Showing papers in "Analyst in 2011"
TL;DR: A 384-well format hanging drop culture plate is described that makes spheroid formation, culture, and subsequent drug testing on the obtained 3D cellular constructs as straightforward to perform and adapt to existing high-throughput screening (HTS) instruments as conventional 2D cultures.
Abstract: Culture of cells as three-dimensional (3D) aggregates can enhance in vitro tests for basic biological research as well as for therapeutics development. Such 3D culture models, however, are often more complicated, cumbersome, and expensive than two-dimensional (2D) cultures. This paper describes a 384-well format hanging drop culture plate that makes spheroid formation, culture, and subsequent drug testing on the obtained 3D cellular constructs as straightforward to perform and adapt to existing high-throughput screening (HTS) instruments as conventional 2D cultures. Using this platform, we show that drugs with different modes of action produce distinct responses in the physiological 3D cell spheroids compared to conventional 2D cell monolayers. Specifically, the anticancer drug 5-fluorouracil (5-FU) has higher anti-proliferative effects on 2D cultures whereas the hypoxia activated drug commonly referred to as tirapazamine (TPZ) are more effective against 3D cultures. The multiplexed 3D hanging drop culture and testing plate provides an efficient way to obtain biological insights that are often lost in 2D platforms.
840 citations
TL;DR: This study demonstrates that wax screen-printing is an easy-to-use and inexpensive alternative fabrication method for µPAD, which will be especially useful in developing countries.
Abstract: Wax screen-printing as a low-cost, simple, and rapid method for fabricating paper-based microfluidic devices (µPADs) is reported here. Solid wax was rubbed through a screen onto paper filters. The printed wax was then melted into the paper to form hydrophobic barriers using only a hot plate. We first studied the relationship between the width of a hydrophobic barrier and the width of the original design line. We also optimized the heating temperature and time and determined the resolution of structures fabricated using this technique. The minimum width of hydrophilic channel and hydrophobic barrier is 650 and 1300 µm, respectively. Next, our fabrication method was compared to a photolithographic method using the reaction between bicinchoninic acid (BCA) and Cu1+ to demonstrate differences in background reactivity. Photolithographically defined channels exhibited a high background while wax printed channels showed a very low background. Finally, the utility of wax screen-printing was demonstrated for the simultaneous determination of glucose and total iron in control human serum samples using an electrochemical method with glucose oxidase and a colorimetric method with 1,10-phenanthroline. This study demonstrates that wax screen-printing is an easy-to-use and inexpensive alternative fabrication method for µPAD, which will be especially useful in developing countries.
524 citations
TL;DR: Starting with their fundamental understanding, through to highlighting new developments in bulk metal and mediator modified electrodes, as well as novel advantageous electrode designs, it is demonstrated the wide and diverse range of applications that sensors based on this fabrication approach have achieved.
Abstract: Screen printing is widely used to fabricate disposable and economical electrochemical sensors and has helped us to establish the route from ‘lab-to-market’ for a plethora of sensors We overview recent developments in the field where screen printed electrochemical sensors are utilised Starting with their fundamental understanding, through to highlighting new developments in bulk metal and mediator modified electrodes, as well as novel advantageous electrode designs, we demonstrate the wide and diverse range of applications that sensors based on this fabrication approach have achieved
397 citations
TL;DR: The roles of several factors affecting the selectivity and sensitivity of the nanosensors toward the analytes are discussed: the size of the Au nanomaterial, the length and sequence of the DNA or the nature of the thiol, the surface density of the recognition ligand, the ionic strength and pH of the buffer solution.
Abstract: Monitoring the levels of potentially toxic metal (PTM) ions (e.g., Hg2+, Pb2+, Cu2+) in aquatic ecosystems is important because these ions can have severe effects on human health and the environment. Gold (Au) nanomaterials are attractive sensing materials because of their unique size- and shape-dependent optical properties. This review focuses on optical assays for Hg2+, Pb2+, and Cu2+ ions using functionalized Au nanomaterials. The syntheses of functionalized Au nanomaterials are discussed. We briefly review sensing approaches based on changes in absorbance resulting from metal ion-induced aggregation of Au nanoparticles (NPs) or direct deposition of metal ions onto Au NPs. The super-quenching properties of Au NPs allow them to be employed in ‘turn on’ and ‘turn off’ fluorescence approaches for the sensitive and selective detection of Hg2+, Pb2+, and Cu2+ ions. We highlight approaches based on fluorescence quenching through analyte-induced aggregation or the formation of metallophilic complexes of Au nanodots (NDs). We discuss the roles of several factors affecting the selectivity and sensitivity of the nanosensors toward the analytes: the size of the Au nanomaterial, the length and sequence of the DNA or the nature of the thiol, the surface density of the recognition ligand, and the ionic strength and pH of the buffer solution. In addition, we emphasize the potential of using new nanomaterials (e.g., fluorescent silver nanoclusters) for the detection of PTM ions.
