Search

Oxidative stress, as an essential cause to many diseases via irreversible biomolecule oxidation, can be induced by casual-contacted nanomaterials, which can generate hydroxyl radical (•OH) due to their active surface capacity. Herein, we report a novel upconversion nanoprobe for ratiometric •OH detection and monitoring titanium oxide nanomaterial-induced oxidative stress. Unlike previously developed nanoprobes, the •OH-responsive acceptor in these nanoprobes can be rapidly prepared and easily recycled. With a detection limit of ~2 nM and a broad linear range of 4 nM—16 μM, the nanoprobe is suitable for both in vitro and in vivo applications. The facile preparation and recyclable strategy provide a new method for developing novel nanoprobes that can directly detect hazardous molecules in biological samples and systems. [ABSTRACT FROM AUTHOR]

In this work, a synchronized polarization induced electrospray ionization (SPI-ESI) method is developed and applied for the analysis of single-cell samples. In SPI-ESI, periodic alternating current square wave voltage (AC-SWV) is applied to induce the bipolar spray and both positive-ion and negative-ion mass spectra are obtained through one measurement by synchronizing the mode of mass analyzer with the bipolar spray process. Compared with conventional nanoelectrospray ionization (nESI, flow rate < 1000 nL/min), ultralow spray flow rate (pico-electrospray ionization, pESI, flow rate < 1000 pL/min) is achieved in SPI-ESI without loss of its sensitivity. The decrease of flow rate prolongs the MS signal duration from single-cell samples to acquire ms2 data for components determination. To our knowledge, this is the first time to successfully achieve comprehensive analysis of single-cell samples by combining both positive-ion and negative-ion mass spectra. Ultimately, 86 components are profiled from single Allium cepa cells and 94 components are profiled from single PC-12 cells. [ABSTRACT FROM AUTHOR]

Smart and versatile nanostructures have demonstrated their effectiveness for biomolecule analysis and show great potential in digging insights into the structural/functional relationships. Herein, a nanoscale molecular self-assembly was constructed for probing the site-specific recognition and conformational changes of human serum albumin (HSA) with tunable size and emission. A tetraphenylethylene derivative TPE-red-COOH was used as the building block for tailoring fluorescence-silent nanoparticles. The highly specific and sensitive response to HSA was witnessed by the fast turn-on of the red fluorescence and simultaneous disassembly of the nanostructures, whereas various endogenous biomolecules cannot induce such response. The mechanism investigation indicates that the combination of multiple noncovalent interactions is the driving force for disassembling and trapping TPE-red-COOH into HSA. The resultant restriction of intramolecular rotation of TPE-red-COOH in the hydrophobic cavity of HSA induces the significant red emission. By using the fluorescence activatable nanosensor as the structural indicator, the stepwise conformational transitions of HSA during denaturing and the partial refolding of subdomain IIA of HSA were facilely visualized. Benefiting from its activatable signaling, sensitivity, and simplicity, such molecular assembly provides a kind of soft nanomaterial for site-specific biomolecule probing and conformational transition detection concerning their structure, function, and biomedical characteristics. [ABSTRACT FROM AUTHOR]

Lanthanide coordination polymer nanoparticles (Ln-CPNs) have been recently demonstrated as excellent platforms for biomolecule detection. In this work, we synthesized novel cerium coordination polymer nanoparticles ATP-Ce-Tris CPNs in a simple and quick way using ATP molecules as the biocompatible ligands to Ce3+ ions in tris(hydroxymethyl)aminomethane hydrochloric (Tris-HCl) solution. In view of the excellent free radical scavenging property of cerium compounds, which is ascribed to the mixed valence state (Ce3+, Ce4+) and the reversible switch from Ce3+ to Ce4+, the synthesized ATP-Ce-Tris CPNs was used as artificial peroxidase to selectively and sensitively detect H2O2. The sensing mechanism depends on the oxidation of the fluorescent ATP-Ce(III)-Tris CPNs to nonfluorescent ATP-Ce(IV)-Tris CPNs by H2O2. Compared with those inorganic cerium oxide sensors, this kind of fluoresence ATP-Ce-Tris CPNs sensor needs no additional organic redox dye, such as ABTS (2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), TMB (3,3,5,5-tetramethylbenzidine), or fluorescein as signal molecules. Moreover, such ATP-Ce-Tris CPNs sensor exhibited a more sensitive response to H2O2 with a detection limit down to 0.6 nM, which is 2 orders of magnitude lower than those of cerium oxide sensors. This sensing platform was further extended to the detection of glucose in combination with the specific catalytic effect of glucose oxidase (GOx) for the oxidation of glucose and formation of H2O2. [ABSTRACT FROM AUTHOR]

As is well-known, graphene shows a remarkable difference in affinity toward nonstructured single-stranded (ss) DNA and double-stranded (ds) DNA. This property makes it popular to prepare DNA-based optical sensors. In this work, taking this unique property of graphene in combination with the sensitive electrochemical transducer, we report a novel affinity-mediated homogeneous electrochemical aptasensor using graphene modified glassy carbon electrode (GCE) as the sensing platform. In this approach, the specific aptamer-target recognition is converted into an ultrasensitive electrochemical signal output with the aid of a novel T7 exonuclease (T7Exo)-assisted target-analog recycling amplification strategy, in which the ingeniously designed methylene blue (MB)-labeled hairpin DNA reporters are digested in the presence of target and, then, converted to numerous MB-labeled long ssDNAs. The distinct difference in differential pulse voltammetry response between the designed hairpin reporters and the generated long ssDNAs on the graphene/GCE allows ultrasensitive detection of target biomolecules. Herein, the design and working principle of this homogeneous electrochemical aptasensor were elucidated, and the working conditions were optimized. The gel electrophoresis results further demonstrate that the designed T7Exo-assisted target-analog recycling amplification strategy can work well. This electrochemical aptasensor realizes the detection of biomolecule in a homogeneous solution without immobilization of any bioprobe on electrode surface. Moreover, this versatile homogeneous electrochemical sensing system was used for the determination of biomolecules in real serum samples with satisfying results. [ABSTRACT FROM AUTHOR]

