Quantification of proteins by functionalized gold nanoparticles using click chemistry

This letter presents a click-chemistry-based assay for proteins (CAP) that allows quantitative determination of the concentration of proteins, using azide- and alkyne-functionalized gold nanoparticles (AuNPs). Compared with conventional methods, CAP has a broader linear range for detection of proteins with good selectivity. CAP enables the analysis of total proteins in various sera and milk samples.     

Interaction of gold nanoparticles with proteins and cells

Abstract

Gold nanoparticles (Au NPs) possess many advantages such as facile synthesis, controllable size and shape, good biocompatibility, and unique optical properties. Au NPs have been widely used in biomedical fields, such as hyperthermia, biocatalysis, imaging, and drug delivery. The broad application range may result in hazards to the environment and human health. Therefore, it is important to predict safety and evaluate therapeutic efficiency of Au NPs. It is necessary to establish proper approaches for the study of toxicity and biomedical effects. In this review, we first focus on the recent progress in biological effects of Au NPs at the molecular and cellular levels, and then introduce key techniques to study the interaction between Au NPs and proteins. Knowledge of the biomedical effects of Au NPs is significant for the rational design of functional nanomaterials and will help predict their safety and potential applications.     

Photosynthesis of gold nanoparticles in presence of proteins

This paper describes the photosynthesis of gold nanoparticles (GNPs) in the presence of bovine serum albumin (BSA). The concentration of NaAuCl4 and the relative ratio of NaAuCl4 to BSA are important parameters for controlling the size of the GNPs. We prepared GNPs having average diameters ranging from 7 to 50 nm by illumination (Hg-Xe lamp) of phosphate-buffered solutions (pH 3.0-11.0) containing 1 mM NaAuCl4 and 10 microM BSA for 9 h. The size distribution of the GNPs synthesized at pH 7.0 is narrower relative to that of those prepared at other values of pH. Based on the observation that there are no GNPs formed at 25 degrees C in the absence of either BSA or illumination, we conclude that photolytic reduction is the main mechanism for the formation of the GNPs and that BSA acts as a capping agent to stabilize the as-synthesized GNPs. In addition to BSA, several other proteins, such as beta-casein, conalbumin, hemoglobin, beta-lactoglobulin, lysozyme, myoglobin, ovalbumin, pepsin, and trypsinogen play the same role.     

ZnO-poly(methyl methacrylate) nanobeads for enriching and desalting low-abundant proteins followed by directly MALDI-TOF MS analysis

A fast solid-phase microextraction method using core-shell ZnO-poly (methyl methacrylate) nanobeads (ZnO-PMMA) as adsorbent was established. This fast method with high enriching efficiency and salt tolerance capability depends on the structure of the core-shell nanobeads. First, the large surface area of the PMMA shell makes the dispersive nanobeads capture samples quickly, by virtue of multi-interactions between ZnO-PMMA and samples except for the interaction with salts. Second, the small nanosize of the ZnO-core (2.1 nm) and the flexible hydrophobic PMMA shell, which can prevent the cores from aggregation, make the nanobeads form a homogeneous layer on the matrix-assisted laser desorption/ionization (MALDI) plate and do not hinder the cocrystallization of the matrix and samples. Third, the ZnO core also prevents PMMA from fragmentation and ionization in mass spectrometer. In this article, approximately 80% bovine serum albumin digests were enriched by ZnO-PMMA from 100 amol/muL solution within 10-min incubation, and the solid phase can be directly analyzed by MALDI mass spectrometry. Mass intensity can be increased 5-10-fold (ZnO-PMMA enrichment vs lyophilization). High-quality mass spectra can be obtained, even with the presence of saturated NaCl (6.2 M), saturated NH 4HCO 3 (2.6 M), or 1 M urea. This method has been successfully applied to human colorectal cancer proteome research, and eight new proteins have been found.     