342 citations
TL;DR: The relationship between experimental gas-phase conformations and those adopted in crystals is considered and an outlook is given on the application of IM-MS as a tool for structural biology.
Abstract: The technique of ion mobility mass spectrometry (IM-MS) has become of increasing interest for rapid analysis of the conformations adopted by biological macromolecules. It is currently used routinely for analysis of explosives and illegal substances in airport and military security. In biophysical research, it can be used to determine the temperature dependent rotationally averaged collision cross section of gas-phase ions of proteins and nucleic acids along with their mass to charge ratios. Nanoelectrospray ionisation allows the gentle transfer of intact biomolecules from solutions in which the native form(s) are present, into the solvent free environment of a mass spectrometer. It is believed by many researchers that the experimental collision cross sections of these molecules should have some relationship to crystal structure coordinates. In this review we outline the different experimental methods that can be used to measure ion mobility; we also describe methods used to calculate collision cross sections from input coordinates. Following this survey of the methodological approaches to IM-MS, we then summarise IM-MS data published to date for some monomeric peptides and small soluble proteins, along with collision cross sections calculated from their crystal structure coordinates. Finally we consider the relationship between experimental gas-phase conformations and those adopted in crystals and give an outlook on the application of IM-MS as a tool for structural biology.
294 citations
TL;DR: To what extent can SVM-based techniques replace ANN-based approaches in real-world (industrial/scientific) applications?
Abstract: In this study, we make a general comparison of the accuracy and robustness of five multivariate calibration models: partial least squares (PLS) regression or projection to latent structures, polynomial partial least squares (Poly-PLS) regression, artificial neural networks (ANNs), and two novel techniques based on support vector machines (SVMs) for multivariate data analysis: support vector regression (SVR) and least-squares support vector machines (LS-SVMs). The comparison is based on fourteen (14) different datasets: seven sets of gasoline data (density, benzene content, and fractional composition/boiling points), two sets of ethanol gasoline fuel data (density and ethanol content), one set of diesel fuel data (total sulfur content), three sets of petroleum (crude oil) macromolecules data (weight percentages of asphaltenes, resins, and paraffins), and one set of petroleum resins data (resins content). Vibrational (near-infrared, NIR) spectroscopic data are used to predict the properties and quality coefficients of gasoline, biofuel/biodiesel, diesel fuel, and other samples of interest. The four systems presented here range greatly in composition, properties, strength of intermolecular interactions (e.g., van der Waals forces, H-bonds), colloid structure, and phase behavior. Due to the high diversity of chemical systems studied, general conclusions about SVM regression methods can be made. We try to answer the following question: to what extent can SVM-based techniques replace ANN-based approaches in real-world (industrial/scientific) applications? The results show that both SVR and LS-SVM methods are comparable to ANNs in accuracy. Due to the much higher robustness of the former, the SVM-based approaches are recommended for practical (industrial) application. This has been shown to be especially true for complicated, highly nonlinear objects.
288 citations
TL;DR: The present study suggests that Cu(2+) induced fluorescence quenching is due to its binding to BSA rather than the fluorescenceQuenching by metal-metal interaction as in the case of Hg(2+).
Abstract: This paper reports the use of fluorescent gold nanoclusters synthesized using bovine serum albumin (Au–BSA) for the sensing of copper ions in live cells. The fluorescence of the clusters was found to be quenched by Cu2+ enabling its detection in cells. The selectivity of the nanosensor was demonstrated in the presence of several cations excluding Hg2+. We did not study the effect of Hg2+ since it was reported earlier. The present study suggests that Cu2+ induced fluorescence quenching is due to its binding to BSA rather than the fluorescence quenching by metal–metal interaction as in the case of Hg2+. The Au–BSA showed excellent selectivity to Cu2+ at various pH conditions. The ‘turn off’ of fluorescence can be retrieved by a Cu2+ chelator glycine. Our results showed that gold clusters can be used as a ‘turn off’ sensor for copper and a ‘turn on’ sensor for glycine. Under the experimental conditions, the probe showed a response for Cu2+ over a range of 100 μM to 5 mM with a detection limit of 50 μM. The role of Cu2+ in the misfolding and disassembly of Prion Protein (PrP) leading to various maladies is well ascertained. The methodology we reported here seems to be useful in supplementing other techniques in predicting disease conditions involving Cu2+.