Photoelectrochemical (PEC) detection is an emerging and promising analytical tool. However, its actual application still faces some challenges like potential damage of biomolecules (caused by itself system) and intrinsic low-throughput detection. To solve the problems, herein we design a novel split-type photoelectrochemical immunoassay (STPIA) for ultrasensitive detection of prostate specific antigen (PSA). Initially, the immunoreaction was performed on a microplate using a secondary antibody/primer-circular DNA-labeled gold nanoparticle as the detection tag. Then, numerously repeated oligonucleotide sequences with many biotin moieties were in situ synthesized on the nanogold tag via RCA reaction. The formed biotin concatamers acted as a powerful scaffold to bind with avidin-alkaline phosphatase (ALP) conjugates and construct a nanoenzyme reactor. By this means, enzymatic hydrolysate (ascorbic acid) was generated to capture the photogenerated holes in the CdS quantum dot-sensitized TiO2 nanotube arrays, resulting in amplification of the photocurrent signal. To elaborate, the microplate-based immunoassay and the high-throughput detection system, a semiautomatic detection cell (installed with a three-electrode system), was employed. Under optimal conditions, the photocurrent increased with the increasing PSA concentration in a dynamic working range from 0.001 to 3 ng mL-1, with a low detection limit (LOD) of 0.32 pg mL-1. Meanwhile, the developed split-type photoelectrochemical immunoassay exhibited high specificity and acceptable accuracy for analysis of human serum specimens in comparison with referenced electrochemiluminescence immunoassay method. Importantly, the system was not only suitable for the sandwich-type immunoassay mode, but also utilized for the detection of small molecules (e.g., aflatoxin B1) with a competitive-type assay format. [ABSTRACT FROM AUTHOR]

In this paper we introduce a low cost rapid prototyping framework for designing Micro-Nano-Micro (MNM) fluidic preconcentration device based on ion concentration polarization (ICP) phenomenon. Xurography-based microchannels are separated by a strip of ion perm-selective Nafion membrane which plays the role of nanofluidic potential barrier for the negatively charged molecules. As a result, by using this rapid and inexpensive fabrication technique, it is possible to get preconcentration plugs as high as 5000 fold with an original symmetric electroosmotic flow (EOF) condition. Due to its simplicity and performance, this device could be implemented in various bioanalysis systems. [ABSTRACT FROM AUTHOR]

Gas-phase hydrogen/deuterium exchange (HDX) is a fast and sensitive, yet unharnessed analytical approach for providing information on the structural properties of biomolecules, in a complementary manner to mass analysis. Here, we describe a simple setup for ND3-mediated millisecond gas-phase HDX inside a mass spectrometer immediately after ESI (gas-phase HDX-MS) and show utility for studying the primary and higher-order structure of peptides and proteins. HDX was achieved by passing N2-gas through a container filled with aqueous deuterated ammonia reagent (ND3/D2O) and admitting the saturated gas immediately upstream or downstream of the primary skimmer cone. The approach was implemented on three commercially available mass spectrometers and required no or minor fully reversible reconfiguration of gas-inlets of the ion source. Results from gas-phase HDX-MS of peptides using the aqueous ND3/D2O as HDX reagent indicate that labeling is facilitated exclusively through gaseous ND3, yielding similar results to the infusion of purified ND3-gas, while circumventing the complications associated with the use of hazardous purified gases. Comparison of the solution-phase- and gas-phase deuterium uptake of Leu-Enkephalin and Glu-Fibrinopeptide B, confirmed that this gas-phase HDX-MS approach allows for labeling of sites (heteroatom-bound non-amide hydrogens located on side-chains, N-terminus and C-terminus) not accessed by classical solution-phase HDX-MS. The simple setup is compatible with liquid chromatography and a chip-based automated nanoESI interface, allowing for online gas-phase HDX-MS analysis of peptides and proteins separated on a liquid chromatographic time scale at increased throughput. Furthermore, online gas-phase HDX-MS could be performed in tandem with ion mobility separation or electron transfer dissociation, thus enabling multiple orthogonal analyses of the structural properties of peptides and proteins in a single automated LC-MS workflow. [ABSTRACT FROM AUTHOR]

A proximity hybridization-triggered signal switch was presented for convenient homogeneous chemiluminescent detection of a wide range of affinity target biomolecules, such as oligonucleotides, protein biomarkers, and aptamer-recognized targets. The presence of the target promoted the formation of a proximate complex via the proximity hybridization of two help DNA strands or the DNA strands labeled to affinity ligands, which subsequently unfolded the self-reporting molecular beacon to switch on the chemiluminescence signal. The response could be further amplified with an in situ enzymatic recycling strategy for highly sensitive chemiluminescence detection. By using an antibody as the affinity ligand, this simple protocol could sensitively detect protein biomarker in a concentration range of 6 orders of magnitude with a detection limit down to 80 fM. With the use of an aptamer as the affinity ligand, a method for homogeneous chemiluminescent detection of thrombin was proposed. The one-step and wash-free assay showed good selectivity and required only 1 μL of sample. Its high sensitivity, acceptable accuracy, and satisfactory versatility of analytes led to various applications in bioanalysis. [ABSTRACT FROM AUTHOR]

Direct assessment of the vascular lesions of model animals in vivo is important for the development of new antiatherosclerotic drugs. Nevertheless, biochemical analysis of the lipid profile in blood in vitro remains the most common way to evaluate the therapeutic effect of drugs targeting atherosclerosis because of an inherent difficulty to access the vascular wall. Using hypercholesterolemic zebrafish, we present an orchestrated application of Raman spectral measurements and confocal fluorescence imaging to interrogate the pharmacological response of atherosclerotic lesions in situ and in vivo. For demonstration, we investigated two commonly prescribed antihyperlipidemic drugs, ezetimibe and atorvastatin. The treatment of ezetimibe or atorvastatin alone decreased effectively the deposition of lipids in the vascular wall, and a combined dose showed a synergistic effect. Atorvastatin exerted a profound antioxidative effect on vascular fatty lesions. Analysis of individual lesions shows further that these lesions exhibited a heterogeneous response to the treatment of atorvastatin; a significant fraction of, but not all, the lesions became nonoxidized after the intervention. Beyond its efficacies in suppressing both the accumulation and oxidation of vascular lipids, atorvastatin expedited the clearance of vascular lipids. The possession of pleotropic (multiple) therapeutic effects on vascular fatty lesions of hypercholesterolemic zebrafish by atorvastatin is notably consistent with the known pharmaceutical effects of this drug on human beings. These results improve our understanding of the antiatherosclerotic effect of drugs. We envisage that our approach has the potential to become a platform to predict the pharmaceutical effects of new drugs aiming to cure human atherosclerotic diseases. [ABSTRACT FROM AUTHOR]

