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Exposure to natural rubber latex (NRL) can result in sensitivity to NRL protein with resulting allergic reactions. Low-cost, portable, simple, sensitive analytical tools for NRL protein measurements are needed for rapid and accurate assessments of allergenic risks at the point-of-care (POC) instead of using traditional methods that require large and expensive instruments, long-time analysis, and complex sample preparation steps. Here, an electrochemical paper-based analytical device (ePAD) is presented by combining sample preparation and electrochemical detection within a single device to offer a one-step NRL analysis. The lack of antibodies and/or enzymes against NRL makes POC analysis difficult. In this work, detection is based on electrochemical measurement of the remaining Cu after in situ protein complexation instead of more complex biological assays for the first time. Graphene screen-printed electrodes modified with 1,10-phenanthroline and Nafion were used in the ePAD to improve Cu signal 18-fold relative to unmodified carbon screen-printed electrodes. The optimum parameters including 1,10-phenanthroline concentration, reaction time between Cu and protein, and the starting Cu concentration were 5% w/v, 1 min, and 600 μg mL−1, respectively. In addition to short analysis time (4 min), the system selectivity indicated no other interfering species affecting protein detection. The proposed ePAD achieved an LOD of 3.0 mg dL−1 and a linear range of 10.0–200.0 mg dL−1. Finally, the proposed sensors were applied for NRL protein detection and the results were not significantly different from the traditional Lowry method at 95% confidence. [ABSTRACT FROM AUTHOR]

Given their simplicity and functionality, paper-based microfluidic systems are considered to be ideal and promising bioassay platforms for use in less developed countries or in point-of-care services. Although a series of innovative techniques have recently been demonstrated for the fabrication of such platforms, development of simple, inexpensive and versatile new strategies are still needed in order to reach their full potential. In this communication, we describe a simple yet facile approach to fabricate paper-based sensor platforms with a desired design through a vapor-phase polymer deposition technique. We also show that the fabricated platforms could be readily employed for the detection of various biological target molecules including glucose, protein, ALP, ALT, and uric acid. The limit of detection for each target molecule was calculated to be 25 mg dL−1 for glucose, 1.04 g L−1 for protein, 7.81 unit per L for ALP, 1.6 nmol L−1 for ALT, and 0.13 mmol L−1 for uric acid. [ABSTRACT FROM AUTHOR]

Two approaches based on intact cell matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (IC-MALDI-ToF MS) have been evaluated in order to discriminate and identify nine former Burkholderia cepaciacomplex (Bcc) species, Burkholderia contaminansbelonging to the novel Taxon K, Burkholderia gladioli, and the most relevant non-fermentative (NF) Gram-negative rods recovered from cystic fibrosis (CF) sputum cultures. In total, 146 clinical isolates and 26 reference strains were analysed. IC mass spectra were obtained with high reproducibility applying a recently developed inactivation protocol which is based on the extraction of microbial proteins by trifluoroacetic acid (TFA). In a first approach, spectral analysis was carried out by means of a gel-view representation of mass spectra, which turned out to be useful to recognize specific identifying biomarker proteins (SIBPs). A series of prominent mass peaks, mainly assigned to constitutively expressed proteins, were selected as SIBPs for identifications at the genus and species level. Two distinctive mass peaks present in B. contaminansspectra (7501 and 7900 Da) were proposed as SIBPs for the identification of this novel species. A second approach of spectral analysis based on data reduction, feature selection and subsequent hierarchical cluster analysis was used to obtain an objective discrimination of all species analysed. Both complementary modalities of analyzing complex IC-MALDI-ToF MS data open the path towards a rapid, accurate and objective means of routine clinical microbiology diagnosis of pathogens from sputum samples of CF patients. [ABSTRACT FROM AUTHOR]

