Antimicrobial peptide-based array for Escherichia coli and Salmonella screening

A method based on solid-phase microextraction (SPME) followed by gas chromatography with microwave-induced plasma atomic emission detection for determining 16 pesticides of different chemical families (organochlorines, organophosphorus compounds and pyrethrins) in honey is proposed. Parameters affecting the sample enrichment step, such as sample mass, ionic strength, absorption and desorption times and temperatures, were carefully optimized in the direct immersion mode. Element-specific detection and quantification was carried out by monitoring the chlorine (479 nm), bromine (478 nm) and sulphur (181 nm) emission lines, which provided nearly specific chromatograms. The matrix effect was evaluated for samples of different floral origin, it being concluded that standard addition calibration was required for quantification purposes. The detection limits ranged from 0.02 to 10 ng g⁻¹, depending on the compound and the honey sample under analysis. The method is reliable and can be considered useful for routine monitoring. None of the honey samples analyzed contained the studied compounds at concentrations above the corresponding detection limits.     

Rapid label-free visual assay for the detection and quantification of viral RNA using peptide nucleic acid (PNA) and gold nanoparticles (AuNPs)

A rapid label-free visual assay for the detection of viral RNA using peptide nucleic acid (PNA) probes and gold nanoparticles (AuNPs) is presented in this study. Diagnosis is a crucial step for the molecular surveillance of diseases, and a rapid visual test with high specificity could play a vital role in the management of viral diseases. In this assay, the specific agglomerative behavior of PNA with gold nanoparticles was manipulated by its complementation with viral RNA. The assay was able to detect 5–10 ng of viral RNA from various biological samples, such as allantoic fluids, cell culture fluids and vaccines, in 100 μl of test solution. The developed assay was more sensitive than a hemagglutination (HA) test, a routine platform test for the detection of Newcastle disease virus (NDV), and the developed assay was able to visually detect NDV with as little as 0.25 HA units of virus. In terms of the specificity, the test could discriminate single nucleotide differences in the target RNA and hence could provide visual viral genotyping/pathotyping. This observation was confirmed by pathotyping different known isolates of NDV. Further, the PNA-induced colorimetric changes in the presence of the target RNA at different RNA to PNA ratios yielded a standard curve with a linear coefficient of R² = 0.990, which was comparable to the value of R² = 0.995 from real-time PCR experiments with the same viral RNA. Therefore, the viral RNA in a given samples could be quantified using a simple visual spectrophotometer available in any clinical laboratory. This assay may find application in diagnostic assays for other RNA viruses, which are well known to undergo mutations, thus presenting challenges for their molecular surveillance, genotyping and quantification.     

Optofluidic microsystem with quasi-3 dimensional gold plasmonic nanostructure arrays for online sensitive and reproducible SERS detection

Practical applications of chemical and biological detections through surface-enhanced Raman scattering (SERS) require high reproducibility, sensitivity, and efficiency, along with low-cost, straightforward fabrication. In this work, we integrated a poly-(dimethylsiloxane) (PDMS) chip with quasi-3D gold plasmonic nanostructure arrays (Q3D-PNAs), which serve as SERS-active substrates, into an optofluidic microsystem for online sensitive and reproducible SERS detections. The Q3D-PNA PDMS chip was fabricated through soft lithography to ensure both precision and low-cost fabrication. The optimal dimension of the Q3D-PNA in PDMS was designed using finite-difference time-domain (FDTD) electromagnetic simulations with a simulated enhancement factor (EF) of 1.6 × 10⁶. The real-time monitoring capability of the SERS-based optofluidic microsystem was investigated by kinetic on/off experiments through alternatively flowing Rhodamine 6G (R6G) and ethanol in the microfluidic channel. A switch-off time of ∼2 min at a flow rate of 0.3 mL min⁻¹ was demonstrated. When applied to the detection of low concentration malathion, the SERS-based optofluidic microsystem with Q3D-PNAs showed high reproducibility, significantly improved efficiency and higher detection sensitivity via increasing the flow rate. The optofluidic microsystem presented in this paper offers a simple and low-cost approach for online, label-free chemical and biological analysis and sensing with high sensitivity, reproducibility, efficiency, and molecular specificity.     

Modified interdigitated arrays by novel poly(1,8-diaminonaphthalene)/carbon nanotubes composite for selective detection of mercury(II)

This study describes a novel type of interdigitated arrays (IDA), microfabricated by electropolymerizing structured Poly(1,8-diaminonaphthalene)/functionalized multi-walled carbon nanotubes (PDAN/CNT) thin film onto a silicon chip for square wave voltammetry (SWV) multi-element heavy metal ion detection. The structure of PDAN/CNT was characterized by Raman, FE-SEM and AFM techniques. Analysed experiments reveal that the uptake of Hg²⁺ by PDAN/CNT is quite specific and it can be used advantageously for electrochemical sensing of Hg²⁺ thanks to original feature of (Hg²⁺/Hg₂²⁺) redox potential with the respect to that of PDAN/CNT. As-developed IDA type electrode can extend its utility in other sensing applications.     

