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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]

Quantification of adsorbed biomolecules (enzymes, proteins) at the cellulose interface is a major challenge in developing eco-friendly biodiagnostics. Here, a novel methodology is developed to visualize and quantify the adsorption of antibody from solution to the cellulose-liquid interface. The concept is to deuterate cellulose by replacing all nonexchangeable hydrogens from the glucose rings with deuterium in order to enhance the scattering contrast between the cellulose film surface and adsorbed antibody molecules. Deuterated cellulose (DC) was obtained from bacterial (Gluconacetobacter xylinus strain) cellulose, which was grown in heavy water (D2O) media with a deuterated glycerol as a carbon source. For comparison, hydrogenated cellulose (HC) was obtained from cellulose acetate. Both HC and DC thin films were prepared on silicon substrate by spin coating. X-ray reflectivity (XR) shows the formation of homogeneous and smooth film. Neutron reflectivity (NR) at the liquid/film interface reveals swelling of the cellulose film by a factor of 2-3× its initial thickness. An Immunoglobulin G (IgG), used as a model antibody, was adsorbed at the liquid-solid interface of cellulose (HC) and deuterated cellulose (DC) films under equilibrium and surface saturation conditions. NR measurements of the IgG antibody layer adsorbed onto the DC film can clearly be visualized, in sharp contrast in comparison to the HC film. The average thickness of the IgG adsorbed layer onto cellulose films is 127 ± 5 Å and a partial monolayer is formed. Visualization and quantification of adsorbed IgG is shown by large difference in scattering length density (SLD) between DC (7.1 × 10-6 Å-2) and IgG (4.1 × 10-6 Å-2) in D2O, which enhanced the scattering contrast in NR. Quartz crystal measurements (QCM-D) were used as a complementary method to NR to quantify the adsorbed IgG over the cellulose interface. [ABSTRACT FROM AUTHOR]

The alternative dissociation pathways initiated by ultraviolet photodissociation (UVPD) compared with collision-induced dissociation (CID) may provide useful diagnostic fragments for biomolecule identification, including glycans. However, underivatized glycans do not commonly demonstrate strong UV absorbance, resulting in low fragmentation yields for UVPD spectra. In contrast to UVPD experiments that leverage covalent modification of glycans, we detail the capacity of metal adduction to yield comparatively rich UVPD fragmentation patterns and enhance separation factors for an isomeric glycan set in a drift tube ion mobility system. Ion mobility and UVPD-MS spectra for two N-acetyl glycan isomers were examined, each adducted with sodium or cobalt cations, with the latter providing fragment yield gains of an order of magnitude versus sodium adducts. Furthermore, our glycan analysis incorporated front-end ion mobility separation such that the structural glycan isomers could still be identified even as a mixture and not simply composite spectra of isomeric standards. Cobalt adduction proved influential in the glycan separation by yielding an isomer resolution of 0.78 when analyzed simultaneously versus no discernable separation obtained with the sodium adducts. It is the combined enhancement of both isomeric drift time separation and isomer distinction with improved UVPD fragment ion yields that further bolster multivalent metal adduction for advancing glycan IM-MS experiments. [ABSTRACT FROM AUTHOR]

Two-dimensional graphene devices are widely used for biomolecule detection. Nevertheless, the surface modification of graphene is critical to achieve the high sensitivity and specificity required for biological detection. Herein, native bovine serum albumin (BSA) in inorganic solution is denatured on the graphene surface by heating, leading to the formation of nanoscale BSA protein films adsorbed on the graphene substrate via π-stacking interactions. This technique yields a controllable, scalable, uniform, and high-coverage method for graphene biosensors. Further, the application of such nanoscale heat-denatured BSA films on graphene as a universal graphene biosensor platform is explored. The thickness of heat-denatured BSA films increased with heating time and BSA concentration but decreased with solvent concentration as confirmed by atomic force microscopy. The noncovalent interaction between denatured BSA films and graphene was investigated by Raman spectroscopy. BSA can act as a p-type and n-type dopant by modulating pH-dependent net charges on the layered BSA-graphene surface, as assessed by current-voltage measurements. Chemical groups of denatured BSA films, including amino and carboxyl groups, were verified by X-ray photoelectron microscopy, attenuated total reflectance-Fourier transform infrared spectra, and fluorescent labeling. The tailoring of the BSA-graphene surfaces through chemical modification, controlled thickness, and doping type via noncovalent interactions provides a controllable, multifunctional biosensor platform for molecular diagnosis without the possibility of nonspecific adsorption on graphene. [ABSTRACT FROM AUTHOR]