Nanoflow liquid chromatography (nano-LC) is an essential technique for highly sensitive analysis of complex biological samples, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is advantageous for rapid identification of proteins and in-depth analysis of post-translational modifications (PTMs). A combination of nano-LC and MALDI-MS (nano-LC/MALDI-MS) is useful for highly sensitive and detailed analysis in life sciences. However, the existing system does not fully utilize the advantages of each technique, especially in the interface of eluate transfer from nano-LC to a MALDI plate. To effectively combine nano-LC with MALDI-MS, we integrated a nano-LC column and a deposition probe for the first time (column probe) and incorporated it into a nano-LC/MALDI-MS system. Spotting nanoliter eluate droplets directly from the column onto the MALDI plate prevents postcolumn diffusion and preserves the chromatographic resolution. A DHB prespotted plate was prepared to suit the fabricated column probe to concentrate the droplets of nano-LC eluate. The performance of the advanced nano-LC/MALDI-MS system was substantiated by analyzing protein digests. When the system was coupled with multidimensional liquid chromatography (MDLC), trace amounts of glycopeptides that spiked into complex samples were successfully detected. Thus, a nano-LC/MALDI-MS direct-spotting system that eliminates postcolumn diffusion was constructed, and the efficacy of the system was demonstrated through highly sensitive analysis of the protein digests or spiked glycopeptides. [ABSTRACT FROM AUTHOR]

Ion mobility-mass spectrometry measurements which describe the gas-phase scaling of molecular size and mass are of both fundamental and pragmatic utility. Fundamentally, such measurements expand our understanding of intrinsic intramolecular folding forces in the absence of solvent. Practically, reproducible transport properties, such as gas-phase collision cross-section (CCS), are analytically useful metrics for identification and characterization purposes. Here, we report 594 CCS values obtained in nitrogen drift gas on an electrostatic drift tube ion mobility-mass spectrometry (IM-MS) instrument. The instrument platform is a newly developed prototype incorporating a uniform-field drift tube bracketed by electrodynamic ion funnels and coupled to a high resolution quadrupole time-of-flight mass spectrometer. The CCS values reported here are of high experimental precision (±0.5% or better) and represent four chemically distinct classes of molecules (quaternary ammonium salts, lipids, peptides, and carbohydrates), which enables structural comparisons to be made between molecules of different chemical compositions for the rapid "omni-omic" characterization of complex biological samples. Comparisons made between helium and nitrogen-derived CCS measurements demonstrate that nitrogen CCS values are systematically larger than helium values; however, general separation trends between chemical classes are retained regardless of the drift gas. These results underscore that, for the highest CCS accuracy, care must be exercised when utilizing helium-derived CCS values to calibrate measurements obtained in nitrogen, as is the common practice in the field. [ABSTRACT FROM AUTHOR]

We develop a new homogeneous method for sensitive detection of various biomolecules on the basis of bioluminescence monitoring the released AMP from the target-triggered hybridization chain reaction-mediated ligation. The introduction of hybridization chain reaction not only improves the sensitivity of DNA assay, but also facilitates the sensitive detection of proteins by designing specific aptamer triggers, providing a universally amplified platform for simultaneous detection of different kinds of biomolecules. Importantly, this bioluminescence assay employs the target-dependent ATP from the ligation byproduct of AMP as the reporter without the requirement for the sophisticated luciferase manipulation, complicated immobilization, and separation steps. The proposed method has significant advantages of simplicity, high sensitivity, low cost, and high throughput, and holds a great promise for practical point-of-care applications. [ABSTRACT FROM AUTHOR]

Single molecule detection schemes promise that one has the ability to reach the ultimate limit of detection: one molecule. In this paper, we use the stochastic luminescence of single semiconductor nanocrystals (quantum dots, QDs) to detect and localize particles as digital counts. These digital counts can be correlated to the concentration of analytes in solution. Here, we use total internal reflection fluorescence (TIRF) microscopy to probe individual QDs immobilized on a functionalized substrate. QDs have found their niche in the bioanalytical community due to their remarkable brightness and stability. Despite their numerous outstanding photophysical properties, QDs at the single particle level display a pronounced intermittent luminescence, posing a challenge for the detection of individual particles. In this paper, we demonstrate a reliable method for detecting QDs that takes advantage of these signal fluctuations by comparing the variations in the QD's fluorescence signals against variations of the background signal. The quantitative methodology developed here results in signal-to-background ratios up to 90:1, which is at least 8-times higher than the ratios obtained using methodologies relying solely on signal integration. This enhanced signal-to-background ratio facilitates a robust thresholding process and results in femtomolar limits of detection. [ABSTRACT FROM AUTHOR]

In this study, we proposed and demonstrated a novel simultaneous analysis system of biosensing by combining a semiconductor-based field effect transistor (FET) with quartz crystal microbalance with a dissipation (QCM-D) monitoring system. Using the combined system, the changes of not only mass and viscoelasticity but also electrical charge for interaction of charged dextran molecules with substrate, recognition of glucose with low molecular weight, and programmed cell death, apoptosis, were simultaneously and quantitatively monitored in a label-free and real-time manner. The combined system will give more detailed information of biomolecule/substrate interface for development of new biomaterial. [ABSTRACT FROM AUTHOR]

We present a novel approach to reference-compensated label-free affinity biosensing in complex media. Unlike conventional approaches that employ surfaces with different biological functionalities in the detection and reference channels to produce a reference-compensated sensor response, the new approach (referred as to single surface referencing (SSR)) uses a single functionalized surface split into the detection and reference channel to which complex sample (detection channel) and complex sample mixed with biomolecules binding to the analyte and thus inhibiting the binding of the analyte to the functionalized surface (reference channel) is introduced. This approach ensures that (i) only the detection channel captures the analyte and (ii) nonspecific binding incurred in the detection and reference channels are the same. We evaluate this approach in a model biosensing experiment, detection of a cancer biomarker carcinoembryonic antigen (CEA) in blood plasma using antibody against CEA and a surface plasmon resonance (SPR) biosensor. We detect CEA in three different blood plasma samples and demonstrate that this novel referencing approach provides more accurate results and lower biological variability than the conventional referencing. [ABSTRACT FROM AUTHOR]