Protein phosphorylation plays an important role in many significant cellular processes, and has thus gained tremendous interest in the field of proteomics. The electro-active tyrosine residue, as an important receptor of phosphorylation in proteins, exhibits electro-inactivity after being phosphorylated on the hydroxy group of its aromatic ring. In this study, the electrochemical oxidation of tyrosine on indium tin oxide (ITO) electrodes was catalyzed with an electron mediator Os(bpy)32+(bpy = 2,2′-bipyridine) and was employed as a signal reporter to differentially detect non-phosphorylated and phosphorylated peptides. A short, tyrosine-containing peptide glu-glu-glu-glu-glu-tyr (EY-6) was immobilized on an ITO surface using the layer-by-layer self-assembly method, and was detected by cyclic voltammetry in an Os(bpy)32+solution. The limit of detection was about 0.23 μg mL−1EY-6 in solution. The phosphorylated peptide glu-glu-glu-glu-glu-tyr-OP (EY-6P) did not produce an appreciable oxidation current on the electrode. Surface plasmon resonance measurements revealed that the amount of EY-6 and EY-6P adsorbed on the sensor chip surface was 269 and 378 pg mm−2, respectively. The poly(glu, tyr) (4 : 1) peptide, a protein tyrosine kinase substrate, was also detected by the same approach, with a detection limit of 0.65 μg mL−1. This new approach offers the possibility of label-free and on-chip detection of protein tyrosine kinase activity. [ABSTRACT FROM AUTHOR]

A surface plasmon resonance imaging method has been developed for high throughput recognition and determination of low level glycoproteins with limited sample volume at least down to 50 nL. Chicken ovalbumin and immunoglobulin G were chosen as model compounds while bovine serum albumin and lysozyme were used as control. Each protein, at a concentration of 0.0080–1.0 mg mL−1, was printed on one gold sensing film, and the films were simultaneously reacted with a probe solution and viewed using a laboratory-built surface plasmon resonance imaging system. The imaging signals were dependent on the concentration and the type of analyte, with a limit of detection down to at least 0.5 ng. The glycoproteins dotted at either 1.0 mg mL−1or 0.010 mg mL−1were easily differentiated from the non-glycoproteins by reaction with 200 nM concanavalin A (con A), giving a limit of recognition down also to 0.5 ng glycoprotein. This imaging method was hence considered a new tool for analyzing glycoproteins. [ABSTRACT FROM AUTHOR]

We present a comprehensive method for proteome analysis that integrates both intact protein separation and proteolytic fragment characterization mass spectrometric approaches. Strong cation exchange chromatography (SCX) was used as the first separation dimension and capillary reversed-phase liquid chromatography (cRPLC) was integrated as the second separation dimension. Fractions from SCX were collected offline and loaded onto cRPLC. Effluents from cRPLC were directly deposited onto the MALDI target plates and further digested by using a rapid on-probe tryptic digestion technique. This approach minimizes the amount of time and extensive labor required for traditional in-solution digestion followed by exhaustive sample cleanup and transfer. MALDI-TOF/TOF was used for subsequent analyses. The sensitivity of on-target digestion is showed by analyzing 0.07 ng of myoglobin, 0.07 ng of cytochrome c and 0.7 ng BSA. The high efficiency of the overall system was demonstrated by the analysis of intact proteins extracted from normal human liver tissue. In total, 458 proteins were identified, which proved the system's promising potential for analysis and application in proteomics. [ABSTRACT FROM AUTHOR]

An amperometric biosensor based on genetically-modified enzymes was used for the in situdetection of trace vapours from a number of explosive compounds. The vapour samples that were generated from a purpose-built vapour generator were collected and pre-concentrated using a trap able to concentrate samples at a rate of 60-fold per minute of sampling. The amperometric biosensor achieved a remarkably low vapour detection limit of 6 parts per trillion from a room temperature sample. The specific activity of the reported enzyme toward a number of explosive compounds was also confirmed using absorbance measurements. [ABSTRACT FROM AUTHOR]