Biomolecular folding often occurs through a cooperative two-state reactant ↔ product transition; the term cooperative does not convey that intermediate structures are nonexistent but rather that these states are not observable by existing experimental techniques. Because of this, few intermediates have been studied and characterized. Recently, ion mobility spectrometry (IMS) measurements revealed that the oligomer polyproline-13 (Pro13, which in propanol (PrOH) favors the right-handed helical PPI structure having adjacent pyrrolidine rings in a cis configuration) folds through six sequential long-lived intermediates as it converts to the all-trans-configured PPII structure that is favored in aqueous solutions. Here, we examine the PPIPrOH → PPIIaq folding transition for a HisPro13 sequence, i.e., Pro13 having a single histidine residue added to the N-terminus. Remarkably, the IMS measurements show that, upon addition of histidine, all of the IMS peaks associated with intermediate structures disappear. Instead, HisPro13 folds via a cooperative two-state transition, delayed by a significant induction period. The induction period is temperature dependentshifting the transition to longer times at lower temperatures. Equilibrium studies show that the HisPro13 PPIPrOH → PPIIaq transition is endothermic but favored entropically. From these clues, we propose a sequential folding mechanism and develop a model that suggests that ∼13-17 long-lived intermediates are likely responsible for the induction period. In this model, intermediates are separated by average individual activation barriers of ∼90 kJ·mol-1, and are entropically favorable. [ABSTRACT FROM AUTHOR]

Conspectus: Air pollution is responsible for the premature death of about 7 million people every year. Ozone (O3) and nitrogen dioxide (NO2•) are the key gaseous pollutants in the troposphere, which predominantly result from combustion processes. Their inhalation leads to reactions with constituents in the airway surface fluids (ASF) of the respiratory tract and/or lungs. ASF contain small molecular-weight antioxidants, which protect the underlying epithelial cells against oxidative damage. When this defense system is overwhelmed, proteins and lipids present on cell surfaces or within the ASF become vulnerable to attack. The resulting highly reactive protein and lipid oxidation products could subsequently damage the epithelial cells through secondary reactions, thereby causing inflammation. While reactions of NO2• with biological molecules are considered to proceed through radical pathways, the biological effect of O3 is attributed to its high reactivity with π systems. Because O3 and NO2• always coexist in the polluted ambient atmosphere, synergistic effects resulting from in situ formed strongly oxidizing nitrate radicals (NO3•) may also require consideration. For example, in vitro product studies revealed that phenylalanine, which is inert not only to oxidants produced through biochemical processes, but also to NO2• or O3 in isolation, is damaged by NO3•. The reaction is initiated by oxidation of the aromatic ring and, depending on the availability of NO2•, leads to formation of nitrophenylalanine or β-nitrooxyphenylalanine, which could serve as marker for NO3•-induced oxidative damage in peptides. More easily oxidizable aromatic amino acids are directly attacked by NO2• and are converted to the same products independent of whether O3 is also present. Remarkably, NO2•-induced oxidative damage in peptides occurs not only through the well-established radical oxidation of peptide side chains, but also through an unprecedented fragmentation/rearrangement of the peptide backbone. This process is initiated by a nonradical N-nitrosation of a peptide bond involving the dimer of NO2•, i.e., N2O4, and contracts the peptide chain in the N → C direction by expelling one amino acid residue with simultaneous fusion of the remaining molecular termini, thereby forming a new peptide bond. This peptide cleavage could potentially be highly relevant for peptide segments with "nonvulnerable" side chains closer to the terminus that are not tied up in complex secondary and tertiary structures and therefore accessible for environmental oxidants. Likewise, NO2• reacts with cholesterol at the CC moiety through an ionic mechanism, which leads to formation of 6-nitrocholesterol in the presence of moisture. Contrary to common belief, this clearly shows that ionic chemistry, in particular nitrosation reactions by intermediately formed NO+, requires consideration when assessing NO2• toxicity. This conclusion is supported by recent work by Colussi et al. (Enami, S.; Hoffmann, M. R.; Colussi, A. J. Absorption of inhaled NO2. J. Phys. Chem. B. 2009, 113, 7977-7981), who showed that anions in the airway surfaces fluids mediate NO2• absorption by catalyzing its hydrolytic disproportionation into NO2-/HNO2 and NO3-. These findings could be the key to our understanding why NO2•, despite its low water solubility, has such pronounced biological effects in vivo. [ABSTRACT FROM AUTHOR]