244 citations
TL;DR: This mini-review article describes the recent uses of ionic liquids in electrochemical sensing applications in the context of voltammetric sensing at solid/liquid, liquid/liquid interfaces and carbon paste electrodes, as well as their use in gas sensing, ion-selective electrodes, and for detecting biological molecules, explosives and chemical warfare agents.
Abstract: Ionic Liquids are salts that are liquid at (or just above) room temperature. They possess several advantageous properties (e.g. high intrinsic conductivity, wide electrochemical windows, low volatility, high thermal stability and good solvating ability), which make them ideal as non-volatile electrolytes in electrochemical sensors. This mini-review article describes the recent uses of ionic liquids in electrochemical sensing applications (covering the last 3 years) in the context of voltammetric sensing at solid/liquid, liquid/liquid interfaces and carbon paste electrodes, as well as their use in gas sensing, ion-selective electrodes, and for detecting biological molecules, explosives and chemical warfare agents. A comment on the future direction and challenges in this field is also presented.
223 citations
TL;DR: A method combining immunomagnetic separation (IMS) and surface-enhanced Raman scattering (SERS) was developed to enumerate Escherichia coli and the ability of the immunoassay to detect E. coli in real water samples was investigated and the results were compared with the experimental results from plate-counting methods.
Abstract: A method combining immunomagnetic separation (IMS) and surface-enhanced Raman scattering (SERS) was developed to enumerate Escherichia coli (E. coli). Gold-coated magnetic spherical nanoparticles were prepared by immobilizing biotin-labeled anti-E. coliantibodies onto avidin-coated magnetic nanoparticles and used in the separation and concentration of the E. colicells. Raman labels have been constructed using rod shaped gold nanoparticles coated with 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) and subsequently with a molecular recognizer. Then DTNB-labeled gold nanorods were interacted with gold-coated magnetic spherical nanoparticle-antibody-E. coli complex. The capture efficiency and calibration graphs were obtained and examined in different E. coli concentrations (101–107 cfu mL−1). The correlation between the concentration of bacteria and SERS signal was found to be linear within the range of 101–104 cfu mL−1 (R2 = 0.992). The limit of detection (LOD) and limit of quantification (LOQ) values of the developed method were found to be 8 and 24 cfu mL−1, respectively. The selectivity of the developed immunoassay was examined with Enterobacter aerogenes, Enterobacter dissolvens, and Salmonella enteriditis which did not produce any significant response. The ability of the immunoassay to detect E. coli in real water samples was also investigated and the results were compared with the experimental results from plate-counting methods. There was no significant difference between the methods that were compared (p > 0.05). This method is rapid and sensitive to target organisms with a total analysis time of less than 70 min.
208 citations
TL;DR: Recent advances in the development and applications of nucleic acid-based biosensors for environmental application are reviewed in this article with special emphasis on functional nucleic Acid elements (aptamers, DNAzymes, aptazymes) and lab-on-a-chip technology.
Abstract: Nucleic acid-based biosensors are finding increasing use for the detection of environmental pollution and toxicity. A biosensor is defined as a compact analytical device incorporating a biological or biologically-derived sensing element either integrated within or intimately associated with a physicochemical transducer. A nucleic acid-based biosensor employs as the sensing element an oligonucleotide, with a known sequence of bases, or a complex structure of DNA or RNA. Nucleic acid biosensors can be used to detect DNA/RNA fragments or either biological or chemical species. In the first application, DNA/RNA is the analyte and it is detected through the hybridization reaction (this kind of biosensor is also called a genosensor). In the second application, DNA/RNA plays the role of the receptor of specific biological and/or chemical species, such as target proteins, pollutants or drugs. Recent advances in the development and applications of nucleic acid-based biosensors for environmental application are reviewed in this article with special emphasis on functional nucleic acid elements (aptamers, DNAzymes, aptazymes) and lab-on-a-chip technology.
201 citations
TL;DR: This review highlights work from the last 2-3 years in the areas of grating-coupled, interferometric, photonic crystal, and microresonator waveguide sensors, with particular emphasis on demonstrations of biosensing using these technologies within microfluidically controlled environments.
Abstract: By leveraging advances in semiconductor microfabrication technologies, chip-integrated optical biosensors are poised to make an impact as scalable and multiplexable bioanalytical measurement tools for lab-on-a-chip applications. In particular, waveguide-based optical sensing technology appears to be exceptionally amenable to chip integration and miniaturization, and, as a result, the recent literature is replete with examples of chip-integrated waveguide sensing platforms developed to address a wide range of contemporary analytical challenges. As an overview of the most recent advances within this dynamic field, this review highlights work from the last 2–3 years in the areas of grating-coupled, interferometric, photonic crystal, and microresonator waveguide sensors. With a focus towards device integration, particular emphasis is placed on demonstrations of biosensing using these technologies within microfluidically controlled environments. In addition, examples of multiplexed detection and sensing within complex matrices—important features for real-world applicability—are given special attention.
