MCH and aptamer reorganization, and thus further drift in RCT. As a result, the RCT change caused by the aptamer-probe DNA hybridization was less than that caused by the hot binding buffer (blank control experiment). Therefore, it is suggested that the use of high temperature in the EIS measurement should be carefully evaluated or avoided. This work provides practical guidelines for the EIS measurements. Moreover, because SAM-functionalized gold electrodes are widely used in biosensors, for example, DNA sensors, an improved understanding of the origin of the observed drift is very important for the development of well-functioning and reproducible biosensors.

Quantification of controlled release leuprolide and triptorelin in rabbit plasma using electromembrane extraction coupled with HPLC-UV.

An electromembrane extraction followed by HPLC-UV technique was developed and validated for quantification of leuprolide and triptorelin in rabbit plasma. The influencing parameters on the extraction efficiency were optimized using experimental design methodology. The optimized conditions were found to be; supported liquid membrane: a mixture of 1-octanol and 2-ethyl hexanol (1:1) containing 10% v/v di(2-ethylhexyl) phosphate, applied voltage: 5 V, extraction time: 5 min, pH of the donor phase: 4.5 and pH of the acceptor phase: 1.0. The optimized method was validated for linearity, intraday and interday precision, and accuracy in rabbit plasma. The range of quantification for both peptides was 0.5-1000 ng/mL with regression coefficients higher than 0.994. Relative recoveries of leuprolide and triptorelin were found to be 80.3 and 75.5%, respectively. Limits of quantification and detection for both peptides were found to be 0.5 and 0.15 ng/mL, respectively. The validated method was successfully applied to pharmacokinetic study of the 1-month depot formulations of each peptide after subcutaneous administration to rabbits.

Resolution of (R,S)-ibuprofen catalyzed by immobilized Novozym40086 in organic phase.

The enantioselective esterification of ibuprofen catalyzed by Novozym40086 was successfully conducted in organic solvent. Removing-water reagent was added into the reaction mixture to remove water produced in the esterification. The effects of temperature, n-hexanol concentration, ibuprofen concentration, and loading of enzymes were investigated. Under the condition of equilibrium, the thermodynamic equilibrium constant (K) of 7.697 and enantioselectivity (E) of 8.512 were obtained. The esterification reaction achieved its equilibrium in approximately 30 hours with conversion of 56% and eeS of 93.78%. The predicted values of X and eeS were 67.90% and 95.60%, respectively. The experimental value is approximately equal to the theoretical value, which indicates the feasibility of ideal models.

Electromembrane extraction of verapamil and riluzole from urine and wastewater samples using a mixture of organic solvents as a supported liquid membrane: Study on electric current variations.

In this study, the application of a mixture of organic solvents as a supported liquid membrane for improving the efficiency of the electromembrane extraction procedure was investigated. The extraction process was followed by high-performance liquid chromatography analysis of two model drugs (verapamil and riluzole). In this research, four organic solvents, including 1-heptanol, 1-octanol, 2-nitrophenyl octyl ether, and 2-ethyl hexanol, were selected as model solvents and different binary mixtures (v/v 2:1, 1:1 and 1:2) were used as the supported liquid membrane. The mixture of 2-ethyl hexanol and 1-otanol (v/v, 2:1) improved the extraction efficiency of model drugs by 1.5 to 12 times. It was found that extraction efficiency is greatly influenced by the level of electric current. In this study, for various mixtures of organic solvents, the electric current fluctuated between 50 and 2500 µA, and the highest extraction efficiencies were obtained with low and stable electric currents. Finally, the optimized extraction condition was validated and applied for the determination of model drugs in urine and wastewater samples.

Aptamer based ratiometric electrochemical sensing of 17ß-estradiol using an electrode modified with gold nanoparticles, thionine, and multiwalled carbon nanotubes.