Conspectus: Biosensors for highly sensitive, selective, and rapid quantification of specific biomolecules make great contributions to biomedical research, especially molecular diagnostics. However, conventional methods for biomolecular assays often suffer from insufficient sensitivity and poor specificity. In some case (e.g., early disease diagnostics), the concentration of target biomolecules is too low to be detected by these routine approaches, and cumbersome procedures are needed to improve the detection sensitivity. Therefore, there is an urgent need for rapid and ultrasensitive analytical tools. In this respect, single-molecule fluorescence approaches may well satisfy the requirement and hold promising potential for the development of ultrasensitive biosensors. Encouragingly, owing to the advances in single-molecule microscopy and spectroscopy over past decades, the detection of single fluorescent molecule comes true, greatly boosting the development of highly sensitive biosensors. By in vitro/in vivo labeling of target biomolecules with proper fluorescent tags, the quantification of certain biomolecule at the single-molecule level is achieved. In comparison with conventional ensemble measurements, single-molecule detection-based analytical methods possess the advantages of ultrahigh sensitivity, good selectivity, rapid analysis time, and low sample consumption. Consequently, single-molecule detection may be potentially employed as an ideal analytical approach to quantify low-abundant biomolecules with rapidity and simplicity. In this Account, we will summarize our efforts for developing a series of ultrasensitive biosensors based on single-molecule counting. Single-molecule counting is a member of single-molecule detection technologies and may be used as a very simple and ultrasensitive method to quantify target molecules by simply counting the individual fluorescent bursts. In the fluorescent sensors, the signals of target biomolecules may be translated to the fluorescence signals by specific in vitro/in vivo fluorescent labeling, and consequently, the fluorescent molecules indicate the presence of target molecules. The resultant fluorescence signals may be simply counted by either microfluidic device-integrated confocal microscopy or total internal reflection fluorescence-based single-molecule imaging. We have developed a series of single-molecule counting-based biosensors which can be classified as separation-free and separation-assisted assays. As a proof-of-concept, we demonstrate the applications of single-molecule counting-based biosensors for sensitive detection of various target biomolecules such as DNAs, miRNAs, proteins, enzymes, and intact cells, which may function as the disease-related biomarkers. Moreover, we give a summary of future directions to expand the usability of single-molecule counting-based biosensors including (1) the development of more user-friendly and automated instruments, (2) the discovery of new fluorescent labels and labeling strategies, and (3) the introduction of new concepts for the design of novel biosensors. Due to their high sensitivity, good selectivity, rapidity, and simplicity, we believe that the single-molecule counting-based fluorescent biosensors will indubitably find wide applications in biological research, clinical diagnostics, and drug discovery. [ABSTRACT FROM AUTHOR]

Technologies that can visualize, capture, and identify subsets of biomolecules that are not encoded by the genome in the context of healthy and diseased cells will offer unique opportunities to uncover the molecular mechanism of a multitude of physiological and disease processes. We describe here a chemical reporter strategy for labeling of cell surface glycoconjugates that takes advantage of recombinant glycosyltransferases and a corresponding sugar nucleotide functionalized by biotin. The exceptional efficiency of this method, termed one-step selective exoenzymatic labeling, or SEEL, greatly improved the ability to enrich and identify large numbers of tagged glycoproteins by LC-MS/MS. We further demonstrated that this labeling method resulted in far superior enrichment and detection of glycoproteins at the plasma membrane compared to a sulfo-NHS-activated biotinylation or two-step SEEL. This new methodology will make it possible to profile cell surface glycoproteomes with unprecedented sensitivity in the context of physiological and disease states. [ABSTRACT FROM AUTHOR]

We describe a novel combination of a responsive polymer brush and a fluorescently labeled biomolecule, where the position of the biomolecule can be switched from inside to outside the brush and vice versa by a change in pH. For this, we grafted ultrathin, amino-terminated poly(acrylic acid) brushes to glass and silicon substrates. Individual bovine serum albumin (BSA) molecules labeled with fluorophore ATTO 488 were covalently end-attached to the polymers in this brush using a bis-N-succinimidyl-(pentaethylene glycol) linker. We investigated the dry layer properties of the brush-protein ensemble, and it is swelling behavior using spectroscopic ellipsometry. Total internal reflection fluorescence (TIRF) microscopy enabled us to study the distance-dependent switching of the fluorescently labeled protein molecules. The fluorescence emission from the labeled proteins ceased (out-state) when the polymer chains stretched away from the interface under basic pH conditions, and fluorescence recurred (in-state) when the chains collapsed under acidic conditions. Moreover, TIRF allowed us to study the fluorescence switching behavior of fluorescently labeled BSA molecules down to the single-molecule level, and we demonstrate that this switching is fast but that the exact intensity during the in-state is the result of a more random process. Control experiments verify that the switching behavior is directly correlated to the responsive behavior of the polymer brush. We propose this system as a platform for switchable sensor applications but also as a method to study the swelling and collapse of individual polymer chains in a responsive polymer brush. [ABSTRACT FROM AUTHOR]