TL;DR: The possibility of exploiting the analytical power of mass spectrometry to image the chemistry of biological and similarly complex materials without the use of tags and with good spatial resolution is seductive.
Abstract: The possibility of exploiting the analytical power of mass spectrometry to image the chemistry of biological and similarly complex materials without the use of tags and with good spatial resolution is seductive. The status, strengths, weaknesses and complementarity of the three main techniques are briefly reviewed and assessed.
TL;DR: The utility of the integrated, single-chamber cassette was demonstrated by detecting the presence of HIV-1 in oral fluids and serving another critical role-enabling the removal of inhibitors that dramatically reduce detection sensitivity.
Abstract: A simple, point of care, inexpensive, disposable cassette for the detection of nucleic acids extracted from pathogens was designed, constructed, and tested. The cassette utilizes a single reaction chamber for isothermal amplification of nucleic acids. The chamber is equipped with an integrated, flow-through, Flinders Technology Associates (Whatman FTA®) membrane for the isolation, concentration, and purification of DNA and/or RNA. The nucleic acids captured by the membrane are used directly as templates for amplification without elution, thus simplifying the cassette's flow control. The FTA membrane also serves another critical role—enabling the removal of inhibitors that dramatically reduce detection sensitivity. Thermal control is provided with a thin film heater external to the cassette. The amplification process was monitored in real time with a portable, compact fluorescent reader. The utility of the integrated, single-chamber cassette was demonstrated by detecting the presence of HIV-1 in oral fluids. The HIV RNA was reverse transcribed and subjected to loop-mediated, isothermal amplification (LAMP). A detection limit of less than 10 HIV particles was demonstrated. The cassette is particularly suitable for resource poor regions, where funds and trained personnel are in short supply. The cassette can be readily modified to detect nucleic acids associated with other pathogens borne in saliva, urine, and other body fluids as well as in water and food.
TL;DR: The P(2)O(7)(4-)-AuNPs bound by Fe(3+) showed excellent selectivity compared to other metal ions, and was used to detect Fe( 3+) in lake water samples, with low interference.
Abstract: A sensitive, selective colorimetric Fe3+ detection method has been developed by using pyrophosphate functionalized gold nanoparticles (P2O74−–AuNPs). Gold nanoparticles were prepared by reducing HAuCl4 with sodium borohydride, in the presence of Na4P2O7. IR spectra suggested that pyrophosphates were capped on the surface of the gold nanoparticles. Aggregation of P2O74−–AuNPs was induced immediately in the presence of Fe3+ ions, yielding a color change from pink to violet. This Fe3+-induced aggregation of P2O74−–AuNPs was monitored using first the naked eye and then UV-vis spectroscopy with a detection limit of 5.6 μM. The P2O74−–AuNPs bound by Fe3+ showed excellent selectivity compared to other metal ions (Ca2+, Cd2+, Co2+, Fe2+, Hg2+, K+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, and Zn2+). The best detection of Fe3+ was achieved in a pH range from 3 to 9. In addition, the P2O74−–AuNPs were also used to detect Fe3+ in lake water samples, with low interference.
TL;DR: The results provide strong evidence that amino acids, carbohydrates, lipids, phosphates, proteins and ions, such as Cl(-) and K(+), leach from tissue sections into the aqueous fixative medium during formalin fixation of the sections.
Abstract: Understanding biochemical mechanisms and changes associated with disease conditions and, therefore, development of improved clinical treatments, is relying increasingly on various biochemical mapping and imaging techniques on tissue sections. However, it is essential to be able to ascertain whether the sampling used provides the full biochemical information relevant to the disease and is free from artefacts. A multi-modal micro-spectroscopic approach, including FTIR imaging and PIXE elemental mapping, has been used to study the molecular and elemental profile within cryofixed and formalin-fixed murine brain tissue sections. The results provide strong evidence that amino acids, carbohydrates, lipids, phosphates, proteins and ions, such as Cl(-) and K(+), leach from tissue sections into the aqueous fixative medium during formalin fixation of the sections. Large changes in the concentrations and distributions of most of these components are also observed by washing in PBS even for short periods. The most likely source of the chemical species lost during fixation is the extra-cellular and intra-cellular fluid of tissues. The results highlight that, at best, analysis of formalin-fixed tissues gives only part of the complete biochemical "picture" of a tissue sample. Further, this investigation has highlighted that significant lipid peroxidation/oxidation may occur during formalin fixation and that the use of standard histological fixation reagents can result in significant and differential metal contamination of different regions of tissue sections. While a consistent and reproducible fixation method may be suitable for diagnostic purposes, the findings of this study strongly question the use of formalin fixation prior to spectroscopic studies of the molecular and elemental composition of biological samples, if the primary purpose is mechanistic studies of disease pathogenesis.