High sequence coverage of proteins isolated from liquid separations of breast cancer cells using capillary electrophoresis-time-of-flight MS and MALDI-TOF MS mapping

A method has been developed for high sequence coverage analysis of proteins isolated from breast cancer cell lines. Intact proteins are isolated using multidimensional liquid-phase separations that permit the collection of individual protein fractions. Protein digests are then analyzed by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting and by capillary electrophoresis-electrospray ionization (CE-ESI)-TOF MS peptide mapping. These methods can be readily interfaced to the relatively clean proteins resulting from liquid-phase fractionation of cell lysates with little sample preparation. Using combined sequence information provided by both mapping methods, 100% sequence coverage is often obtained for smaller proteins, while for larger proteins up to 75 kDa, over 90% coverage can be obtained. Furthermore, an accurate intact protein MW value (within 150 ppm) can be obtained from ESI-TOF MS. The intact MW together with high coverage sequence information provides accurate identification. More notably the high sequence coverage of CE-ESI-TOF MS together with the MS/MS information provided by the ion trap/reTOF MS elucidates posttranslational modifications, sequence changes, truncations, and isoforms that may otherwise go undetected when standard MALDI-MS peptide fingerprinting is used. This capability is critical in the analysis of human cancer cells where large numbers of expressed proteins are modified, and these modifications may play an important role in the cancer process.     

Signal amplification using "spot-on-a-chip" technology for the identification of proteins via MALDI-TOF MS

The presented "spot-on-a-chip" technology enables easy enrichment of samples in the low nanomolar (1-5 nM) range and provides a fast and reliable automated sample preparation method for performing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis with high sensitivity and throughput. Through microdispensing, which allows accurate deposition of 60-pL droplets, dilute samples were enriched by making multiple droplet depositions in nanovials. The sample was confined to a defined spot area (300 x 300 microm), and multiple depositions increase the surface density of analyte in the nanovial, thereby providing detection of low attomole levels. The impact of the nanovial geometry with respect to the MALDI-TOF MS resolution for peptides deposited in the microfabricated silicon vials was investigated and the optimal geometry and size were determined. The spot-on-a-chip technology, that is, the combination of microdispensing, micromachined silicon nanovials and on-spot enrichment provides a signal amplification of at least 10-50 times as compared to an ordinary sample preparation. The linearity of the enrichment effect is shown by the analysis of a peptide mixture at the 5 nM level. The signal amplification provided by the spot-on-a-chip enrichment is demonstrated by the analysis of relevant biological samples, interleukin-8 from a spiked cell supernatant, and by successful protein identification of an excised spot from a high-sensitivity silver-stained two-dimensional electrophoresis gel separation.     

Coumarin tags for improved analysis of peptides by MALDI-TOF MS and MS/MS. 1. Enhancement in MALDI MS signal intensities

The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF MS and MS/MS. The proposed tags, commercially available fluorescent derivatives of coumarin, can be advantageous for peptide analysis in both MS and MS/MS modes. This paper, part 1, will focus on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. Labeling peptides with tags containing the coumarin core was found to enhance the intensities of peptide peaks (in some cases over 40-fold) in MALDI-TOF MS using CHCA and 2,5-DHAP matrixes. The signal enhancement was found to be peptide- and matrix-dependent, being the most pronounced for hydrophilic peptides. No correlation was found between the UV absorptivity of the tags at the excitation wavelengths typical for UV-MALDI and the magnitude of the signal enhancement. Interestingly, peptides labeled with Alexa Fluor 350, a coumarin derivative containing a sulfo group (i.e., bearing strong negative charge), showed a 5-15-fold increase in intensity of MALDI MS signal in the positive ion mode, relative to the underivatized peptides, when 2,5-DHAP was used as the matrix. The Alexa Fluor 350 tag yielded a significantly higher signal relative to that for the CAF tag, likely due to the increased hydrophobicity of the coumarin structure. With 2,5-DHB, a decrease of MALDI MS signal was observed for all coumarin-labeled peptides, again relative to the unlabeled species. These findings support the hypothesis that derivatization with coumarin, a relatively hydrophobic structure, improves incorporation of hydrophilic peptides into hydrophobic MALDI matrixes, such as CHCA and 2,5-DHAP.     

Visual sensing of proteins using gold nanoparticles coated with polyphenolic glycoside

Gold nanoparticles combined with a polyphenolic glycoside (α-glucosylrutin) were prepared and applied for the selective detection of proteins. Glucosylrutin was coated onto gold nanoparticles with a particle size of 40 nm, without aggregates and color changes. The glucosylrutin-coated gold surface was preferentially adsorbed with concanavalin A, which has specificity against glucosides and mannosides. When the glucosylrutin-coated gold nanoparticles were mixed with concanavalin A, the color of the dispersion changed from red to reddish violet. The level of chromatic change was dependent on the concentration of concanavalin A. When other proteins (bovine serum albumin and peanut agglutinin) were added to the dispersion, no color change was observed. The molecular recognition site for detection of concanavalin A was the glycoside moiety, because the catechin-coated gold nanoparticles have no function in the detection of concanavalin A. Urchin-shaped glucosylrutin-coated gold nanoparticles were also useful for the visual sensing of concanavalin A.     