Rapid progress in identifying biomarkers that are hallmarks of disease has increased demand for high-performance detection technologies. Implementation of electrochemical methods in clinical analysis may provide an effective answer to the growing need for rapid, specific, inexpensive, and fully automated means of biomarker analysis. This Review summarizes advances from the past 5 years in the development of electrochemical sensors for clinically relevant biomolecules, including small molecules, nucleic acids, and proteins. Various sensing strategies are assessed according to their potential for reaching relevant limits of sensitivity, specificity, and degrees of multiplexing. Furthermore, we address the remaining challenges and opportunities to integrate electrochemical sensing platforms into point-of-care solutions. [ABSTRACT FROM AUTHOR]

The n → π* interaction is a weak but important noncovalent interaction present in biomolecules and other compounds. Complexes between 7-azaindole and 2,6-difluorinated pyridines were demonstrated earlier to interact not only via an expected strong hydrogen bond but also by a weaker and unexpected n → π* interaction between the nucleophilic nitrogen atom of the 7-azaindole and the electrophilic π-system of the pyridine ring. This system provides a unique and convenient framework upon which to investigate the effect that distal substitution on the 7-azaindole ring has on the strength of the n → π* interaction. Herein we describe our thorough analysis of these effects by applying a variety of diverse methods including NBO, ETS-NOCV, and AIM. Very good agreement in trends was observed among all these diverse methods of analysis. Substitution at the position para to the nucleophilic nitrogen atom of the 7-azaindole ring with electron-donating groups weakened the hydrogen bond interaction with the 2,6-difluoropyridine but enhanced the n → π* interaction. Substitution with electron-withdrawing groups had the opposite effect. In addition, good correlation of the results of the calculations with the substituents' Hammett σp values was observed. Energy decomposition analysis (EDA) corroborated the conclusions derived by the other methods of analysis. [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]

Quantitating and understanding the physical forces responsible for the interactions of biomolecules are fundamental to the biological sciences. This is especially challenging for membrane proteins because they are embedded within cellular bilayers that provide a unique medium in which hydrophobic sequences must fold. Knowledge of the energetics of protein-lipid interactions is thus vital to understand cellular processes involving membrane proteins. Here we used a host-guest mutational strategy to calculate the Gibbs free energy changes of water-to-lipid transfer for the aromatic side chains Trp, Tyr, and Phe as a function of depth in the membrane. This work reveals an energetic gradient in the transfer free energies for Trp and Tyr, where transfer was most favorable to the membrane interfacial region and comparatively less favorable into the bilayer center. The transfer energetics follows the concentration gradient of polar atoms across the bilayer normal that naturally occurs in biological membranes. Additional measurements revealed nearest-neighbor coupling in the data set are influenced by a network of aromatic side chains in the host protein. Taken together, these results show that aromatic side chains contribute significantly to membrane protein stability through either aromatic-aromatic interactions or placement at the membrane interface. [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]

The molecular characterization of the air/water interface is a key step in understanding fundamental multiphase phenomena ranging from heterogeneous chemical processes in the atmosphere to the hydration of biomolecules. The apparent simplicity of the air/water interface, however, masks an underlying complexity associated with the dynamic nature of the water hydrogen-bond network that has so far hindered an unambiguous characterization of its microscopic properties. Here, we demonstrate that the application of quantum many-body molecular dynamics, which enables spectroscopically accurate simulations of water from the gas to the condensed phase, leads to a definitive molecular-level picture of the interface region. For the first time, excellent agreement is obtained between the simulated vibrational sum-frequency generation spectrum and the most recent state-of-the-art measurements, without requiring any empirical frequency shift or ad hoc scaling of the spectral intensity. A systematic dissection of the spectral features demonstrates that a rigorous representation of nuclear quantum effects as well as of many-body energy and electrostatic contributions is necessary for a quantitative reproduction of the experimental data. The unprecedented accuracy of the simulations presented here indicates that quantum many-body molecular dynamics can enable predictive studies of aqueous interfaces, which by complementing analogous experimental measurements will provide unique molecular insights into multiphase and heterogeneous processes of relevance in chemistry, biology, materials science, and environmental research. [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]

Fabrication of metal-organic frameworks (MOFs) with large apertures triggers a brand-new research area for selective encapsulation of biomolecules within MOF nanopores. The underlying inclusion mechanism is yet to be clarified however. Here we report a molecular dynamics study on the mechanism of protein encapsulation in MOFs. Evaluation for the binding of amino acid side chain analogues reveals that van der Waals interaction is the main driving force for the binding and that guest size acts as a key factor predicting protein binding with MOFs. Analysis on the conformation and thermodynamic stability of the miniprotein Trp-cage encapsulated in a series of MOFs with varying pore apertures and surface chemistries indicates that protein encapsulation can be achieved via maintaining a polar/nonpolar balance in the MOF surface through tunable modification of organic linkers and Mg-O chelating moieties. Such modifications endow MOFs with a more biocompatible confinement. This work provides guidelines for selective inclusion of biomolecules within MOFs and facilitates MOF functions as a new class of host materials and molecular chaperones. [ABSTRACT FROM AUTHOR]