We report on the first development of a simple distance-based β-amyloid (Aβ) protein quantification using a paper-based device (dPAD) to screen for Alzheimer's disease (AD) and to subsequently follow up on its influence, i.e., clinical dementia. This sensor method is based on the transformation of a free acid form and its binding with a basic form of bromocresol purple (BCP) through its electrostatic interaction with an Aβ protein. This sensor can measure the length of color change from yellow to blue-green on a paper strip, with this change proportional to the amount of Aβ protein level. We found that the linearity for Aβ protein monitoring was in the range from 0.50 to 10.0 ng mL−1, and the subsequent naked-eye detection limit for Aβ was 0.20 ng mL−1. This system also provided high reproducibility and with no apparent interference effect for Aβ protein analysis in human urine samples. Furthermore, our developed dPAD constituted an accurate and effective device to precisely determine an Aβ protein concentration in real samples, with percentage recoveries in the range of 97–103%, and with the highest relative standard deviation of 5.41%. Subsequently, the validation of our assay was assessed by comparison with a commercial ELISA approach, with favorable results. Finally, the proposed dPAD was successfully applied to the determination of an Aβ protein in human urine samples and showed more benefits for the unskilled user, such as cost-efficiency, simplicity, low reagent usage, and low time consumption. It is also suitable for point-of-care monitoring. [ABSTRACT FROM AUTHOR]

Biopharmaceuticals are used to treat a range of diseases from arthritis to cancer, however, since the advent of these highly specific, effective drugs, there have been challenges involved in their production. The most common biopharmaceuticals, monoclonal antibodies (mAbs), are vulnerable to aggregation and precipitation during processing. Freeze thaw cycles (FTCs), which can be required for storage and transportation, can lead to a substantial loss of product, and contributes to the high cost of antibody production. It is therefore necessary to monitor aggregation levels at susceptible points in the production pathway, such as during purification and transportation, thus contributing to a fuller understanding of mAb aggregation and providing a basis for rational optimisation of the production process. This paper uses attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and spectroscopic imaging to investigate the effect of these potentially detrimental FTCs on protein secondary structure in both static wells and under flowing conditions, using lysozyme as a model protein. The results revealed that the amount of protein close to the surface of the ATR crystal, and hence level of aggregates, increased with increasing FTCs. This was observed both within wells and under flow conditions, using conventional ATR-FTIR spectroscopy and ATR-FTIR spectroscopic imaging. Interestingly, we also observed changes in the Amide I band shape indicating an increase in β-sheet contribution, and therefore an increase in aggregates, with increasing number of FTCs. These results show for the first time how ATR-FTIR spectroscopy can be successfully applied to study the effect of FTC cycles on protein samples. This could have numerous broader applications, such as in biopharmaceutical production and rapid diagnostic testing. [ABSTRACT FROM AUTHOR]

Adsorption is the most common approach to immobilize biorecognition elements on the surface of paper-based devices. Adsorption is also the route selected to coat the substrate with albumin, therefore minimizing the interaction of other proteins. While similar in nature, the structure of the selected proteins as well as the conditions selected from the immobilization have a significant effect on the amount and distribution of the resulting composites. To illustrate these differences and provide general guidelines to efficiently prepare these devices, this article explores the interaction (adsorption and desorption) of BSA with 3MM chromatography paper. The experimental conditions investigated were the protein concentration, the interaction time, the number of times the protein was spotted, the pH of buffer solution, and the ionic strength of the buffer solution. The proposed approach mimics the steps involved in the fabrication (adsorption) and use (rinsing induced by the sample) of paper-based microfluidic devices. To identify the protein location following the rinsing step, the protein was fixed by dehydration in a convection oven and then stained using Coomassie Blue. The color intensity, which was found to be proportional to the amount of protein immobilized, was determined using a desktop scanner. To highlight the importance of understanding the adsorption process to the rational development of μPADs, results were complemented by experiments performed with lysozyme and immunoglobulin G. [ABSTRACT FROM AUTHOR]

High electrostability and long life-time of planar chip technology are crucial for electrophysiological measurements such as ionic current recording through ion channel proteins embedded in biological cell membrane. In this paper, we propose a novel planar microchip integrated with microelectrochemical cell array toward to a feasible solution for ion channel screening with high resolution and long life-time. In order to reduce the interference from the leakage currents, a synthetic lipid bilayer is applied to form a high sealing resistance. The whole-cell electrical access can be constructed via electroporating the lipid bilayer in close proximity to the cell membrane. Parameters of electroporation including amplitude and time scale are firstly optimized by using parallel electroporation to the lipid bilayers. In this approach, individual cells can be trapped to the target positions by applying dielectrophoresis (DEP) manipulation. Poly(ethylene glycol) (PEG) is employed with low concentration to facilitate the closed contact between the cells and the lipid bilayer to increase the efficiency of the whole-cell mode construction. Through this chip based method, stable current recordings through inward rectifier potassium (Kir) ion channels embedded in rat basophilic leukemia (RBL-1) cell membrane are achieved with high electrical sealing resistance (over 1 GΩ). In addition, without need for complex fluidic connections, this method allows for an easy operation and further miniaturization of the measuring system. [ABSTRACT FROM AUTHOR]

