Species In Complex Samples Research Articles

Towards single molecule biosensors using super-resolution fluorescence microscopy

Conventional immunosensors require many binding events to give a single transducer output which represents the concentration of the analyte in the sample. Because of the requirements to selectively detect species in complex samples, immunosensing interfaces must allow immobilisation of antibodies while repelling nonspecific adsorption of other species. These requirements lead to quite sophisticated interfacial design, often with molecular level control, but we have no tools to characterise how well these interfaces work at the molecular level. The work reported herein is an initial feasibility study to show that antibody-antigen binding events can be monitored at the single molecule level using single molecule localisation microscopy (SMLM). The steps to achieve this first requires showing that indium tin oxide surfaces can be used for SMLM, then that these surfaces can be modified with self-assembled monolayers using organophosphonic acid derivatives, that the amount of antigens and antibodies on the surface can be controlled and monitored at the single molecule level and finally antibody binding to antigen modified surfaces can be monitored. The results show the amount of antibody that binds to an antigen modified surface is dependent on both the concentration of antigen on the surface and the concentration of antibody in solution. This study demonstrates the potential of SMLM for characterising biosensing interfaces and as the transducer in a massively parallel, wide field, single molecule detection scheme for quantitative analysis.

Advances in DNA metabarcoding for food and wildlife forensic species identification.

Species identification using DNA barcodes has been widely adopted by forensic scientists as an effective molecular tool for tracking adulterations in food and for analysing samples from alleged wildlife crime incidents. DNA barcoding is an approach that involves sequencing of short DNA sequences from standardized regions and comparison to a reference database as a molecular diagnostic tool in species identification. In recent years, remarkable progress has been made towards developing DNA metabarcoding strategies, which involves next-generation sequencing of DNA barcodes for the simultaneous detection of multiple species in complex samples. Metabarcoding strategies can be used in processed materials containing highly degraded DNA e.g. for the identification of endangered and hazardous species in traditional medicine. This review aims to provide insight into advances of plant and animal DNA barcoding and highlights current practices and recent developments for DNA metabarcoding of food and wildlife forensic samples from a practical point of view. Special emphasis is placed on new developments for identifying species listed in the Convention on International Trade of Endangered Species (CITES) appendices for which reliable methods for species identification may signal and/or prevent illegal trade. Current technological developments and challenges of DNA metabarcoding for forensic scientists will be assessed in the light of stakeholders’ needs.

Fluorescence Detection of H5N1 Virus Gene Sequences Based on Optical Tweezers with Two-Photon Excitation Using a Single Near Infrared Nanosecond Pulse Laser.

We present an analytical platform by combining near-infrared optical tweezers with two-photon excitation for fluorescence detection of H5N1 virus gene sequences. A heterogeneous enrichment strategy, which involved polystyrene (PS) microsphere and quantum dots (QDs), was adopted. The final hybrid-conjugate microspheres were prepared by a facile one-step hybridization procedure by using PS microspheres capturing target DNA and QDs tagging, respectively. Quantitative detection was achieved by the optical tweezers setup with a low-cost 1064 nm nanosecond pulse laser for both optical trapping and two-photon excitation for the same hybrid-conjugate microsphere. The detection limits for both neuraminidase (NA) gene sequences and hemagglutinin (HA) gene sequences are 16-19 pM with good selectivity for one-base mismatch, which is approximately 1 order of magnitude lower than the most existing fluorescence-based analysis method. Besides, because of the fact that only signal from the trapped particle is detected upon two-photon excitation, this approach showed extremely low background in fluorescence detection and was successfully applied to directly detect target DNA in human whole serum without any separation steps and the corresponding results are very close to that in buffer solution, indicating the strong anti-interference ability of this method. Therefore, it can be expected to be an emerging alternative for straightforward detecting target species in complex samples with a simple procedure and high-throughput.

Multidimensional mapping method using an arrayed sensing system for cross-reactivity screening.