Direct analysis in real time mass spectrometry (DART-MS) is used to analyze the surface chemical composition of nanometer-sized organic aerosol partides in real time at atmospheric pressure. By introducing a stream of partides in between the DART ionization source and the atmospheric pressure inlet of the mass spectrometer, the aerosol is exposed to a thermal flow of helium or nitrogen gas containing some fraction of metastable helium atoms or nitrogen molecules, in this configuration, the molecular constituents of organic partides are desorbed, ionized, and detected with reduced molecular ion fragmentation, allowing for compositional identification. Aerosol particles detected include alkanes, alkenes, acids, esters, alcohols, aldehydes, and amino acids. The ion signal produced by DART-MS scales with the aerosol surface area rather than volume, suggesting that DART-MS is a viable technique to measure the chemical composition of the partide interface. For oleic acid, particle size measurements of the aerosol stream exiting the ionization region suggest that the probing depth depends upon the desorption temperature, and the probing depth is estimated to be on the order of S nm for a 185 run diameter partide at a DART heater temperature of 500 °C with nitrogen as the DART gas. The reaction of ozone with submicrometer oleic acid particles is measured to demonstrate the ability of this technique to identify products and quantify reaction rates in a heterogeneous reaction. [ABSTRACT FROM AUTHOR]

A novel strategy for ultrasensitive detection of model protein based on the integration of tyramide signal amplification (TSA) and polymerization-assisted signal amplification was proposed. The surface-initiated atom transfer radical polymerization (SI-ATRP) of glycidyl methacrylate (GMA) was triggered by the initiator-coupled protein immobilized on the electrode surface through sandwiched immunoreactions. Growth of long chain polymeric materials provided numerous epoxy groups for subsequent coupling of horseradish peroxidase (HRP), which in turn significantly increased the loading of quantum dots (QDs) labeled tyramide in the presence of hydrogen peroxide. As a result, electrochemiluminescence (ECL) and square-wave voltammetric (SWV) measurements showed 9.4- and 10.5-fold increase in detection signal in comparison with the unamplified method, respectively. To demonstrate the feasibility of this approach, human immunoglobulin G antigen (IgG) as a model target protein was employed and the detection limits were 0.73 and 0.09 pg mL–1 for ECL and SWV, respectively. The results showed that sensitivity of the presented immunoassay significantly increased by one-order of magnitude and offered great application promises in providing a sensitive, specific, and potent method for biological detection. [ABSTRACT FROM AUTHOR]

Analysis of chemical or biomolecular contents in a tiny amount of specimen presents a significant challenge in many biochemical studies and diagnostic applications. In this work, we present a single-layer, optofluidic device for real-time, high-throughput, quantitative analysis of droplet contents. Our device integrates an optical fiber-based, on-chip detection unit with a droplet-based microfluidic unit. It can quantitatively analyze the contents of individual droplets in real-time. It also achieves a detection throughput of 2000 droplets per second, a detection limit of 20 nM, and an excellent reproducibility in its detection results. In a proof-of-concept study, we demonstrate that our device can be used to perform detection of DNA and its mutations by monitoring the fluorescent signal changes of the target DNA/molecular beacon complex in single droplets. Our approach can be immediately extended to a real-time, high-throughput detection of other biomolecules (such as proteins and viruses) in droplets. With its advantages in throughput, functionality, cost, size, and reliability, the droplet-based optofluidic device presented here can be a valuable tool for many medical diagnostic applications. [ABSTRACT FROM AUTHOR]

Semitransparent porous silicon substrates have been developed for pairing nanostructure-initiator mass spectrometry (NIMS) imaging with traditional optical-based microscopy techniques. Substrates were optimized to generate the largest NIMS signal while maintaining sufficient transparency to allow visible light to pass through for optical microscopy. Using these substrates, both phase-contrast and NIMS images of phospholipids from a scratch-wounded cell monolayer were obtained. NIMS images were generated using a spatial resolution of 14 µm. Coupled with further improvements in spatial resolution, this approach may allow for the localization of intact biological molecules within cells without the need for labeling. [ABSTRACT FROM AUTHOR]

Label-free sensing strategies are an intensely studied and increasingly used alternative to signal amplification via fluorescent labels and enzymatic methods. This article discusses one class of optical sensors, termed "photonic crystals", that effectively amplify binding events (such as analyte capture) via strong light-matter interactions. [ABSTRACT FROM AUTHOR]

Tip enhanced Raman scattering (TERS) microscopy is used to image antibody conjugated nanoparticles on intact cellular membranes. The combination of plasmonic coupling and the resultant electric field obtained from intermediate focusing of a radially polarized source gives rise to Raman images with spatial resolution below 50 nm. Finite element method calculations are used to explain the origins of the observed image resolution and spectroscopic signals. The observed Raman scattering provides information about the biomolecules present near the nanoparticle probes. The results show that aggregates of nanoparticles produce spectroscopic results similar to those reported from other surface enhanced Raman spectroscopies, e.g., shell isolated nanoparticle enhanced Raman spectroscopy (SHINERS) and aggregated nanoparticles; however, TERS enables the detection of isolated nanoparticles on cell membranes where the observed spectra provide information about the interaction of the specific biomolecule conjugated to the nanoparticle probe. These measurements present a new technique for exploring biomolecular interactions on the surface of cells and tissue. [ABSTRACT FROM AUTHOR]

Differential ion mobility spectrometry (FAIMS) integrated with mass spectrometry (MS) is a powerful new tool for biological and environmental analyses. Large proteins occupy regions of FAIMS spectra distinct from peptides, lipids, or other medium-size biomolecules, likely because strong electric fields align huge dipoles common to macroions. Here we confirm this phenomenon in separations of proteins at extreme fields using FAIMS chips coupled to MS and demonstrate their use to detect even minor amounts of large proteins in complex matrixes of smaller proteins and peptides. [ABSTRACT FROM AUTHOR]