Enrichment and identification of cysteine-containing peptides from tryptic digests of performic oxidized proteins by strong cation exchange LC and MALDI-TOF/TOF MS

The extreme complexity of sample and uninformative fragmentation of peptides in MS/MS experiments are two of several real challenges faced by proteomics. In this work, a strategy aimed at tackling these two problems is presented. Briefly, proteins were first oxidized by performic acid to cleave the disulfide bonds and simultaneously convert cysteine residue into its sulfonic form. Then the resultant sulfonic peptides were enriched by SCX chromatography, exploiting the negative solution charge of sulfonic group. The sulfonic peptide could be easily detected by MALDI-MS in negative mode and showed both enhanced fragmentation efficiency and a simplified spectrum in MALDI-MS/MS experiment in positive mode. The strength of the strategy was demonstrated by applying it to bovine serum albumin. Potential use of the strategy in proteomics was also discussed.     

HPLC and MALDI-TOF MS analysis of highly branched polystyrene: resolution enhancement by branching

Temperature gradient interaction chromatography (TGIC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were applied for the characterization of highly branched polystyrenes (PS) prepared by linking living polystyryl anions using 4-chlorodimethylsilylstyrene. Reversed-phase (RP)-TGIC showed an unexpectedly high resolution according to the number of branches despite significant overlap of the molecular weight as confirmed by MALDI-TOF MS. The enhancement of the resolution is ascribed to the contribution of the nonpolar groups in the branched PS: the dimethylsilyl groups in the branching unit as well as the sec-butyl initiator groups. As the number of branches increases, the number of nonpolar groups increases, which in turn increases the RP-TGIC retention synergistically with increasing molecular weight. In contrast, a poorer resolution was found in normal-phase-TGIC, in which the nonpolar groups reduce the retention. The resolution in RP-TGIC appears superior to that of liquid chromatography at the chromatographic critical condition (LCCC) of PS. It is seemingly due to the synergistic contribution of the incremental PS molecular weight to the functionality in the branched PS in RP-TGIC while only the functionality contributes to the separation in LCCC. This type of resolution enhancement could be utilized efficiently for the analysis of highly branched polymers such as dendrimers or hyperbranched polymers.     

Fabrication of gold micro- and nanostructures by photolithographic exposure of thiol-stabilized gold nanoparticles

Exposure of thiol-stabilized gold nanoparticles supported on silicon wafers to UV light leads to oxidation of the thiol molecules and coagulation of the nanoparticles, forming densified structures that are resistant to removal by solvent exposure. Unoxidized particles may, in contrast, readily be removed leaving gold structures behind at the surface. This process provides a convenient and simple route for the fabrication of gold structures with dimensions ranging from micrometers to nanometers. The use of masks enables micrometer-scale structures to be fabricated rapidly. Exposure of nanoparticles to light from a near-field scanning optical microscope (NSOM) leads to the formation of gold nanowires. The dimensions of these nanowires depend on the method of preparation of the film: for spin-cast films, a width of 200 nm was achieved. However, this was reduced significantly, to 60 nm, for Langmuir-Schaeffer films.     

Grafting of gold nanoparticles and nanorods on plasma-treated polymers by thiols

Grafting of gold nanoparticles and nanorods on the surface of polymers, modified by plasma discharge, is studied with the aim to create structures with potential applications in electronics or tissue engineering. Surfaces of polyethyleneterephthalate and polytetrafluoroethylene were modified by plasma discharge and subsequently, grafted with 2-mercaptoethanol, 4,4′-biphenyldithiol, and cysteamine. The thiols are expected to be fixed via one of –OH, –SH or –NH₂ groups to reactive places on the polymer surface created by the plasma treatment. “Free” –SH groups are allowed to interact (graft) with gold nanoparticles and nanorods. Gold nano-objects were characterized before grafting by transmission electron microscopy and UV–Vis spectroscopy. X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), and electrokinetic analysis (zeta potential determination) were used for the characterization of polymer surface at different modification phases. It was proved by FTIR and XPS measurements that the thiols were chemically bonded on the surface of the plasma-treated polymers, and they mediate subsequent grafting of the gold nano-objects. On the surfaces, modified polymers were indicated some objects by AFM, size of which was dramatically larger in comparison with that of original nanoparticles and nanorods. This result and the other results of UV–Vis spectroscopy indicate an aggregation of deposited gold nano-objects.     