TL;DR: The Fe(2)O(3) nanowire array modified glucose sensor exhibited excellent biocatalytic performance towards the oxidation of glucose and a high sensing selectivity towards glucose oxidation in the presence of ascorbic acid and dopamine has also been obtained at their maximum physiological concentrations.
Abstract: Fe2O3 was generally considered to be biologically and electrochemically inert, and its electrocatalytic functionality has been rarely realized directly in the past. In this work, Fe2O3 nanowire arrays were synthesized and electrochemically characterized. The as prepared Fe2O3 nanomaterial was proved to be an ideal electrode material due to the intrinsic peroxidase-like catalytic activity. The Fe2O3 nanowire array modified glucose sensor exhibited excellent biocatalytic performance towards the oxidation of glucose with a response time of <6 s, a linear range between 0.015–8 mM, and sensitivity of 726.9 μA mM−1cm−1. Additionally, a high sensing selectivity towards glucose oxidation in the presence of ascorbic acid (AA) and dopamine (DA) has also been obtained at their maximum physiological concentrations, which makes the Fe2O3 nanomaterial promising for the development of effective electrochemical sensors for practical applications.
TL;DR: The covalent-orientated immobilization strategy was the best for SPR-based HFA immunoassay and can detect 0.6-20.0 ng/mL of HFA in less than 10 min.
Abstract: Antibody immobilization strategies (random, covalent, orientated and combinations of each) were examined to determine their performance in a surface plasmon resonance-based immunoassay using human fetuin A (HFA) as the model antigen system. The random antibody immobilization strategy selected was based on passive adsorption of anti-HFA antibody on 3-aminopropyltriethoxysilane (APTES)-functionalized gold (Au) chips. The covalent strategy employed covalent crosslinking of anti-HFA antibody on APTES-functionalized chips using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and sulfo-N-hydroxysuccinimide (SNHS). The orientation strategy used passive adsorption of protein A (PrA) on Au chips, with subsequent binding of the anti-HFA antibody in an orientated fashion via its fragment crystallisable (Fc) region. In the covalent-orientated strategy, PrA was first bound covalently, to the surface, which in turn, then binds the anti-HFA antibody in an orientated manner. Finally, in the most widely used strategy, covalent binding of anti-HFA antibody to carboxymethyldextran (CM5-dextran) was employed. This immobilization strategy gave the highest anti-HFA antibody immobilization density, whereas the highest HFA response was obtained with the covalent-orientated immobilization strategy. Therefore, the covalent-orientated strategy was the best for SPR-based HFA immunoassay and can detect 0.6–20.0 ng/mL of HFA in less than 10 min.
TL;DR: This review focuses on bio-impedance based measurement for monitoring the behaviours in real time and without using labels in cell-substrate interactions for anticancer drug development.
Abstract: Cell-substrate interactions are investigated in a number of studies for drug targets including angiogenesis, arteriosclerosis, chronic inflammatory diseases and carcinogenesis. One characteristic of malignant cancerous cells is their ability to invade tissue. Cell adhesion and cytoskeletal activity have served as valuable indicators for understanding the cancer cell behaviours, such as proliferation, migration and invasion. This review focuses on bio-impedance based measurement for monitoring the behaviours in real time and without using labels. Electric cell-substrate impedance sensing (ECIS) provides rich information about cell-substrate interactions, cell-cell communication and cell adhesion. High sensitivity of the ECIS method allows for observing events down to single-cell level and achieving nanoscale resolution of cell-substrate distances. Recently, its miniaturization and integration with fluorescent detection techniques have been highlighted as a new tool to deliver a high-content platform for anticancer drug development.
TL;DR: Results of SEM and SPR studies indicate that the maximum host bacterial capture is obtained when 1.5 × 10(11) pfu ml(-1) concentration of T4 phages was used for immobilization, and have implications for the development of online bioassays for the detection of various food and water borne pathogens using the inherent selectivity of bacteriophage recognition.