The development of versatile biofunctional surfaces is a fundamental prerequisite in designing Lab on a Chip (LOC) devices for applications in biosensing interfaces and microbioreactors. The current paper presents a rapid combinatorial approach to create multiplex protein patterns in a single microfluidic channel. This approach consists of coupling microcontact printing with microfluidic patterning, where microcontact printing is employed for silanization using (3-Aminopropyl) triethoxysilane (APTES), followed by microfluidic patterning of multiple antibodies. As a result, the biomolecules of choice could be covalently attached to the microchannel surface, thus creating a durable and highly resistant functional interface. Moreover, the experimental procedure was designed to create a microfluidic platform that maintains functionality at high flow rates. The functionalized surfaces were characterized using X-ray photoelectron spectroscopy (XPS) and monitored with fluorescence microscopy at each step of functionalization. To illustrate the possibility of patterning multiple biomolecules along the cross section of a single microfluidic channel, microarrays of five different primary antibodies were patterned onto a single channel and their functionality was evaluated accordingly through a multiplex immunoassay using secondary antibodies specific to each patterned primary antibody. The resulting patterns remained stable at shear stresses of up to 50 dyn/cm². The overall findings suggest that the developed multiplex functional interface on a single channel can successfully lead to highly resistant multiplex functional surfaces for high throughput biological assays. [ABSTRACT FROM AUTHOR]

While the importance of microRNAs (miRNAs) in cancer treatment or manipulation of genetic expression has been increasingly recognized for developing miRNA-based therapies, the controlled delivery of miRNAs into specific cells constitutes a challenging task. This report describes preliminary findings from an investigation of the conjugation of gold nanoparticles with miRNAs (miRNA-AuNPs) and their cell transfection. The immobilization of miRNAs on the AuNPs was detected, and the surface stability was substantiated by gel electrophoresis assessment of the highly charged characteristics of miRNA-AuNPs and their surface-exchange inactivity with a highly charged surfactant. The miRNA-AuNPs were tested in cell transfection using multiple myeloma cells, demonstrating efficient knockdown in the functional luciferase assay. The findings have important implications for understanding the mechanistic details of cell transfection involving miRNA-conjugated nanoparticles as biosensing or targeting probes. [ABSTRACT FROM AUTHOR]

Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) provides the highest mass resolving power and mass measurement accuracy for unambiguous identification of biomolecules. Previously, the highest-mass protein for which FTICR unit mass resolution had been obtained was 115 kDa at 7 T. Here, we present baseline resolution for an intact 147.7 kDa monoclonal antibody (mAb), by prior dissociation of noncovalent adducts, optimization of detected total ion number, and optimization of ICR cell parameters to minimize space charge shifts, peak coalescence, and destructive ion cloud Coulombic interactions. The resultant long ICR transient lifetime (as high as 20 s) results in magnitude-mode mass resolving power of 420?000 at m/z 2?593 for the 57+ charge state (the highest mass for which baseline unit mass resolution has been achieved), auguring for future characterization of even larger intact proteins and protein complexes by FTICR MS. We also demonstrate up to 80% higher resolving power by phase correction to yield an absorption-mode mass spectrum. [ABSTRACT FROM AUTHOR]

We present a configuration for fluorescence spectroscopy that exploits the optical properties of semitransparent gold films and widely available instrumentation. This method enables monitoring of biomolecule interactions with small molecules tethered on substrates in multicomponent environments. The neurotransmitter serotonin (5-hydroxytryptamine) was covalently attached to self-assembled monolayers on thin gold films at low density to facilitate antibody recognition. Protein-binding studies were performed in a fluorescently labeled immunoassay format. We find that the use of this method enables evaluation of nonspecific binding and relative quantification of specific binding between competing binding partners. This fluorescence spectroscopy technique has the potential to assess biosensor or medical device responses in complex biological matrices. [ABSTRACT FROM AUTHOR]

Gaseous biogenic amines such as putrescine, spennidine, aniline, and trimethylamine are important biomolecules that play many crucial rules in metabolism and medical diagnostics. A chemodosimetric detection assay has been developed for those gaseous amines by R&'-Eu" heterobimetallic complexes, K([Ru°Bubpy)(CN)4]2Eum(l12O)4l (where tBubpy = 4,4'-di-tert-butyl-2,2'-bipyridine). Synthesis, X-ray crystal characterization, and spectroscopic properties of this Ru"-E&11 heterobimetallic complex were reported. Binding properties of the Ru"-Eu" complex with common gases revealed that this complex is very selective to gaseous amine molecules. Sensitivity of this complex toward the amines was found as ∼log k = 4.5-4.8. Real time monitoring of gaseous biogenic amines was applied to real fish samples (Atlantic mack. erel) by studying the spectrofluorinietric responses of the Ru"-E&" complex toward different biogenic amine concentration. GC/MS studies were also used as a reference for the studies. A linear spectrofluorimetric response was found toward biogenic amine concentration in real fish samples. This complex was found to respond specifically to those biogenic amines dowa to 10 ppb. [ABSTRACT FROM AUTHOR]

Glycosaminoglycans heparin and heparan sulfate are biologically active polysulfated carbohydrates that are among the most challenging biopolymers with regards to their structural analysis and functional assessment. Fragmentation of oligosaccharides and sulfate loss are important hindrance to their analysis by mass spectrometry (MS), requiting thus soft ionization methods. The recently introduced soft ionization method desorption electrospray ionization (DESI) has been applied here to heparin and heparan sulfate oligosaccharides, showing that DESI-MS is well suited for the detection of such fragile biomolecules in their intact form. Characterization of complicated oligosaccharides such as synthetic heparin octadecasulfated dodecasaccharide was successfully achieved. The use of water for a spray solvent instead of denaturing organic solvents allowed the first DESI-MS detection of noncovalent biomolecular complexes between heparin oligosaccharides and the chemokine Stromal Cell-derived Factor-1. The hyphenation of the DESI ion source with the high-resolution LTQ-Orbitrap MS analyzer led to high accuracy of mass measurement and enabled unambiguous detennination of the protein-bound sulfated oligosaccharide. [ABSTRACT FROM AUTHOR]