When measuring chemical information in biological fluids, challenges of cross-reactivity arise, especially in sensing applications where no biological recognition elements exist. An understanding of the cross-reactions involved in these complex matrices is necessary to guide the design of appropriate sensing systems. This work presents a methodology for investigating cross-reactions in complex fluids. First, a systematic screening of matrix components is demonstrated in buffer-based solutions. Second, to account for the effect of the simultaneous presence of these species in complex samples, the responses of buffer-based simulated mixtures of these species were characterized using an arrayed sensing system. We demonstrate that the sensor array, consisting of electrochemical sensors with varying input parameters, generated differential responses that provide synergistic information of sample. By mapping the sensing array response onto multidimensional heat maps, characteristic signatures were compared across sensors in the array and across different matrices. Lastly, the arrayed sensing system was applied to complex biological samples to discern and match characteristic signatures between the simulated mixtures and the complex sample responses. As an example, this methodology was applied to screen interfering species relevant to the application of schizophrenia management. Specifically, blood serum measurement of antipsychotic clozapine and antioxidant species can provide useful information regarding therapeutic efficacy and psychiatric symptoms. This work proposes an investigational tool that can guide multi-analyte sensor design, chemometric modeling and biomarker discovery.

Analysis of tissue specimens by matrix-assisted laser desorption/ionization imaging mass spectrometry in biological and clinical research.

Analysis of tissue specimens by matrix-assisted laser desorption/ionization imaging mass spectrometry in biological and clinical research.

Strategies to avoid electrode fouling for nimesulide detection using unmodified electrodes

Voltammetric quantification of nimesulide presents a serious drawback due to surface fouling on unmodified electrodes. In the anodic region, the electrode fouling effects are critical and alternative procedures are required to bypass this effect. In the present study, new strategies for nimesulide quantification in pharmaceutical agents in the anodic region were developed. We propose a simple procedure to overcome this problem: just doing an electrochemical conditioning process before measurement. This was achieved by differential pulse voltammetry (DPV) and multiple pulsed amperometry (MPA), using batch and in flow injection analyses, respectively. These studies were performed in the presence of 0.1 mol L−1 H2SO4 and the application of 1.6 V to the electrode surface at short time intervals was effective both for electrode surface cleaning and for attaining a steady state regime. The new procedures proposed here allow for quantification of nimesulide in both anodic and cathodic regions, favouring the quantification of this species in complex samples. The results obtained here were in good agreement with the official spectrophotometric method recommended in the Brazilian Pharmacopoeia.

Noncovalent Antibody Immobilization on Porous Silicon Combined with Miniaturized Solid-Phase Extraction (SPE) for Array Based ImmunoMALDI Assays

This paper presents a new strategy to combine the power of antibody based capturing of target species in complex samples with the benefits of microfluidic reverse phase sample preparation on an integrated sample enrichment target (RP-ISET) and the analysis speed of MALDI MS. The immunoaffinity step is performed on an in-house developed 3D-structured high surface area porous silicon (PSi) matrix, which allows efficient antibody immobilization by surface adsorption without any coupling agents in 30-60 min. The hydrophilic nature of the porous silicon surface at the molecular level displays a low adsorption of background peptides when exposed to complex digests or plasma samples, improving the conditions for the antigen specific extraction and subsequent readout. At the same time, the hydrophobic behavior, due to the nanostructured surface, of the PSi material facilitates liquid confinement during the assay. Using a footprint conforming to the standard for 384 well microplates, direct adaption of the protocol into standard sample handling robots is possible. The performance of the proposed immunoaffinity PSi-ISET immunoMALDI (iMALDI) assay was evaluated by specific detection of angiotensin I at a 10 femtomol level in diluted plasma samples (10 μL, 1 nM).

Identification of gasoline adulteration using comprehensive two-dimensional gas chromatography combined to multivariate data processing

A method to detect potential adulteration of commercial gasoline (Type C gasoline, available in Brazil and containing 25% (v/v) ethanol) is presented here. Comprehensive two-dimensional gas chromatography with flame ionization detection (GC × GC–FID) data and multivariate calibration (multi-way partial least squares regression, N-PLS) were combined to obtain regression models correlating the concentration of gasoline on samples from chromatographic data. Blends of gasoline and white spirit, kerosene and paint thinner (adopted as model adulterants) were used for calibration; the regression models were evaluated using samples of Type C gasoline spiked with these solvents, as well as with ethanol. The method was also checked with real samples collected from gas stations and analyzed using the official method. The root mean square error of prediction (RMSEP) for gasoline concentrations on test samples calculated using the regression model ranged from 3.3% (v/v) to 8.2% (v/v), depending on the composition of the blends; in addition, the results for the real samples agree with the official method. These observations suggest that GC × GC–FID and N-PLS can be an alternative for routine monitoring of fuel adulteration, as well as to solve several other similar analytical problems where mixtures should be detected and quantified as single species in complex samples.