Abstract: A bioassay platform using T4 bacteriophage (T4) as the specific receptor and surface plasmon resonance (SPR) as the transduction technique has been developed for the detection ofEscherichia coliK12 bacteria. The T4 phages have been covalently immobilized onto gold surfaces using a self-assembled monolayer of dithiobis(succinimidyl propionate) (DTSP). Substrates of BSA/EA-T4/DTSP/Au prepared using different T4 phage concentrations have been characterized using scanning electron microscopy (SEM). The studies reveal that the use of DTSP results in a uniform binding of T4 phages onto the surface. The SPR analysis demonstrates that these BSA/EA-T4/DTSP/Au interfaces can detect the E. coliK12 with high specificity against non-host E. coliNP10 and NP30. Results of SEM and SPR studies indicate that the maximum host bacterial capture is obtained when 1.5 × 1011 pfu ml−1 concentration of T4 phages was used for immobilization. The surface of these chemically anchored phage substrates can be regenerated for repeated detection ofE. coliK12 and can be used for detection in 7 × 102 to 7 × 108 cfu ml−1 range. The results of these studies have implications for the development of online bioassays for the detection of various food and water borne pathogens using the inherent selectivity of bacteriophage recognition.
TL;DR: The attractive features of the new micromachine-based target isolation and signal transduction protocols reviewed in this article offer numerous potential applications in biomedical diagnostics, environmental monitoring, and forensic analysis.
Abstract: Electrochemically-propelled nanomotors offer considerable promise for developing new and novel bioanalytical and biosensing strategies based on the direct isolation of target biomolecules or changes in their movement in the presence of target analytes. For example, receptor-functionalized nanomotors offer direct and rapid target isolation from raw biological samples without preparatory and washing steps. Microtube engines functionalized with ss-DNA, aptamer or antibody receptors are particularly useful for the direct isolation of nucleic acids, proteins or cancer cells, respectively. A new nanomotor-based signal transduction involving measurement of speed and distance travelled by nanomotors, offers highly sensitive, rapid, simple and low cost detection of target biomarkers, and a new dimension of analytical information based on motion. The resulting distance signals can be easily visualized by optical microscope (without any sophisticated analytical instrument) to reveal the target presence and concentration. The attractive features of the new micromachine-based target isolation and signal transduction protocols reviewed in this article offer numerous potential applications in biomedical diagnostics, environmental monitoring, and forensic analysis.
TL;DR: This method has developed a method to identify and quantify isomeric native glycans using nanoflow liquid chromatography (nano-LC)/mass spectrometry and provides comprehensive, selective, and quantitative glycan profiling.
Abstract: Glycosylation is highly sensitive to the biochemical environment and has been implicated in many diseases including cancer. Glycan compositional profiling of human serum with mass spectrometry has already identified potential biomarkers for several types of cancer and diseases; however, composition alone does not fully describe glycan stereo- and regioisomeric diversity. The vast structural heterogeneity of glycans presents a formidable analytical challenge. We have developed a method to identify and quantify isomeric native glycans using nanoflow liquid chromatography (nano-LC)/mass spectrometry. A microfluidic chip packed with graphitized carbon was used to chromatographically separate the glycans. To determine the utility of this method for structure-specific biomarker discovery, we analyzed serum samples from two groups of prostate cancer patients with different prognoses. More than 300 N-glycan species (including isomeric structures) were identified, corresponding to over 100 N-glycan compositions. Statistical tests established significant differences in glycan abundances between patient groups. This method provides comprehensive, selective, and quantitative glycan profiling.
TL;DR: A simple, rapid, and sensitive method for quantitative detection of influenza A virus using dynamic light scattering (DLS) and gold nanoparticle (AuNP) labels and it was determined that decreasing the Au NP probe concentration improves the detection limit while the effect of changing the AuNP size is minimal.
Abstract: Herein we detail the development of a simple, rapid, and sensitive method for quantitative detection of influenza A virus using dynamic light scattering (DLS) and gold nanoparticle (AuNP) labels. Influenza-specific antibodies are conjugated to AuNPs, and aggregation of the AuNP probes is induced upon addition of the target virus. DLS is used to measure the extent of aggregation and the mean hydrodynamic diameter is correlated to virus concentration. The effects of nanoparticle concentration and size on the analytical performance of the assay were systematically investigated. It was determined that decreasing the AuNP probe concentration improves the detection limit while the effect of changing the AuNP size is minimal. Optimization of the assay provided a detection limit of <100 TCID50/mL which is 1–2 orders of magnitude improved over commercial diagnostic kits without increasing the assay time or complexity. Additionally, this assay was demonstrated to perform equivalently for influenza virus prepared in different biological matrices.
TL;DR: The performance characteristics of a second-generation traveling-wave ion mobility separator are assessed, focusing on those figures of merit that lead to making measurements of collision cross-section having both high precision and high accuracy.