Although matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis is an important tool for analyzing and characterizing biomolecules of varying complexity, the sensitivity of MALDI-TOFMS is dependent on proper preparation of the sample, a process that is oftentimes problematic and requires considerable expertise. In this study, we have developed a radiate microstructure chip on which samples can be concentrated for analysis by MALDI-TOFMS. The sample/matrix mixture was deposited onto the central space of the well on the chip and allowed to dry. Microscopic analysis confirmed that the applied samples were confined to the central zone. Sample spots focused on the chip were much smaller than those on an unmodified plate with the same total volume. Optimizing processes of several preparation factors were also performed to ensure matrix homogeneity in our chip. Analysis of the samples with MALDI-TOFMS showed that the signals from samples on our chip were significantly greater than those on the unmodified plate. The feasibility of using this chip to detect peptides and phosphopeptides was also demonstrated. [ABSTRACT FROM AUTHOR]

There are two major array formats used in life science research and biomedical analysis. The first is the micro-well plate format with millimeter-sized wells each with microliter capacity addressed individually and repeatedly during experiments. The second is the microarray format with micrometer-sized spots that are patterned initially but not addressable individually thereafter. Here, we present an addressable nanoliter-well plate with micrometer sized wells that combines the advantages of the two array formats. The nanowells are formed by reversibly sealing a steel stencil featuring an array of micrometer-scale openings to an optically transparent substrate. The nano-wells have a capacity of ∼1 nL, are ∼140 μm in diameter, and are arrayed at a density of 1600 wells cm-2. A soft polymer is patterned photolithographically around each opening so as to form a microgasket for pressure sensitive, liquid tight, and reversible sealing to any type of smooth substrate, either hydrophilic or hydrophobic. The rigidity of the steel prevents the distortion that occurs in soft, all-polymeric stencils and permits accurate registration across the entire array, which in turn allows for repeated, individual addressing of wells using an inkjet spotter. The stencils are used to pattern cells, make protein microarrays, and create nanowells on surfaces to study reverse transfection by first spotting plasmids encoding fluorescent proteins into the wells, seeding cells, and monitoring the transfection of the cells in real time using lime-lapse imaging. The hybrid elastomer-metal stencils (HEMSs) are versatile and useful for multiplexed analysis of drugs, biomolecules, and cells with microarray density. [ABSTRACT FROM AUTHOR]

Ultrasensitive detection of proteins is of great importance to proteomics studies. We report here a method to enhance detection sensitivity in surface plasmon resonance (SPR) spectroscopy by coupling a polymerization initiator to a biospecific interaction and inducing inline atom transfer radical polymerization (AThP) for amplifying SPR response. Bacterial cholera toxin (CI) is chosen as the model protein that has been covalently immobilized on the surface for demonstrating the principle. The specific recognition is achieved by use of biotinylated anti-CF, which allows initiators nith a biotin tag to be fixed at the protein binding site through a neutravidin bridge and triggers the localized growth of polymer brushes of poly-(hydroxyl-ethyl methacrylate) (PHEMA) via an ATRP mechanism. To further enhance the signal, a second ATRP reaction is conducted that takes advantage of the hydroxyl groups of PHEMA brushes from the first step to form hypethranched polymers onto the sensing surface. The two consecutive ATRP steps significantly improve SPR detection, allowing low amounts of CF that yield no direct measurement to be quantified with large signals. The resulting polymer film has been characterized by optical and atomic force microscopy. Ascorbic acid (AA) is employed as deoxygen reagent in the catalyst mixture that effectively suppresses oxygen interference, shortening the reaction time and making it possible for applying this ATRP approach to flow injection based SPR detection. A calibration curve of PHEMA amplification for CF detection based on suiface coverage has been obtained that displays a correlation in a range from 8.23 x 10-15 to 3.61 x 10-12 mol/cm2 with a limit of detection of 6.27 x 10-15 mol/cm2. The versatile biotin-neutravidin interaction used here should allow adaptation of ATRP enhancement to many other systems that include DNA, RNA, peptides, and carbohydrates, opening new avenues for ultrasensitive analysis of biomolecules with flow-injection assay and SPR spectroscopy. [ABSTRACT FROM AUTHOR]

An alternative version of fluorescence correlation spectroscopy is presented, where the signal from a medium surrounding the particles of interest is analyzed, as opposed to a signal from the particles themselves. This allows for analysis of unlabeled particles and potentially of biomolecules. Here, the concept together with principal experiments on polystyrene beads of 100, 200, 400, and 800 nm diameter in an aqueous solution of alexa 488- fluorophores are presented. The use of photo detectors allowing higher photon fluxes, or of reduced detection volumes, should enable analysis of significantly smaller particles or even biomolecules. [ABSTRACT FROM AUTHOR]

With the rapid growth of complex heterogeneous biological molecules, effective techniques that are capable of rapid characterization of biologics are essential to ensure the desired product characteristics. To address this need, we have developed a method for analysis of intact glycoproteins based on high-resolution capillary electrophoretic separation coupled to an LTQ-FT mass spectrometer. We evaluated the performance of this method on the a subunit of mouse cell line-derived recombinant human chomionic gonadotmpbin (r-αhCG), a protein that is glycosylated at two sites and is part of the clinically relevant gonadotrophin family. Analysis of r-αhCG, using capillary electrophoresis (CE) with a separation time under 20 mm, resulted in the identification of over 60 different glycofonos with up to nine sialic acids. High-resolution CE-Fourier transform mass spectrometiy (FT-MS) allowed separation and analysis of not only intact glycofonns wills different numbers of sialic acids but also intact glycoforms that differed by the number and extent of neutral monosaccharides. The high mass resolution of the FT-MS enabled a limited mass range to be targeted for the examination of the protein glycoforms, simplifying the analysis without sacrificing accuracy. In addition, the limited mass range resulted in a fast scan speed that enhanced the reproducibility of the relative quantitation of individual glycofonos. The intact glycoprotein analysis was complemented with the analysis of the titic glycopeptides and glycans of r-αhCG to enable the assignment of glycan structures to individual sites, resulting in a detailed characterization of the protein. Samples of r-αhCG obtained fiom a CHO cell line were also analysed and briefly shosos to be significantly different from the murine cell line product. Taken together, the results suggest that the CE coupled to high-resolution FT-MS can be one of the effective tools for in-process monitoring as well as for final product characterization. [ABSTRACT FROM AUTHOR]