Highly sensitive detection of molecular biomarkers plays a significant role in diagnosing various types of diseases at the early stage. We demonstrated in this paper an ultrasensitive aptamer-based fluorescence method for detecting mucin 1 (MUC1) in human serum via a cascaded multiple recycling signal amplification strategy. The MUC1 target molecules present in the samples cause structure switching of the hairpin aptamer probes, which initiates three cascaded recycling cycles for the cleavage of the fluorescently quenched signal probes to recover significant fluorescence for highly sensitive detection of MUC1. The developed method has a linear range from 100 fM to 1 nM for MUC1 detection. Besides, owing to the substantial signal amplification by the integrated and cascaded recycling cycles, a low detection limit of 35 fM is achieved with high selectivity. Moreover, the monitoring of trace MUC1 in human serum can also be realized with such a method, indicating its great potential for highly sensitive detection of different disease biomarkers. [ABSTRACT FROM AUTHOR]

Near-infrared (near-IR) spectroscopy has been investigated for use in direct measurements of human tissue for the purpose of quantifying clinically relevant analytes such as glucose. Spectra collected by transmitting near-IR light through human tissue reveal the presence of both aqueous components and solid structures within the optical path of the measurement. Developing technology for use in making these measurements requires either the availability of human subjects or an in vitro experimental platform that can effectively simulate the spectroscopic properties of tissue. This paper describes the preparation and testing of films of collagen and keratin, the two proteins that comprise the bulk of the solid material in the human epidermis and dermis. By placing these films in the optical path of a near-IR spectrometer together with an aqueous sample cell, a phantom can be constructed that allows experiments to be performed that simulate noninvasive measurements of tissue. In this work, both constant and variable thickness films are prepared and evaluated by use of a regression fit to spectra of human tissue. The stability and spectral reproducibility of the prepared films are also assessed. The regression fits to the human subject spectra yield values of R2 ranging from 0.97 to 0.99 and the films are found to yield spectra that vary by less than a 2% relative standard deviation under three different reproducibility tests. [ABSTRACT FROM AUTHOR]

Biosensor chips for immune-based assay systems have been investigated for their application in early diagnostics. The development of such systems strongly depends on the effective protein immobilization on polymer substrates. In order to achieve this complex heterogeneous interaction the polymer surface must be functionalized with chemical groups that are reactive towards proteins in a way that surface functional groups (such as carboxyl, –COOH; amine, –NH2; and hydroxyl, –OH) chemically or physically anchor the proteins to the polymer platform. Since the proteins are very sensitive towards their environment and can easily lose their activity when brought in close proximity to the solid surface, effective surface functionalization and high level of control over surface chemistry present the most important steps in the fabrication of biosensors. This paper reviews recent developments in surface functionalization and preparation of polymethacrylates for protein immobilization. Due to their versatility and cost effectiveness, this particular group of plastic polymers is widely used both in research and in industry. [ABSTRACT FROM AUTHOR]

The paper presents a new multi-parametric protein microarray embracing the multi-analyte capabilities of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The combination of high throughput reverse phase protein microarrays with element tagged antibodies and LA-ICP-MS makes it possible to detect and quantify many proteins or biomarkers in multiple samples simultaneously. A proof of concept experiment is performed for the analysis of cytochromes particularly of cytochrome P450 enzymes, which play an important role in the metabolism of xenobiotics such as toxicants and drugs. With the aid of the LA-ICP-MS based multi-parametric reverse phase protein microarray it was possible to analyse 8 cytochromes in 14 different proteomes in one run. The methodology shows excellent detection limits in the lower amol range and a very good linearity of R2≥ 0.9996 which is a prerequisite for the development of further quantification strategies. [ABSTRACT FROM AUTHOR]