Gold nanoparticles (AuNPs) with a size of ~3 nm were placed on a thionine-multiwalled carbon nanotube (Thi-CNTs) conjugate to form a novel AuNP-Thi-CNTs nanocomposite. Its morphology and composition were characterized by transmission electron microscopy, atomic force microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The nanocomposite was placed on an electrode and used as a redox-active signaling interface to fabricate a ratiometric electrochemical aptasensor for 17ß-estradiol (E2). The potentiostatic insertion method was applied to insert an aptamer against E2 into a thin alkane monolayer to warrant an adequate distance between aptamers. The aptamer against E2 acts as both a collector and separator to specifically bind E2. The electrode displays two peak signals (at +0.50 V vs. SCE for E2; and at -0.32 V for Thi) which increase and decrease, respectively, in the 12 pM to 60 nM E2 concentration range. Therefore, the current ratio can be used to reliably, reproducibly, and sensitively quantify the concentration of E2. Graphical abstract Schematic presentation of a novel AuNP-Thi-CNTs nanocomposite. AuNP-Thi-CNTs showed good electrocatalytic oxidation to E2. AuNP-Thi-CNTs was used as self-redox signal interface to fabricate aptasensor. Dual signals of extrinsic E2 and inner Thi was applied to monitor the concentration of E2.

High-Speed Microscopy of Diffusion in Pore-Spanning Lipid Membranes.

Pore-spanning membranes (PSMs) provide a highly attractive model system for investigating fundamental processes in lipid bilayers. We measure and compare lipid diffusion in the supported and suspended regions of PSMs prepared on a microfabricated porous substrate. Although some properties of the suspended regions in PSMs have been characterized using fluorescence studies, it has not been possible to examine the mobility of membrane components on the supported membrane parts. Here, we resolve this issue by employing interferometric scattering microscopy (iSCAT). We study the location-dependent diffusion of DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) lipids (DOPE) labeled with gold nanoparticles in (1,2-dioleoyl-sn-glycero-3-phosphocholine) (DOPC) bilayers prepared on holey silicon nitride substrates that were either (i) oxygen-plasma-treated or (ii) functionalized with gold and 6-mercapto-1-hexanol. For both substrate treatments, diffusion in regions suspended on pores with diameters of 5 µm is found to be free. In the case of functionalization with gold and 6-mercapto-1-hexanol, similar diffusion coefficients are obtained for both the suspended and the supported regions, whereas for oxygen-plasma-treated surfaces, diffusion is almost 4 times slower in the supported parts of the membranes. We attribute this reduced diffusion on the supported parts in the case of oxygen-plasma-treated surfaces to larger membrane-substrate interactions, which lead to a higher membrane tension in the freestanding membrane parts. Furthermore, we find clear indications for a decrease of the diffusion constant in the freestanding regions away from the pore center. We provide a detailed characterization of the diffusion behavior in these membrane systems and discuss future directions.

A new biosensor for osteoporosis detection.

Osteoporosis is a disease that is characterized by deterioration of bone tissue and increased risk of fracture as it leads to a decrease in bone mineral density, which is an important public health problem. Today, bone mineral density is measured by radiological techniques. Alternative techniques are needed because of the disadvantages such as excessive radiation intake, the cost of radiological techniques, and the necessity for specialist personnel for the devices. The quantitative determination of biochemical markers that play a role in bone mineralization may be a good alternative for the osteoporosis diagnosis and especially in the follow-up of treatment. In this study, a specific and sensitive immunological biosensor, which quantitatively determines the osteocalcin molecule, has been developed to be used in the early osteoporosis diagnosis and to evaluate the response to the drug treatment. Anti-osteocalcin antibody was immobilized onto gold electrode surface via covalent immobilization method by using 6-mercaptohexanol, 1,4-butanedioldiglycidyl ether, ethanolamine, and glutaraldehyde. Immobilization steps and biosensor characterization were specified by cyclic voltammetry and electrochemical impedance spectroscopy. The detection time and range of Ocn biosensor were determined as 45 min and 10-60 pg µL-1 Ocn concentration, respectively. The Ocn biosensor was successfully applied in artificial serum samples spiked with Ocn.

Characterisation of the Convective Hot-Air Drying and Vacuum Microwave Drying of Cassia alata: Antioxidant Activity, Essential Oil Volatile Composition and Quality Studies.