Abstract: High-accuracy, high-resolution ion mobility measurements enable a vast array of important contemporary applications in biological chemistry With the recent advent of both new, widely available commercial instrumentation and also new calibration datasets tailored for the aforementioned commercial instrumentation, the possibilities for extending such high performance measurements to a diverse set of applications have never been greater Here, we assess the performance characteristics of a second-generation traveling-wave ion mobility separator, focusing on those figures of merit that lead to making measurements of collision cross-section having both high precision and high accuracy Through performing a comprehensive survey of instrument parameters and settings, we find instrument conditions for optimized drift time resolution, cross-section resolution, and cross-section accuracy for a range of peptide, protein and multi-protein complex ions Moreover, the conditions for high accuracy IM results are significantly different from those optimized for separation resolution, indicating that a balance between these two metrics must be attained for traveling wave IM separations of biomolecules We also assess the effect of ion heating during IM separation on instrument performance
TL;DR: A simple, sensitive and reliable assay for melamine in raw milk with dopamine-stabilized silver nanoparticles (AgNPs) as a colorimetric reader is proposed and can be used for rapid monitoring of raw milk quality.
Abstract: In this work, we propose a simple, sensitive and reliable assay for melamine in raw milk with dopamine-stabilized silver nanoparticles (AgNPs) as a colorimetric reader. Dopamine can reduce Ag(+) and functionalize the produced AgNPs to form monodispersed AgNPs. The coexisting melamine in reaction solution could bind dopamine through Michael addition and Schiff base reactions, which leads to the aggregation of AgNPs and induces a colorimetric response. The one-step assay is simple, rapid and highly sensitive. The color-change is quantitatively correlated with the concentration of melamine in the range of 10 ppb to 1.26 ppm, which is below the safety limit in China (1.0 ppm) and EU (2.0 ppm). The coexisting substances including phenylalanine, dl-leucine, l-glutamate, sulfanilic acid, Mg(2+), galactose, lysine, urea and glucose do not affect the determination of melamine. The colorimetric sensor can be used for rapid monitoring of raw milk quality.
TL;DR: The present upconversion luminescent bioassay offers a promising approach for the screening of small molecular mycotoxins because it is simple, rapid, highly sensitive, specific, does not require sample pre-concentration and lacks interference from autofluorescence of other biomolecules.
Abstract: A sensitive luminescent bioassay for the detection of ochratoxin A (OTA), a small molecular mycotoxin, was developed using aptamer-conjugated magnetic nanoparticles (MNPs) as the recognition and concentration element and upconversion nanoparticles (UCNPs) as highly sensitive labels. The bioassay system was fabricated by immobilizing aptamer DNA 1 sequence onto the surface of Fe3O4 MNPs, which were implemented to capture and concentrate OTA from bulk samples. The aptamer DNA 1 sequence then hybridized with UCNPs modified with DNA 2 sequence, which could dissociate from DNA 1 and result in a decreased luminescent signal when aptamer DNA 1 recognized and bound to target OTA. Under the optimal conditions, the decreased luminescent intensity (ΔI) is proportional to the concentration of OTA in the range of 1 × 10−13 to 1 × 10−9 g mL−1 with a detection limit of 1 × 10−13 g mL−1. The proposed method then was successfully applied to measure OTA in naturally contaminated maize samples and validated by a commercially available enzyme-linked immunosorbent assay (ELISA) method. Benefiting from the magnetic separation and concentration effect of MNPs, the high sensitivity of UCNPs, as well as the selectivity and stability of the aptamer, the present upconversion luminescent bioassay offers a promising approach for the screening of small molecular mycotoxins because it is simple, rapid, highly sensitive, specific, does not require sample pre-concentration and lacks interference from autofluorescence of other biomolecules.
TL;DR: A sensitive assay for melamine in complex matrices is built using cysteamine-modified gold nanoparticles (AuNPs) and an effective sample pretreatment protocol and could be an alternative means for onsite detection of melamine without costly instruments.
Abstract: A sensitive assay for melamine in complex matrices is built using cysteamine-modified gold nanoparticles (AuNPs) and an effective sample pretreatment protocol. Citrate-stabilized AuNPs were modified by cysteamine in order to weaken the electrostatic repulsion force between the gold nanoparticles. Detection sensitivity gained through this modification increased about 100 fold compared with the result using the unmodified AuNPs. Direct colorimetric visualizations of melamine in milk products, eggs and feeds was successfully demonstrated within the linear ranges of 1–200 mg L−1 and detection limits below 1 mg L−1. The proposed scheme could be an alternative means for onsite detection of melamine without costly instruments.
TL;DR: An overview of the methodologies in top-down mass spectrometry is provided and applications covering protein posttranslational modifications, protein biophysics, DNAs/RNAs, and protein assemblies are reviewed.