All stakeholders in cultural heritage share an interest in fabrication methods and material technology. Until now methods for analysis of organic materials, particularly proteins, have not been widely available to researchers at cultural institutions. This paper will describe an analytical method for the identification of collagen-based materials from soft tissue sources and show examples of its application to diverse museum objects. The method, peptide mass fingerprinting (PMF), uses enzymatic digestion of extracted proteins to produce a mixture of peptides. The mass spectrum of the mixture contains characteristic marker ions―a peptide mass fingerprint―which are compared to species-specific markers from references as the basis of identification. Preliminary results indicate that analysis of materials from aged samples, several different tissue types, and tanned or untanned materials yields comparable PMF results. Significantly, PMF is simple, rapid, sensitive and specific, has been implemented in a museum laboratory, and is being practiced successfully by non-specialists. [ABSTRACT FROM AUTHOR]

Several methods can be used to improve the enrichment of phosphorylated proteins. In this paper, phosphopeptides were enriched using magnetic iron(ii,iii) oxide (magnetite, Fe3O4) nanoparticles (NPs) on a radiate microstructure silicon chip and then analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) without further purification processes. We have developed a radiate microstructure chip on which samples can be concentrated for analysis by MALDI-TOFMS. The phosphoprotein digests and magnetic iron oxide NPs aqueous solution were deposited onto the central zone of the radiate microstructure silicon chip and enabled the on-chip enrichment of phosphopeptides. Microscopic analysis confirmed that the applied samples were confined to the central zone. Sample spots focused on the chip were much smaller than those on an unmodified plate with the same total volume. Different additives were used and optimized processes were performed to minimize non-phosphopeptides interference. These data collectively demonstrate that our on-chip phosphopeptide enrichment protocol is a rapid and easy-to-use method for phosphoproteome analysis. [ABSTRACT FROM AUTHOR]

This paper describes an ultrasensitive surface plasmon resonance (SPR) detection method using biofunctionalized gold nanorods for the direct detection of protein biomarkers. Immunoglobulin E (IgE), which has separate antibody and DNA aptamer binding sites, was chosen as a model protein for which a sandwich assay platform was designed. Detection was achieved viathe specific adsorption of unlabelled IgE proteins onto the surface immobilized aptamer followed by the specific adsorption of anti-IgE coated gold nanorods (Au-NRs). Using the biofunctionalized nanorods in conjunction with SPR, we were able to directly measure IgE proteins at attomolar concentrations. This is a remarkable 108enhancement compared to conventional SPR measurements of the same surface sandwich assay format ‘anti-IgE/IgE/surface bound IgE-aptamer’ in the absence of gold nanorod signal amplification. [ABSTRACT FROM AUTHOR]

The cationic surfactants didodecyldimethylammonium bromide (DDAB) and dioctadecyldimethyl-ammonium bromide (DODAB) have previously been shown to form semi-permanent coatings that effectively prevent adsorption of cationic proteins in fused silica capillaries with inner diameters of 25–75 µm. This paper investigates the impact that narrower capillary diameters (≤25 µm) have on the stability of surfactant bilayer coatings and the efficiency of separations of model cationic proteins and neurotransmitters. Using a DODAB-coated 5 µm i.d. capillary 210 consecutive protein separations (1050 min) were performed without recoating the capillary between runs. Separation efficiencies of 1 400 000–2 000 000 plates per m (340 000–430 000 plates) were obtained. Migration time reproducibilites of 6.8% RSD were observed for 300 injections performed over a 30 day period without any regeneration of the coating. Neurotransmitters were separated with efficiencies ranging from 470 000-–610 000 plates per m (110 000–140 000 plates) in a 5 µm capillary. [ABSTRACT FROM AUTHOR]

This paper reports a simple, rapid molecular binding scheme for the detection and quantification of the transcription factor nuclear factor kappa B and other DNA binding proteins without any separation or immobilization step. [ABSTRACT FROM AUTHOR]