Mass spectrometry based structural proteomics approaches for probing protein structures are increasingly gaining in popularity. The potential for such studies is limited because of the lack of analytical techniques for the automated interpretation of resulting data. In this article, a suite of algorithms called ProtMapMS is developed, integrated, and implemented specifically for the comprehensive automatic analysis of mass spectrometmy data obtained for protein structure studies using covalent labeling. The functions include data format conversion, mass spectrum interpretation, detection, and verification of all peptide species, confirmation of the modified pep- tide products, and quantification of the extent of peptide modification. The results thus obtained provide valuable data for use in combination with computational approaches for protein structure modeling. The structures of both monomeric and hexameric forms of insulin were investigated by oxidative protein footprinting followed by Is-resolution mass spectrometmy. The resultant data was analyzed both manually and using ProtMapMS without any manual intervention. The results obtained using the two methods were found to be in close agreement and overall were consistent with predictions from the czystallographic structure. [ABSTRACT FROM AUTHOR]

A compact, high-sensitivity, dual-channel flow cytometer (HSDCFCM) was developed for the individual analysis of nanosized particles and biomolecules. A hydrodynamic focusing technique was applied to confine the sample stream and enable small probe volume. Fluorescence bursts from single R-phycoerythrin (R-PE) molecules passing through the laser beam were well resolved from the background with signal-to-noise ratio of 17. Excellent size discrimination was demonstrated with a mixture of three sizes of polystyrene nanoparticles. Simultaneous measurement of fluorescence and light scattering signals from individual nanoparticles was demonstrated with the 100 nm fluorescent latex beads. Doxorubicin-loaded ZrO2 nanoparticles and fluorescently stained Escherichia coli ER2738 cells were analyzed successfully with dual-channel detection. Particle counting is demonstrated with the 210 nm fluorescent latex beads, and excellent correlation (R² > 0.998) between the manufacturer-reported concentrations and those measured by HSDCFCM enumeration was obtained. The measured sample detection efficiency was ∼90% on average for particle concentrations ranging from 1.62 × 105 to 3.93 × 107 particles/mL. Sample mixtures with varying proportions of fluorescently labeled and unlabeled nanoparticles were also analyzed with good ratio correspondence. By providing rapid, quantitative, and multiparameter characterization of nanoparticles, it is believed that the HSDCFCM will find many applications in the fields of bionanotechnology, bioanalytical chemistry, and biomedicine. [ABSTRACT FROM AUTHOR]

Label-free biomolecular binding measurement methods, such as surface plasmon resonance (SPR), are becoming increasingly more important for the estimation of real-lime binding kinetics. Recent advances in surface plasmon resonance imaging (iSPR) are emerging for label-free microarray-based assay applications, where multiple biomolecular interactions can be measured simultaneously. However, conventional iSPR microarray systems rely on protein printing techniques for ligand immobilization to the gold imaging surface and external pumps for analyte transport. In this article, we present an integrated microfluidics and iSPR platform that uses only electrokinelic transport and guiding of ligands and analytes and, therefore, requires only electrical inputs for sample transport. An important advantage of this new approach, compared to conventional systems, is the ability to direct a single analyte to a specific ligand location in the microarray, which can facilitate analysis parallelization. Additionally, this simple approach does not require complicated microfluidic channel arrangements, external pumps, or valves. As a demonstration, kinetics and affinity have been extracted from measured binding responses of human IgG and goat antihuman IgG using a simple 1:1 model and compared to responses measured with conventional pressure driven analyte transport. The measured results indicate similar binding kinetics and affinity between the electrokinetic and pressure-driven sample manipulation methods and no cross contamination to adjacent measurement locations has been observed. [ABSTRACT FROM AUTHOR]

Polyhedral boron clusters are proposed as new, chemically and biologically stable, versatile redox labels for electrochemical DNA hybridization sensors. Selective and sensitive detection of the redox labeled DNA-probe was achieved by means of covalently attached electroactive marker 7,8-dicarba-nido-undekaborate group. A nanomolar concentration of boron cluster-labeled DNA was recognized. High specificity of the analysis with the boron cluster-labeled DNA probe, including detection of single-base mismatch, was demonstrated. The above findings, together with proposed earlier use of metallacarboranes as an electrochemical label for biomolecules opens the door for a "multicolor" electrochemical coding of DNA with boron clusters and simultaneous detection of several DNA targets. [ABSTRACT FROM AUTHOR]

A quartz crystal microbalance was integrated into an AFM in order to monitor the adsorption of biomolecules to the resonator surface with both atomic force microscopy and microgravimetry. The comparison between the two techniques allows the fractional coverage of the surface, θ, to be correlated with the frequency shift of the resonator, Δf: The adsorbed material was ferritin, which is a spherical protein with a diameter of ∼12 nm. Even at a coverage below 50%, the protein layer exhibits Sauerbrey-like behavior, meaning that the magnitude in the frequency shift ∣Δf∣ much exceeds the shift in bandwidth and that the normalized frequency shift, Δf/n (n the overtone order), is similar on the different overtones. The relation between coverage and frequency shift was found to be nonlinear. In order to model this situation, we performed finite element method calculations based on the incompressible Navier-Stokes equation. The comparison between the model and the experiment suggests that the deformation of the protein upon adsorption is small. For low coverage, the volume of the trapped solvent exceeds the volume of the adsorbate itself. The ratio of the two decreases with increasing coverage. This is the cause of the experimentally observed nonlinear relationship between the surface coverage and frequency shift. Comparing frequency shifts at different overtones, one finds that Δf/n slightly decreases with overtone order. Such a behavior is typically attributed to softness. The model shows that, for the adsorbed spheres, this apparent softness arises through a rocking motion of the spheres at the surface instead of the shear deformation. Also, there is a hydrodynamic contribution (related to roughness) to the overtone dependence of Δf/n. [ABSTRACT FROM AUTHOR]