Most of the protein-based diseases are caused by anomalies in the functionality and stability of these molecules. Experimental and theoretical studies of the conformational dynamics of proteins are becoming in this respect essential to understand the origin of these anomalies. However, a description of the conformational dynamics of proteins based on mechano-energetic principles still remains elusive because of the intrinsic high flexibility of the peptide chains, the participation of weak noncovalent interactions, and the role of the ubiquitous water solvent. In this work, the conformational dynamics of trialanine dissolved in water (D2O) is investigated through Molecular Dynamics (MD) simulations combined with instantaneous normal modes (INMs) analysis both at equilibrium and after the vibrational excitation of the C-terminal amide I mode. The conformational equilibrium between a and pPII conformers is found to be altered by the intramolecular relaxation of the amide I mode as a consequence of the different relaxation pathways of each conformer which modify the amount of vibrational energy stored in the torsional motions of the tripeptide, so the a ? pPII and pPII ? a conversion rates are increased differently. The selectivity of the process comes from the shifts of the vibrational frequencies with the conformational changes that modify the resonance conditions driving the intramolecular energy flows. [ABSTRACT FROM AUTHOR]

A series of diseases, ranging from cholera via travelers' diarrhea to hamburger disease, are caused by bacterially produced toxic proteins. In particular, a toxic protein unit is brought into the host cell upon binding to specific membrane-bound oligosaccharides on the host cell membrane. For example, the protein that causes cholera, cholera toxin (CT), has five identical, symmetrically placed binding pockets (B proteins), on top of which the toxic A protein resides. A promising strategy to counteract the devastating biological effects of this AB5 protein involves the development of inhibitors that can act as mimics of membrane-bound GM1 molecules, i.e., that can bind CT strongly and selectively. To reach this goal, two features are essential: First of all, the inhibitor should display oligosaccharides that resemble as much as possible the naturally occurring cell-surface pentasaccharide onto which CT normally binds, the so-called GM1 sugar (the oligosaccharide part of which is then labeled GM1os). Second, the inhibitor should be able to bind CT via multivalent interactions so as to bind CT as strongly as possible to allow for a real competition with the cell-membrane-bound GM1 molecules. In this Account, we present elements of the path that leads to strong CT inhibition by outlining the roles of multivalency and the development and use of GM1 mimics. First, multivalency effects were investigated using "sugar-coated" platforms, ranging from dendritic structures with up to eight oligosaccharides to platforms that mimicked the fivefold symmetry of CT itself. The latter goal was reached either via synthetic scaffolds like corannulene or calix[5]arene or via the development of a neolectin CT mimic that itself carries five GM1os groups. Second, the effect of the nature of the oligosaccharide appended to this platform was investigated via the use of oligosaccharides of increasing complexity, from galactose and lactose to the tetrasaccharide GM2os and eventually to GM1os itself. The combination of these threads gives rise to a series of inhibitors that can strongly bind CT, with IC50 values below 100 pM, and in some cases can even bind one-on-one. [ABSTRACT FROM AUTHOR]

Only α1 and α2 domains of the α-chain of the major histocompatibility complex (MHC) directly bind peptide antigens (Ag-s) and the T-cell receptor (TCR). Significant plasticity was found in the TCR but only minor in (α1 + α2). The α3-domain position variation was noted only in connection to its binding the coreceptor CD8. We apply our methods for identifying functional conformational changes in proteins to a systematic study of similarities between 43 X-ray structures of the entire α chains of MHC-I HLA-A2. Out of 903 different αHLA-A2 pairs 203 show similarities within the earlier determined uncertainty threshold and unexpectedly form three similarity clusters (SCs) with all/most structures in a cluster similar within the uncertainty threshold. Pairs from different SCs always differ above the threshold, mainly due to variations in the α3 position/structure. All structures in SC3 cannot bind the CD8 coreceptor. Strong hydrogen bonds between (α1 + α2) and α3 differ between SC1 and SC2 but are nearly invariant within each SC. Small conformational changes in the (α1 + α2), caused by Ag-s differences, act as an α3 "allosteric switch" between SC2 and SC1. Binding of CD8 to SC2-HLA-A2 (Tax-type Ag-s) changes it to SC1-HLA-A2 (HuD-type Ag-s). HuD binding to HLA-A2 is much less stable than Tax binding. CD8-liganded HLA-A2 preference for binding HuD suggests that CD8-HLA-A2 may present a weakly binding peptide for TCR recognition, supporting the hypothesis that CD8 increases TCR avidity to weak Ag-s. Other HLA-A2 functions may involve α3. TCR-A6-liganded-Tax-type-HLA-A2s form two small clusters, similar to either A6-liganded-HuD or A6-liganded-native-Tax HLA-A2s. [ABSTRACT FROM AUTHOR]