The preservation of active constituents in Cassia alata through the removal of moisture is crucial in producing a final product with high antioxidant activity. This study aims to determine the influences of various drying methods and drying conditions on the antioxidant activity, volatiles and phytosterols content of C. alata. The drying methods used were convective drying (CD) at 40 °C, 50 °C and 60 °C; freeze drying; vacuum microwave drying (VMD) at 6, 9 and 12 W/g; and two-stage convective pre-drying followed by vacuum microwave finish drying (CPD-VMFD) at 50 °C and 9 W/g. The drying kinetics of C. alata are best described by the thin-layer model (modified Page model). The highest antioxidant activity, TPC and volatile concentration were achieved with CD at 40 °C. GC-MS analysis identified the presence of 51 volatiles, which were mostly present in all samples but with quantitative variation. The dominant volatiles in fresh C. alata are 2-hexenal (60.28 mg 100 g-1 db), 1-hexanol (18.70 mg 100 g-1 db) and salicylic acid (15.05 mg 100 g-1 db). The concentration of phytosterols in fresh sample was 3647.48 mg 100 g-1 db, and the major phytosterols present in fresh and dried samples were ß-sitosterol (1162.24 mg 100 g-1 db). CPD-VMFD was effective in ensuring the preservation of higher phytosterol content in comparison with CD at 50 °C. The final recommendation of a suitable drying method to dehydrate C. alata leaves is CD at 40 °C.

High-Throughput/High-Precision Sampling of Single Cells into ICP-MS for Elucidating Cellular Nanoparticles.

In single-cell analysis with ICP-MS it is highly important to ensure precise single-cell sampling into ICP. For this purpose, a simple configured pressure-resistant MicroCross interface is developed for high-throughput/high-precision microdroplet generation and single-cell encapsulation. Aqueous cell suspension is ejected and sheared into droplets by tangent-flowing hexanol-continuous phases in the flow-focusing geometry of MicroCross, wherein to precisely trap a single cell into a droplet, with an extremely low probability of <0.005% for a single droplet encapsulating two cells. MicroCross interface is coupled with time-resolved ICP-MS (TRA-ICP-MS) for quantifying nanoparticles in single MCF-7 cells. At the optimal conditions, sufficient temporal-spatial resolution of the microdroplets is achieved facilitating high-throughput sampling of single cells into ICP. For solving the serious carbon deposition on the sampling cone and the unstable plasma torch caused by incomplete oxidation of hexanol phase in ICP, dimethyl carbonate (DMC) is for the first time used as a superb oxygen compensation reagent, which ensures adequate oxidation of hexanol, effectively eliminates the carbon deposition, and maintains a stable plasma. The single-cell analysis results indicated a remarkable discrepancy of the number of nanoparticles among the individual cells, falling into a range of 130-584 per MCF-7 cell in the case of AuNPs.

An impedimetric aptasensor for Shigella dysenteriae using a gold nanoparticle-modified glassy carbon electrode.

This work describes an aptasensor for the foodborne pathogen Shigella dysenteriae (S. dysenteriae). A glassy carbon electrode (GCE) was modified with gold nanoparticles (AuNPs) by electrodeposition. Then, thiolated aptamer for S. dysenteriae detection was self-assembled on the surface of the modified GCE, and any free residual AuNPs were blocked with 6-mercapto-1-hexanol. The size, morphology, and distribution of the AuNPs were characterized by field emission scanning electron microscopy. Detection of S. dysenteriae was performed measurement of the charge transfer resistance (Rct) before and after addition of S. dysenteriae using hexacyanoferrate as an electrochemical probe. The interaction between the aptamer and outer-membrane proteins of S. dysenteriae lead to an increase in the Rct of the sensor. The assay has a linear dynamic range that extends from 101 to 106 CFU.mL-1 and a limit of detection of 100 CFU.mL-1. It can differentiate between alive S. dysenteriae and other pathogens. Dead S. dysenteriae cells do not have any effect on selectivity. Unpasteurized and pasteurized skim milk and some water samples were spiked with S. dysenteriae and then successfully examined by this method. The results were validated by real-time PCR. The method is fast, low-cost, highly sensitive, and specific. Hence, it represents a valuable tool in food quality control. Graphical abstract Schematic presentation of a label free impedimetric aptasensor for Shigella dysenteriae using a glassy carbon electrode modified with gold nanoparticles (AuNPs) and 6-mercapto-1-hexanol (MCH). The limit of detection of this aptasensor is as low as 1 CFU.mL-1 for target bacteria.