Quartz crystal microbalance with dissipation monitoring (QCM-D) has become a popular tool to investigate biomolecular adsorption phenomena at surfaces. In contrast to optical mass-sensitive techniques, which commonly detect the adsorbed nonhydrated mass, the mechanically coupled mass measured by QCM-D includes a significant amount of water. A mechanistic and quantitative picture of how the surrounding liquid couples to the deposited solutes has so far been elusive for apparently simple phenomena like the random adsorption of nanometer-sized particles on a planar surface. Using a setup that enables simultaneous measurements by reflectometry and QCM-D on the same support, we have quantified the variations in coupled water, as sensed by the QCM frequency response, as a function of coverage for the formation of monolayers of globular proteins, virus particles, and small unilamellar vesicles. We found a close-to-linear relationship between the surface coverage and the relative contribution of water to the frequency response for these adsorption scenarios. The experimental hydration curves could be reproduced quantitatively using a theoretical model that assigns a pyramid-shaped hydration coat to each adsorbed particle and that accounts for the random distribution of adsorbents on the surface. This simple model fits the experimental data well and provides insight into the parameters that affect hydration. [ABSTRACT FROM AUTHOR]

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) can be utilized to map the distribution of various molecules on a surface with submicrometer resolution. Much of its biological application has been in the study of membrane lipids, such as phospholipids and cholesterol. Cholesterol is a particularly interesting molecule due to its involvement in numerous biological processes. For many studies, the effectiveness of chemical mapping is limited by low signal intensity from various biomolecules. Because of the high energy nature of the SIMS ionization process, many molecules are identified by detection of characteristic fragments. Commonly, fragments of a molecule are identified using standard samples, and those fragments are used to map the location of the molecule. In this work, MS/MS data obtained from a prototype C60+/quadrupole time-of-flight mass spectrometer was used in conjunction with indium LMIG imaging to map previously unrecognized cholesterol fragments in single cells. A model system of J774 macrophages doped with cholesterol was used to show that these fragments are derived from cholesterol in cell imaging experiments. Examination of relative quantification experiments reveals that m/z 147 is the most specific diagnostic fragment and offers a 3-fold signal enhancement. These findings greatly increase the prospects for cholesterol mapping experiments in biological samples, particularly with single cell experiments. In addition, these findings demonstrate the wealth of information that is hidden in the traditional TOF-SIMS spectrum. [ABSTRACT FROM AUTHOR]

This paper presents a study of supported lipid bilayer (SLB) formation and subsequent protein binding using a sensor that combines localized surface plasmon resonance (LSPR) and quartz crystal microbalance with dissipation (QCM-D) monitoring. The LSPR activity arises from silicon oxide (SiO~) coated nanometric apertures in a thin gold film, which also serves as the active electrode of a QCM-D crystal. Both transducer principles provide signatures for the formation of a SIB upon adsorption and subsequent rupture of adsorbed lipid vesicles. However, the two techniques are sensitive over different regions of the sample: LSPR primarily inside and on the rim of the holes and QCM-D primarily on the planar areas between the holes. Although the dimension of the lipid vesicles is on the same order as the dimension of the nanoholes, it is concluded from the response of the combined system that vesicle rupture in the nanoholes and on the planar region between the holes is synchronized. Furthermore, by determining the thickness of the SIB from the QCM-D response, the characteristic decay length of the LSPR field intensity could be determined. This made it possible not only to determine the mass and refractive index of the homogeneous SIB but also to postulate a generic means to quantify the LSPR response in terms of mass-uptake also for nonhomogeneous films. This is exemplified by measuring the adsorbed lipid mass during vesicle adsorption, yielding the critical lipid vesicle coverage at which spontaneous rupture into a planar bilayer occurs. The generic applicability and versatility of the method is demonstrated from specific protein binding to a functionalized SLB. From the absolute refractive index of the protein, provided from the LSPR data alone, it was possible to determine both the effective thickness of the protein film and the molecular mass (or number) of bound protein. [ABSTRACT FROM AUTHOR]

Small molecules are difficult to detect by conventional SPR technique directly because the changes in the refractive index resulting from the binding processes of small biomolecules are often small. In order to extend the application of SPR biosensor in detecting a small molecule, we combine the advantage of aptamer technique with the amplifying effect of Au nanoparticles to design a sensitive SPR sensor for detecting small molecules. The principle of this sensor is based on surface inhibition detection. The aptamer is first immobilized on SPR gold film with its ss-DNA structure. The aptamer possessing this structure can be hybridized with Au nanoparticles-tagged complementary ss-DNA and result in a large change of SPR signal. However, the aptamer will change its structure from ss-DNA to tertiary structure after adenosine is added to the SPR cell. The aptamer possessing tertiary structure could not hybridize with Au nanoparticles-tagged complementary ss-DNA. Thus, the change of SPR signal resulted in the hybridization reaction between aptamer and Au nanoparticles-tagged complementary ss-DNA will decrease with the increase of the number of aptamers possessing tertiary structure, which is proportional to the concentration of the small molecule. Based on this principle, we choose a simple system (antiadenosine aptamer/adenosine) to detect the sensing ability of this SPR biosensor for a small molecule. The experimental results confirm that the SPR sensor we developed possesses a good sensitivity and a high selectivity for adenosine. The detection range for adenosine is from 1 × 10-9 to 1 × 10-6 M. More significantly, it is fairly easy to generalize this strategy to detect a spectrum of small molecules by SPR spectroscopy using different aptamers. Therefore, it is expected that this method may offer a new direction in designing high-performance SPR biosensors for sensitive and selective detection of a wide spectrum of small molecules. [ABSTRACT FROM AUTHOR]

In this paper, we report on steady-state and time-resolved single-molecule fluorescence measurements performed on a phycobiliprotein, R-phycoerythrin (RPE), assembled on silver nanostructures. Single-molecule measurements clearly show that RPE molecules display a 10-fold increase in fluorescence intensity, with a 7-fold decrease in lifetime when they are assembled on silver nanostructured surfaces, as compared to control glass slides. The emission spectrum of individual RPE molecules also displays a significant fluorescence enhancement on silver nanostructures as compared to glass. From intensity and lifetime histograms, it is clear that the intensities as well as lifetimes of individual RPE molecules on silver nanostructures are more heterogeneously distributed than that on glass. This single-molecule study provides further insight on the heterogeneity in the fluorescence intensity and lifetimes of the RPE molecules on both glass and SiFs surfaces, which is otherwise not possible to observe using ensemble measurements. Finite-difference time-domain calculations have been performed to study the enhanced near-fields induced around silver nanoparticles by a radiating excited-state fluorophore, and the effect of such enhanced fields on the fluorescence enhancement observed is discussed. [ABSTRACT FROM AUTHOR]