Methoxy poly(ethylene glycol)-b-poly(valerolactone) diblock polymeric micelles for enhanced encapsulation and protection of camptothecin

Amphiphilic block copolymers, methoxy poly(ethylene glycol)-b-poly(valerolactone) (mPEG-b-PVL), were synthesized via ring opening polymerization of δ-valerolactone in the presence of methoxy poly(ethylene glycol) (mPEG). The copolymers form micelle-like nanoparticles by their amphiphilic characteristics and their structures were examined by Nuclear Magnetic Resonance (NMR). The sizes of nanoparticles ranged from 60 to 120 nm as measured by dynamic light scattering detection, and were larger with higher molecular weight of the copolymers. The Critical Micelle Concentration (CMC) of these nanoparticles in water decreased with increasing molecular weight of hydrophobic segment. Stability analysis showed that the micellar solutions maintain their sizes at 37 °C for six weeks without aggregation or dissociation. The lyophilization method was better than the evaporation method when camptothecin (CPT) was incorporated to the micelles. The former method yielded higher CPT loading efficiency and lower aggregation. The loading efficiency of CPT could be more than 96% and a steady release rate of CPT was kept for twenty six days. Moreover, the mPEG-b-PVL polymeric micelles offered good protection of CPT lactone form at 37 °C for sixteen days. The copolymers showed no cytotoxicity towards L929 mouse muscular cells when incubated for one day. Taken together, the mPEG-b-PVL copolymer has potential to be used for the delivery of CPT or other similar drugs.     

A highly specific immunoassay for microcystin-LR detection based on a monoclonal antibody

Microcystins (MC) are cyanobacterial hepatotoxins responsible for animal-poisoning and human health incidents. Immunoassays provide a sensitive and fast means to detect these toxins, but cross-reactivity (CR) characteristic of different antibodies was variable. Here, we have produced and characterized a monoclonal antibody (Clone MC8C10) with highly specificity against the most frequent and most toxic variant of microcystins, MC-LR. MC8C10 is more specific against MC-LR among the reported antibodies before. The immunogen was synthesized from the modified MC-LR and bovine serum albumin (BSA). An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) with MC8C10 was established to detect the MCs in waters, which showed highly specificity with MC-LR, and have a detection limit for MC-LR 0.1 μg L⁻¹, the 50% inhibition concentration (IC₅₀) for MC-LR was 1.8 ± 0.1 μg L⁻¹ and the quantitative detection range was from 0.3 to 10 μg L⁻¹. The [4-arginine] microcystins and the nodularin-R showed lower cross-reactivates (CR < 10%), and other MCs such as MC-LF and MC-LW are not recognized (CR < 10⁻⁴). The analysis results of real water samples with ic-ELISA showed that all the coefficients of variation were less than 15%, and the recovery was (100.3 ± 5.9)%. So the highly specific ic-ELISA will commendably suit for sensitive analysis for MC-LR in surface water as well as drinking water.     

Functionalized self-assembled monolayers for measuring single molecule lectin carbohydrate interactions

The specific interactions between sugar-binding proteins (lectins) and their complementary carbohydrates mediate several complex biological functions. There is a great deal of interest in uncovering the molecular basis of these interactions. In this study, we demonstrate the use of an efficient one-step amination reaction strategy to fabricate carbohydrate arrays based on mixed self-assembled monolayers. These allow specific lectin carbohydrate interactions to be interrogated at the single molecule level via AFM. The force required to directly rupture the multivalent bonds between Concanavalin A (Con A) and mannose were subsequently determined by chemical force microscopy. The mixed self-assembled monolayer provides a versatile platform with active groups to attach a 1-amino-1-deoxy sugar or a protein (Con A) while minimizing non-specific adhesion enabling quick and reliable detection of rupture forces. By altering the pH of the environment, the aggregation state of Con A was regulated, resulting in different dominant rupture forces, corresponding to di-, tri- and multiple unbinding events. We estimate the value of the rupture force for a single Con A-mannose bond to be 95 ± 10 pN. The rupture force is consistent even when the positions of the binding molecules are switched. We show that this synthesis strategy in conjunction with a mixed SAM allows determination of single molecules bond with high specificity, and may be used to investigate lectin carbohydrate interactions in the form of carbohydrate arrays as well as lectin arrays.     