Abstract: Top-down mass spectrometry is an emerging approach for the analysis of intact proteins. The term was coined as a contrast with the better-established, bottom-up strategy for analysis of peptide fragments derived from digestion, either enzymatically or chemically, of intact proteins. Although the term top-down originates from proteomics, it can also be applied to mass spectrometric analysis of intact large biomolecules that are constituents of protein assemblies or complexes. Traditionally, mass spectrometry has usually started with intact molecules, and in this regard, top-down approaches reflect the spirit of mass spectrometry. This article provides an overview of the methodologies in top-down mass spectrometry and then reviews applications covering protein posttranslational modifications, protein biophysics, DNAs/RNAs, and protein assemblies. Finally, challenges and future directions are discussed.
TL;DR: The design and characterization of a microneedle array-based carbon paste electrode towards minimally invasive electrochemical sensing are described and the operational stability of the array is demonstrated as well as the interference-free detection of lactate in the presence of physiologically relevant levels of ascorbic acid, uric Acid, and acetaminophen.
Abstract: The design and characterization of a microneedle array-based carbon paste electrode towards minimally invasive electrochemical sensing are described. Arrays consisting of 3 × 3 pyramidal microneedle structures, each with an opening of 425 µm, were loaded with a metallized carbon paste transducer. The renewable nature of carbon paste electrodes enables the convenient packing of hollow non-planar microneedles with pastes that contain assorted catalysts and biocatalysts. Smoothing the surface results in good microelectrode-to-microelectrode uniformity. Optical and scanning electron micrographs shed useful insights into the surface morphology at the microneedle apertures. The attractive performance of the novel microneedle electrode arrays is illustrated in vitro for the low-potential detection of hydrogen peroxide at rhodium-dispersed carbon paste microneedles and for lactate biosensing by the inclusion of lactate oxidase in the metallized carbon paste matrix. Highly repeatable sensing is observed following consecutive cycles of packing/unpacking the carbon paste. The operational stability of the array is demonstrated as well as the interference-free detection of lactate in the presence of physiologically relevant levels of ascorbic acid, uric acid, and acetaminophen. Upon addressing the biofouling effects associated with on-body sensing, the microneedle carbon paste platform would be attractive for the subcutaneous electrochemical monitoring of a number of physiologically relevant analytes.
TL;DR: The ability to use a solution based, direct readout SERS method as a quantitative tool for the detection of ultra-low levels of thiram, a dithiocarbamate fungicide, is demonstrated.
Abstract: We report the use of two different sizes of dogbone shaped gold nanoparticles as colloidal substrates for surface enhanced Raman spectroscopy (SERS) based detection of ultra-low levels of thiram, a dithiocarbamate fungicide. We demonstrate the ability to use a solution based, direct readout SERS method as a quantitative tool for the detection of ultra-low levels of thiram. The two different sizes of dogbone shaped gold nanoparticles are synthesized by using the seed-mediated growth method and characterized by using UV-visible spectroscopy and transmission electron microscopy (TEM). The smaller dogbone shaped nanoparticles have an average size of 43 ± 13 nm. The larger dogbone shaped gold nanoparticles have an average size of 65 ± 15 nm. The nanoparticle concentration is 1.25 × 1011nanoparticles per mL for the smaller dogbone shaped gold nanoparticles and is 1.13 × 1011nanoparticles per mL for the larger dogbone shaped gold nanoparticles. Different concentrations of thiram are allowed to bind to the two different sizes of dogbone shaped gold nanoparticles and the SERS spectra are obtained. From the calibration curve, the limit of detection for thiram is 43.9 ± 6.2 nM when the smaller dogbone shaped gold nanoparticles are used as colloidal SERS substrates In the case of the larger dogbone shaped gold nanoparticles, the limit of detection for thiram is 11.8 ± 3.2 nM. The lower limit of detection obtained by using the larger dogbone shaped gold nanoparticles as colloidal substrates is due to the lightning rod effect, higher contributions from the electromagnetic enhancement effect, and larger number of surface sites for thiram to bind.
TL;DR: The current advances in the field of graphene electroanalytical chemistry are presented, including the modern methods of graphene production, and graphene functionalization, and electrochemical (bio) sensing developments using graphene and graphene-based materials are summarized.
Abstract: Similar to its popular older cousins of fullerene and carbon nanotubes (CNTs), the latest form of nanocarbon, graphene, is inspiring intensive research efforts in its own right. As an atomically thin layer of sp2-hybridized carbon, graphene possesses spectacular electronic, optical, magnetic, thermal and mechanical properties, which make it an exciting material in a variety of important applications. In this review, we present the current advances in the field of graphene electroanalytical chemistry, including the modern methods of graphene production, and graphene functionalization. Electrochemical (bio) sensing developments using graphene and graphene-based materials are summarized in more detail, and we also speculate on their future and discuss potential progress for their applications in electroanalytical chemistry.