Effective detection of low molecular weight compounds in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is often hindered by matrix interferences in the low m/z region of the mass spectrum. Here, we show that monolayer-protected gold nanoparticles (AuNPs) can serve as alternate matrices for the very sensitive detection of low molecular weight compounds such as amino acids. Amino acids can be detected at low fmol levels with minimal interferences by properly choosing the AuNP deposition method, density, size, and monolayer surface chemistry. By inkjet-printing AuNPs at various densities, we find that AuNP clusters are essential for obtaining the greatest sensitivity. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The article discusses research concerning the evolution of diagnostic and therapeutic nanoparticles for biomedical applications in medicine, science and engineering. Information about the structural and functional properties of fundamental-based nanometer-sized particles, is examined. Also investigated is the use of biomolecular targeting ligands such as monoclonal antibodies, peptides, or small molecules.

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]

An effective strategy has been documentedto combine both carbohydrateand amino acid biomolecules in a single synthetic polymeric systemvia a reversible addition–fragmentation chain transfer (RAFT)polymerization technique. The resultant unique block copolymer wasengineered to form uniform micelles with the desired projection ofeither selective or both amino acid/sugar residues on the outer surfacewith multivalency, providing pH-based stimuli-responsiveness and/orlectin recognition. The self-assembly process was studied in detailby field emission scanning electron microscopy (FE-SEM), dynamic lightscattering (DLS), and UV–visible spectroscopy. The enhancedlectin binding behavior was observed for glyconanoparticles with bothamino acid/sugar entities on the shell as compared to the only glycopolymernanoparticle because of the higher steric hindrance factor in thecase of only the glycopolymer nanoparticle. Fluorophore conjugationby postpolymerization functionalization was further exploited by fluorescencespectroscopy for evidencing the lectin recognition process. [ABSTRACT FROM AUTHOR]

The adherence of serum protein onconjugated polymer is a majorbottleneck in the application of the latter for selective sensingof small biomolecules in blood serum. In this report, we present newpolyfluorenes with d-glucuronic acid appendage that is anonreceptor for any serum protein, thereby providing a platform forselective sensing of free bilirubin in the clinically relevant rangeof <25 to >50 μmol/L in human blood serum. The appended d-glucuronic acid formed noncovalent interactions with bilirubin,which in conjunction with favorable spectral overlap between the polymersand bilirubin facilitated efficient FRET process in aqueous solutions.Addition of bilirubin resulted in the quenching of the polyfluoreneemission with simultaneous appearance of bilirubin emission exhibitingvisual emission color change from blue to light green. The polymerremained stable in serum even under severe basic conditions and exhibitedhigh selectivity with visual sensitivity only toward free bilirubinin human serum in the presence of crucial interferences such as hemoglobin,proteins, biliverdin, glucose, cholesterol, and metal ions. Nanomolarsensing of bilirubin could also be demonstrated successfully usingone of the d-glucuronic acid appended polymer (PF-Ph-GlcA),which could sense ∼150 nm of bilirubin in human serum. Thecombined role of energy transfer and noncovalent interaction highlightsthe potential of the new polymer design for highly selective sensingactivity in complex biofluids. [ABSTRACT FROM AUTHOR]

Biomolecular systems are able to respond to their chemical environment through reversible, selective, noncovalent intermolecular interactions. Typically, these interactions induce conformational changes that initiate a signaling cascade, allowing the regulation of biochemical pathways. In this work, we describe an artificial molecular system that mimics this ability to translate selective noncovalent interactions into reversible conformational changes. An achiral but helical foldamer carrying a basic binding site interacts selectively with the most acidic member of a suite of chiral ligands. As a consequence of this noncovalent interaction, a global absolute screw sense preference, detectable by 13C NMR, is induced in the foldamer. Addition of base, or acid, to the mixture of ligands competitively modulates their interaction with the binding site, and reversibly switches the foldamer chain between its left and right-handed conformations. As a result, the foldamer-ligand mixture behaves as a biomimetic chemical system with emergent properties, functioning as a "proton-counting" molecular device capable of providing a tunable, pH-dependent conformational response to its environment. [ABSTRACT FROM AUTHOR]