Coumarin tags for analysis of peptides by MALDI-TOF MS and MS/MS. 2. Alexa Fluor 350 tag for increased peptide and protein Identification by LC-MALDI-TOF/TOF MS

The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF/TOF MS. Part 1 of the study was focused on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. In part 2, various derivatization approaches for the improvement of peptide fragmentation efficiency in MALDI-TOF/TOF MS are explored. We demonstrate that permanent cation tags, while significantly improving signal intensity in the MS mode, lead to severe suppression of MS/MS fragmentation, making these tags unsuitable for high-throughput MALDI-TOF/TOF MS analysis. In the present work, it was found that labeling with Alexa Fluor 350, a coumarin tag containing a sulfo group, along with guanidation of epsilon-amino groups of Lys, could enhance unimolecular fragmentation of peptides with the formation of a high-intensity y-ion series, while the peptide intensities in the MS mode were not severely affected. LC-MALDI-TOF/TOF MS analysis of tryptic peptides from the SCX fractions of an E. coli lysate revealed improved peptide scores, a doubling of the total number of peptides, and a 30% increase in the number of proteins identified, as a result of labeling. Furthermore, by combining the data from native and labeled samples, confidence in correct identification was increased, as many proteins were identified by different peptides in the native and labeled data sets. Additionally, derivatization was found not to impair chromatographic behavior of peptides. All these factors suggest that labeling with Alexa Fluor 350 is a promising approach to the high-throughput LC-MALDI-TOF/TOF MS analysis of proteomic samples.     

Studies on the preparation and characterization of gold nanoparticles protected by dendrons

The preparation and characterization of a group of new composites, namely, gold nanoparticles-cored dendrimers were reported. These materials were obtained by the reduction of hydrogen tetrachloroaurate phase-transferred into organic phase in the presence of poly(benzyl ether) alcohol dendrons with generation 2. These materials, possessing nanometer-sized gold particles at the core and dendritic wedges radially connected to the core by Au-O bonds, were analyzed by UV-vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD), energy dispersive X-ray (EDX) and Fourier transform infrared spectroscopy (FTIR). A possible mechanism of the formation of Gold nanoparticles-cored dendrimers was proposed.     

Method for screening and MALDI-TOF MS sequencing of encoded combinatorial libraries

We describe a new method for encoded synthesis, efficient on-resin screening, and rapid unambiguous sequencing of combinatorial peptide libraries. An improved binary tag system for encoding peptide libraries during synthesis was designed to facilitate unequivocal assignment of isobaric residues by MALDI-TOF MS analysis. The improved method for encoded library synthesis was combined with a new versatile on-resin screening strategy that permitted multiple stages and types of screening to be employed successively on one library under mild conditions. The new method facilitated a combinatorial study of transglutaminase (TGase) enzyme substrate peptides, revealing new details of the effect of amino acid composition on TGase substrates. The approach was first demonstrated for an encoded library (130,321 compounds) of lysine pentapeptide substrates of TGase, synthesized using the "split-mix" method. The library was reacted on-resin with TGase enzyme and a soluble desthiobiotin labeled glutamine substrate. Initial screening was performed by adsorbing streptavidin-coated magnetic microparticles onto library beads, followed by magnetic separation. The differential binding affinities of desthiobiotin and biotin for streptavidin were exploited to release the magnetic microparticles and regenerate the desthiobiotin-labeled resin beads for further screening by flow-cytometry-based automated bead sorting, resulting in 345 beads that were sequenced by MALDI-TOF MS analysis. A second library consisted of encoded glutamine hexapeptide substrates, which was reacted on-resin with TGase enzyme and a soluble desthiobiotin-labeled cadaverine. Two-stage screening identified 267 glutamine peptides as TGase-reactive, of which 21 were further analyzed by solution-phase enzyme kinetics. Kinetic results indicated that the peptide PQQQYV from the library has a 68-fold greater substrate specificity than the best known glutamine substrate QQIV. The new encoding and screening strategies described here are expected to be broadly applicable to synthesis and screening of combinatorial peptide libraries in the future.     