Pretreatment procedures for characterization of arsenic and selenium species in complex samples utilizing coupled techniques with mass spectrometric detection

Research interest in analyzing arsenic and selenium is dictated by their species-dependent behavior in the environment and in living organisms. Different analytical methodologies for known species in relatively simple chemical systems are well established, yet the analysis of complex samples is still a challenge. Owing to the complex matrix and low concentrations of target species that may be chemically labile, suitable pretreatment of the sample becomes a critical step in any speciation procedure. In this paper, the pretreatment procedures used for arsenic and selenium speciation are reviewed with the emphasis on the link between the analytical protocol applied and the biologically-significant information provided by the results obtained. In the first approach, the aim of pretreatment is to convert the original sample into a form that can be analyzed by a coupled (hyphenated) technique, preventing possible losses and/or species interconversion. Common techniques include different leaching and extraction modes, enzymatic hydrolysis, species volatilization, and so on, with or without species preconcentration. On the other hand, if the speciation analysis is performed for elucidation of elemental pathways and specific functions in a living system, more conscious pretreatment and/or fractionation is needed. The macroscopic separation of organs and tissues, isolation of certain types of cells, cell disruption and separation of sub-cellular fractions, as well as isolation of a specific biomolecules become important. Furthermore, to understand molecular mechanisms, the identification of intermediate-often highly instable--metabolites is necessary. Real life applications are reviewed in this work for aquatic samples, soils and sediments, plants, yeast, and urine.

Monitoring Exposure to Polynuclear Aromatic Compounds via Room Temperature Phosphorescence from Solid Substrates

Room Temperature Phosphorescence (RTP) Spectroscopy is a relatively new solid state luminescence technique for monitoring polynuclear aromatic (PNA) compounds. The RTP technique can detect a variety of PNA pollutants at subnanogram levels. Practical applications include rapid and cost-effective screening of PNA species in complex samples and direct reading detection of airborne PNA compound collected on a new type of personnel vapor dosemeter.

Measuring blood sugar.

<h3>Abstract</h3> <i>Francisella tularensis</i>, the bacterium that causes the zoonosis tularemia, and its genetic near neighbor species, can be difficult or impossible to cultivate from complex samples. Thus, there is a lack of genomic information for these species that has, among other things, limited the development of robust detection assays for <i>F. tularensis</i> that are both specific and sensitive. The objective of this study was to develop and validate approaches to capture, enrich, sequence, and analyze <i>Francisella</i> DNA present in DNA extracts generated from complex samples. RNA capture probes were designed based upon the known pan genome of <i>F. tularensis</i> and other diverse species in the family <i>Francisellaceae</i>. Probes that targeted genomic regions also present in non-<i>Francisellaceae</i> species were excluded, and probes specific to particular <i>Francisella</i> species or phylogenetic clades were identified. The capture-enrichment system was then applied to diverse, complex DNA extracts containing low-level <i>Francisella</i> DNA, including human clinical tularemia samples, environmental samples (<i>i.e.</i>, animal tissue and air filters), and whole ticks/tick cell lines, which was followed by sequencing of the enriched samples. Analysis of the resulting data facilitated rigorous and unambiguous confirmation of the detection of <i>F. tularensis</i> or other <i>Francisella</i> species in complex samples, identification of mixtures of different <i>Francisella</i> species in the same sample, analysis of gene content (<i>e.g.</i>, known virulence and antimicrobial resistance loci), and high-resolution whole genome-based genotyping. The benefits of this capture-enrichment system include: even very low target DNA can be amplified; it is culture-independent, reducing exposure for research and/or clinical personnel and allowing genomic information to be obtained from samples that do not yield isolates; and the resulting comprehensive data not only provide robust means to confirm the presence of a target species in a sample, but also can provide data useful for source attribution, which is important from a genomic epidemiology perspective.