A dual column system using agarose-based adsorbents for preconcentration and speciation of chromium in water

Three different agarose-based chelating adsorbents with, respectively, iminodiacetic acid (IDA), tris(2-aminoethyl)amine (TREN) and dipicolylamine (DPA) functional groups and an agarose-based anion exchanger (Q-Sepharose), were studied for the separation and preconcentration of Cr(III) and Cr(VI) species in water. Column recoveries of all the adsorbents were plotted against pH, and it was found that at pH 3.0 the IDA adsorbent selectively adsorbs Cr(III), with a 100 ± 1.0% recovery. The Q-Sepharose, on the other hand, accumulated only Cr(VI) at this pH, again with a recovery of 100 ± 1.0%. A dual column system was accordingly designed, using the two adsorbents in tandem, for the separation and preconcentration of the chromium species.The effects of pH, sample flow rate, column length, eluent type, eluent volume, acid concentration and interfering ions on the recoveries of Cr(III) and Cr(VI) were carefully studied. It was shown that by passing test solutions, at pH 3.0; through the dual column system, the two chromium species could be individually collected on the columns, respectively, and eluted, one after the other. A portion of 2 mol l⁻¹ hydrochloric acid was used for elution of each column before final measurement by flame AAS method. A preconcentration factor of 12, a detection limit of 7.7 ± 0.1 μg l⁻¹ and a precision expressed as relative standard deviation of 0.4% (at 0.3 mg l⁻¹) were achieved for six replicates.Application of the developed method to the determination of chromium species in spiked river and tap water and wastewater samples, from a dye production plant, resulted in excellent agreements with accepted concentrations.     

Molecularly imprinted polymer for selective extraction of domoic acid from seafood coupled with high-performance liquid chromatographic determination

In this work, a highly selective sample cleanup procedure that combining molecular imprinting technique (MIT) and solid phase extraction (SPE) was developed for the isolation of domoic acid (a fascinating marine toxin) from seafood samples. The molecular imprinting polymer (MIP) for domoic acid was prepared using 1,3,5-pentanetricarboxylic acid as the template molecule instead of domoic acid. 4-Vinyl pyridine was used as the functional monomer and ethylene glycol dimethacrylate was used as the cross-linking monomer. The obtained imprinted polymer showed high affinity to domoic acid and was used as selective sorbent for the SPE of domoic acid from seafood samples. An off-line molecularly imprinted solid phase extraction (MISPE) method followed by high-performance liquid chromatography (HPLC) with diode-array detection for the detection of domoic acid was also established. Good linearity was obtained from 0.5 mg L⁻¹ to 25 mg L⁻¹ (R² > 0.99) with a quantitation limit of 0.1 mg L⁻¹, which was sufficient to determine domoic acid at the maximum level permitted by several authorities. The mean recoveries of domoic acid from mussel extracts were 93.4-96.7%. It was demonstrated that the proposed MISPE-HPLC method could be applied to direct determination of domoic acid from seafood samples.     

Development of a liquid chromatography-based screening methodology for proteolytic enzyme activity

A new methodology for the detection and isolation of serine proteases in complex mixtures has been developed. It combines the characterization of crude samples by electrospray tandem mass spectrometry (ESI-MS/MS) in a multi-substrate assay and the differentiated sensitive detection of the responsible enzymes by means of liquid chromatography hyphenated online to biochemical detection (BCD). First, active samples are identified in the multi-substrate assay monitoring the conversion of eight substrates in multiple reaction monitoring in parallel within 60 s. Hereby, the product patterns are investigated and the suitable peptide as substrate for BCD analysis is selected. Subsequently, the active proteases are identified online in the continuous-flow reactor serving as BCD after non-denaturing separation by size-exclusion chromatography and ion-exchange chromatography. For BCD, the selected para-nitroaniline (pNA) labeled peptide is added post-column and is cleaved by eluting proteases under release of the coloured pNA in a reaction coil (reaction time 5 min). The method was optimized and the figures of merit were characterized with trypsin and chymotrypsin serving as the model proteases. For trypsin, a limit of detection in LC–BCD of 0.1 U/mL corresponding to an injected amount of 0.4 ng protein (∼18 fmol) was observed. The BCD signal remained linear for an injected enzyme concentration of 0.3–10 U/mL (1.3–42 ng enzyme). The method was applied to the characterization of the crude venom of the pit viper Bothrops moojeni and the extracellular protease of the pathogenic amoeba Acanthamoeba castellanii. In the two samples, fractions with proteolytic activity potentially interfering with the blood coagulation cascade were identified. The described methodology represents a tool for serine protease screening in complex mixtures by a fast ESI-MS/MS identification of active samples followed by the separation and isolation of active sample constituents in LC–BCD.