Measuring the flexibility of immunoglobulin by gold nanoparticles

We measured the flexibility of Fab and Fc arms of immunoglobulin using gold nanoparticles (GNPs). Enzyme-linked immunosorbent assay was performed to measure the affinity of anti-5 nm GNP antiserum against various sizes of GNPs. The flexibility of Fc was also measured by electron microscopy. The restricted binding affinity indicated that only a very limited amount of freedom was allowed for the Fab-Fab hinge, while Fab-Fc showed a much larger degree of freedom.     

Quantification of single fluid inclusions by combining synchrotron radiation-induced micro-X-ray fluorescence and transmission

Fluid inclusions represent the only direct samples of ancient fluids in many crustal rocks; precise knowledge of their chemical composition provides crucial information to model paleofluid-rock interactions and hydrothermal transport processes. Owing to its nondestructive character, micrometer-scale spatial resolution, and high sensitivity, synchrotron radiation-induced micro-X-ray fluorescence has received great interest for the in situ multielement analysis of individual fluid inclusions. Major uncertainties associated with the quantitative analysis of single fluid inclusions arise from the inclusion depth and the volume of fluid sampled by the incident beam. While the depth can be extracted directly from the fluorescence spectrum, its volume remains a major source of uncertainty. The present study performed on natural and synthetic inclusions shows that the inclusion thickness can be accurately evaluated from transmission line scans. Experimental data matched numerical simulations based on an elliptical inclusion geometry. However, for one nonelliptical inclusion, the experimental data were confirmed using a computed absorption tomography reconstruction. Good agreement between the imaging and scanning techniques implies that the latter provides reliable fluid thickness values independent of the shape of the inclusion. Taking into consideration the incident angle, the incident beam energy, the inclusion fluid salinity, and the transmission measurement stability resulted in errors of 0.3-2 microm on calculated fluid inclusion thicknesses.     

Biosynthesis of gold nanoparticles

Synthesis of nanoparticles with interesting physico-chemical properties using efficient as well as eco-friendly technology is one of the main objectives of nanotechnology. Biological systems have been reported to synthesize inorganic materials under certain circumstances. Exploiting the biosynthetic potential of different organisms, nanoparticles of varying morphologies and sizes have been synthesized. Among the nanomaterials, gold has received considerable attention owing to its varied applications in the fields of nano-medicine, catalysis, electronics, and optics. This review gives an account on the biosynthesis of gold nanoparticles from microorganisms, plants, and other biological sources, with particular emphasis on the probable mechanisms leading to the formation of gold nanoparticles and the extent of control over nanoparticle properties that has been achieved so far in the biosynthetic protocols. It has been speculated that enzymes and/or proteins secreted by the organisms are involved in the bio-reduction and stabilization of the nanoparticles. The biosynthetic procedures could compete with existing solvent-based chemical synthetic procedures in order to achieve stable and monodisperse gold nanoparticles in large scale.     

A MALDI-TOF MS study of oligomeric poly(m-phenyleneisophthalamide)

MALDI-TOF MS was used to study the end-group distribution of a series of poly(m-phenyleneisophthalamide) oligomers which were synthesized using various mole percent ratios of diamine to diacid chloride (90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, and 10:90) to clarify results obtained in previous work published in this journal. Oligomers synthesized with excess diamine or excess diacid chloride were found to contain abundances of amine or carboxylate end groups, respectively, as expected. Oligomers synthesized with equal molar ratios of reactants produced cyclic species which were also found in a previous publication as an oligomer in commercially produced, high molecular mass Nomex.     

Conductive thin film multilayers of gold on glass formed by self-assembly of multiple size gold nanoparticles

Highly conductive thin films of gold have been fabricated on glass substrates by the deposition of gold nanoparticles of two different diameters. A deposition sequence, alternating between 2.6-nm and 12-nm diameter particles, was used whereby the 2.6-nm particles served to fill in the gaps created by the assembly of the larger 12-nm diameter particles. The resulting thin films, with thicknesses of less than 35 nm, displayed high conductivities, yet were fabricated in substantially fewer deposition cycles than required by previous methods. Analysis of surface morphology performed by atomic force microscopy and scanning electron microscopy showed that the high conductivity is the result of a less porous surface structure than can be achieved through the layering of a single size nanoparticle. Conductivity analysis was performed by 4-point probe with resistivities of 5.00 ± 0.4 × 10^− 6 Ω m for 5 layers and 4.49 ± 0.2 × 10^− 6 Ω m for the 6-layer films.