Freeenergy calculations are used to study how strongly potentialdrug molecules interact with their target receptors. The accuracyof these calculations depends on the accuracy of the molecular dynamics(MD) force field as well as proper sampling of the major conformationsof each molecule. However, proper sampling of ligand conformationscan be difficult when there are large barriers separating the majorligand conformations. An example of this is for ligands with an asymmetricallysubstituted phenyl ring, where the presence of protein loops hindersthe proper sampling of the different ring conformations. These ringconformations become more difficult to sample when the size of thefunctional groups attached to the ring increases. The Adaptive IntegrationMethod (AIM) has been developed, which adaptively changes the alchemicalcoupling parameter λ during the MD simulation so that conformationssampled at one λ can aid sampling at the other λ values.The Accelerated Adaptive Integration Method (AcclAIM) builds on AIMby lowering potential barriers for specific degrees of freedom atintermediate λ values. However, these methods may not work whenthere are very large barriers separating the major ligand conformations.In this work, we describe a modification to AIM that improves samplingof the different ring conformations, even when there is a very largebarrier between them. This method combines AIM with conformationalMonte Carlo sampling, giving improved convergence of ring populationsand the resulting free energy. This method, called AIM/MC, is appliedto study the relative binding free energy for a pair of ligands thatbind to thrombin and a different pair of ligands that bind to aspartylprotease β-APP cleaving enzyme 1 (BACE1). These protein–ligandbinding free energy calculations illustrate the improvements in conformationalsampling and the convergence of the free energy compared to both AIMand AcclAIM. [ABSTRACT FROM AUTHOR]

Residual dipolar couplings (RDCs) are important probes in structural biology, but their analysis is often complicated by the determination of an alignment tensor or its associated assumptions. We here apply the maximum entropy principle to derive a tensor-free formalism which allows for direct, dynamic analysis of RDCs and holds the classic tensor formalism as a special case. Specifically, the framework enables us to robustly analyze data regardless of whether a clear separation of internal and overall dynamics is possible. Such a separation is often difficult in the core subjects of current structural biology, which include multidomain and intrinsically disordered proteins as well as nucleic acids. We demonstrate the method is tractable and self-consistent and generalizes to data sets comprised of observations from multiple different alignment conditions. [ABSTRACT FROM AUTHOR]

Anharmonicvibrational frequencies and intensities (infrared andRaman) of an isolated free-base porphin molecule are predicted fromthe quantum mechanical (QM) geometry, the “semi-diagonal”quartic force field, and dipole moment and polarizability surfaces.The second-order vibrational perturbation theory plus the numericaldiagonalization of the Hamiltonian matrix containing off-diagonalFermi and Darling–Dennison resonance couplings (VPT2+WK) was used. The QM calculations were carried out withthe Becke–Lee–Yang–Parr composite exchange–correlationfunctional (B3LYP) and with the 6-31+G(d,p) basis set. The harmonicforce field for the equilibrium configuration was transformed intononredundant local symmetry internal coordinates, and normal coordinateswere defined. The semi-diagonal quartic rectilinear normal coordinatepotential energy surface (PES), as well as the cubic surfaces of dipolemoment (p) and polarizability (α) components,needed for the VPT2+WKcalculation, were constructedby a five-point finite differentiation of Hessians (for PES) and ofthe values and first derivatives of pand α.They were obtained at the point of equilibrium and for 432 displacedconfigurations. This theoretical approach provides very good agreementbetween the predicted and experimental frequencies and intensities.However, the favorable result can be partly attributed to error cancellationwithin the B3LYP/6-31+G(d,p) QM model, as observed in earlier studies.Reassignments of some observed bands are proposed. [ABSTRACT FROM AUTHOR]

We used molecular dynamics simulationsof a typical monomeric protein, SNase, in combination with Voronoi–Delaunaytessellation to study and analyze the temperature dependence of theapparent volume, Vapp, of the solute.We show that the void volume, VB, createdin the boundary region between solute and solvent, determines thetemperature dependence of Vappto a majorextent. The less pronounced but still significant temperature dependenceof the molecular volume of the solute, VM, is essentially the result of the expansivity of its internal voids,as the van der Waals contribution to VMis practically independent of temperature. Results for polypeptidesof different chemical nature feature a similar temperature behavior,suggesting that the boundary/hydration contribution seems to be auniversal part of the temperature dependence of Vapp. The results presented here shine new light on thediscussion surrounding the physical basis for understanding and decomposingthe volumetric properties of proteins and biomolecules in general. [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 commonly used electrolyte in electrospray mass spectrometry (ESI-MS) of biomolecules is ammonium acetate (NHOAc). Although some nucleic acid structures such as duplexes require only proper physiological ionic strength (whatever the monovalent ions) to be properly folded in ESI-MS conditions, the folding of some other nucleic acid structures such as DNA G-quadruplexes also depends on direct binding of specific cations. Here, we developed ESI-MS compatible conditions that allow one to observe DNA G-quaduplexes with K ions specifically bound between G-quartets. NHOAc was replaced with trimethylammonium acetate (TMAA), at concentrations up to 150 mM to provide physiological ionic strength, and the solution was doped with KCl at concentrations up to 1 mM. The trimethylammonium ion is too large to coordinate between G-quartets, where only K ions bind. Compared with the equivalent NHOAc/KCl mixtures, the TMAA/KCl mixtures provide cleaner spectra by suppressing the nonspecific adducts, and favor the formation of similar stacking arrangements as in 100 mM KCl (physiologically relevant cation) for the polymorphic human telomeric DNA G-quadruplexes. This new sample preparation method can be exploited to determine the number of potassium binding sites in new sequences, to screen ligand binding to the structures favored in potassium, and to transfer potassium-bound G-quadruplexes to the mass spectrometer for gas-phase structural probing, as illustrated herein with ion mobility spectrometry experiments. [Figure not available: see fulltext.] [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]

Wereport a versatile analysis platform, based on a set of nanogapelectrodes, for the manipulation and sensing of biomolecules, as demonstratedhere for low-copy number protein detection. An array of Ti nanogapelectrode with sub-10 nm gap size function as templates for alternatingcurrent dielectrophoresis-based molecular trapping, hot spots forsurface-enhanced Raman spectroscopy as well as electronic measurements,and fluorescence imaging. During molecular trapping, recorded Ramanspectra, conductance measurements across the nanogaps, and fluorescenceimaging show unambiguously the presence and characteristics of thetrapped proteins. Our platform opens up a simple way for multifunctionallow-concentration heterogeneous sample analysis without the need fortarget preconcentration. [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]

The article focuses on the use of high-speed atomic force microscopy (HS-AFM) to analyze single-molecular dynamics of proteins. Topics include use of HS-AFM technique to study DNA- binding proteins, inability of AFM technique to resolve transition states or intermediates of biomolecules in action, and the use of HS-AFM in the analysis of structural changes of motor proteins including dynein C and myosin V.

Solid state nuclear magnetic resonance (NMR) measurements at low temperatures have been common in physical sciences for many years and are becoming increasingly important in studies of biomolecular systems. This Account reviews a diverse set of projects from my laboratory, dating back to the early 1990s, that illustrate the motivations for low-temperature solid state NMR, the types of information that are available from the measurements, and likely directions for future research. These projects include NMR studies of both physical and biological systems, performed at low (cooled with nitrogen, down to 77 K) and ultralow (cooled with helium, below 77 K) temperatures, and performed with and without magic-angle spinning (MAS). [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]

There is an increasing interest in the use of bioorthogonal ligation to advance biomedical research through selective labeling of biomolecules in living systems. Accordingly, discovering new reactions to expand the toolbox of bioorthogonal chemistry is of particular interest to chemical biologists. Herein we report a new bioorthogonal ligation enabled by click hetero-Diels–Alder (HDA) cycloaddition of in situ-generated o-quinolinone quinone methides and vinyl thioethers. This reaction is highly selective and proceeds smoothly under aqueous conditions. The functionalized vinyl thioethers are small and chemically stable in vivo, making them suitable for use as bioorthogonal chemical reporters that can be effectively coupled to various biomolecules. We utilized this bioorthogonal ligation for site-specific labeling of proteins as well as imaging of bioactive small molecules inside live cells. [ABSTRACT FROM AUTHOR]

The article discusses the advancements in metabolomics and also reflects on its link with the separation of low molecular weight compounds such as biofluid or organism. It states that the metabolite profiling uses several techniques such as nuclear magnetic resonance (NMR) and mass spectrometry (MS). It discusses several separation techniques such as chromatographic methods and electrophoresis.

The translational hydration dynamics within 0.5–1.5 nm of the surface of a DPPC liposome, a model biomacromolecular surface, is analyzed by the recently developed Overhauser dynamic nuclear polarization (ODNP) technique. We find that dramatic changes to the bulk solvent cause only weak changes in the surface hydration dynamics. Specifically, both a >10-fold increase in bulk viscosity and the restriction of diffusion by confinement on a multiple nm length-scale change the local translational diffusion coefficient of the surface water surrounding the lipid bilayer by <2.5-fold. By contrast, previous ODNP studies have shown that changes to the biomacromolecular surface induced by folding, binding, or aggregation can cause local hydration dynamics to vary by factors of up to 30. We suggest that the surface topology and chemistry at the ≤1.5 nm scale, rather than the characteristics of the solvent, nearly exclusively determine the macromolecule's surface hydration dynamics. [ABSTRACT FROM AUTHOR]

Copper represents one of the most important biological metal ions due to its role as a catalytic cofactor in a multitude of proteins. However, an excess of copper is highly toxic. Thus, copper is heavily regulated, and copper homeostasis is controlled by many metalloregulatory proteins in various organisms. Here we report a genetically encoded copper(I) probe capable of monitoring copper fluctuations inside living cells. We insert the copper regulatory protein Ace1 into a yellow fluorescent protein, which selectively binds copper(I) and generates improved copper(I) probes. [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]