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A concept integrating sampling and protein digestion is introduced here combining fast and simple fabrication by wax printing on filter paper with trypsin immobilized polymer beads. The paper reactors showed promising results with a high degree of protein digestion within fifty minutes in model protein mixtures as well as in human blood. The model protein mixture was used for the evaluation of performance both with and without a reduction and alkylation step. The paper reactors without reduction and alkylation showed between 46% and 75% protein sequence coverage and between five and 20 high confidence peptides (one and five zero missed cleavage peptides, respectively). Compared to a conventional in-solution approach, the paper reactor showed 10% less protein sequence coverage, 29% fewer high confidence peptides and 19% fewer high confidence peptides with zero missed cleavages. Placement of the protein reduction and alkylation step (before or after protein digestion) was shown to be of low importance. The storage stability of the paper reactors with (six weeks) and without (twelve weeks) tryptic peptides was satisfactory. The ability of the paper reactors to digest complex biological samples was investigated by comparison with human whole blood samples prepared using a conventional dried blood spot (DBS) procedure with overnight digestion in non-targeted analysis. The reactors showed a comparable performance with 75 ± 25 for the protein groups compared to 76 ± 5 for the DBS samples. Additionally, 267 ± 72 and 335 ± 11 unique peptides (high confidence) were identified for on-paper digestion and DBS, respectively.

A green and facile method is presented for in situ synthesis of a novel photoluminescence-quenching nanopaper with a highly-efficient quenching ability, rapid reaction time and long-term storage. The as-prepared nanopaper is further used to construct an aptasensor platform with high performance, rapidness and robustness.

A method for patterning narrow lines of biomolecules onto nitrocellulose membranes using laboratory syringe pumps is described. One syringe pump is used to drive the biomolecule solution through a needle, while another modified syringe pump acts as a one-dimensional translation stage, moving the needle across the membrane much like a pen. This method consumes very small volumes of reagents, and is a viable option for laboratory-scale fabrication and prototyping of point-of-care rapid diagnostic test strips.

Paper has been proposed as an inexpensive and versatile carrier for microfluidics devices with abilities well beyond simple capillary action for pregnancy tests and the like. Unlike standard microfluidics devices, extracting a fluid from the paper is a challenge and a drawback to its broader use. Here, we extract fluid from narrow paper strips using surface acoustic wave (SAW) irradiation that subsequently atomizes the extracted fluid into a monodisperse aerosol for use in mass spectroscopy, medical diagnostics, and drug delivery applications. Two protein molecules, ovalbumin and bovine serum albumin (BSA), have been preserved in paper and then extracted using atomized mist through SAW excitation; protein electrophoresis shows there is less than 1% degradation of either protein molecule in this process. Finally, a solution of live yeast cells was infused into paper, which was subsequently dried for preservation then remoistened to extract the cells via SAW atomization, yielding live cells at the completion of the process. The successful preservation and extraction of fluids, proteins and yeast cells significantly expands the usefulness of paper in microfluidics.

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]

Paper has been proposed as an inexpensive and versatile carrier for microfluidics devices with abilities well beyond simple capillary action for pregnancy tests and the like. Unlike standard microfluidics devices, extracting a fluid from the paper is a challenge and a drawback to its broader use. Here, we extract fluid from narrow paper strips using surface acoustic wave (SAW) irradiation that subsequently atomizes the extracted fluid into a monodisperse aerosol for use in mass spectroscopy, medical diagnostics, and drug delivery applications. Two protein molecules, ovalbumin and bovine serum albumin (BSA), have been preserved in paper and then extracted using atomized mist through SAW excitation; protein electrophoresis shows there is less than 1% degradation of either protein molecule in this process. Finally, a solution of live yeast cells was infused into paper, which was subsequently dried for preservation then remoistened to extract the cells viaSAW atomization, yielding live cells at the completion of the process. The successful preservation and extraction of fluids, proteins and yeast cells significantly expands the usefulness of paper in microfluidics. [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]

The goal of the presented study was to provide preliminary information on cobalt speciation in chicken eggs. Simulation of a digestive system was set up to acquire information about bioavailability of cobalt. Additionally, it was found that cobalt recovery by means of enzymatic digestion was decreasing with longer boiling time from 100 to 62%. Size exclusion, silica based column was coupled to ICP MS and used for separation of B12analogues from their degradation products and adducts with proteins extracted from egg tissue. Silica based column offered detection limits down to 0.1 ng mL−1due to higher recovery of cobalt from stationary phase in comparison to most commonly used Superdex (DL 10 ng mL−1). Phosphitin and ovalbumin showed good affinity to B12and they were assigned to cobalophilins responsible for protection of cobalamins from enzymatic, acidic or thermal degradation. [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]

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]

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]

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]

Aggregated t-CuP binds to poly-l-glutamate through supramolecular interactions, revealing itself to be an extremely sensitive probe for the major conformations of the polymeric scaffold. [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]

Protein-ligand binding affinity prediction plays an important role in the field of drug discovery. Existing deep learning-based approaches have significantly improved the efficiency of protein-ligand binding affinity prediction through their excellent inductive bias capability. However, these methods only focus on fragmented three-dimensional data, which truncates the integrity of pocket data, leading to the neglect of potential long-range interactions. In this paper, we propose a dual-stream framework, with amino acid sequence assisting the atomic data fusion for graph neural network (termed SadNet), to fuse both 3D atomic data and sequence data for more accurate prediction results. In detail, SadNet consists of a pocket module and a sequence module. The sequence module expands the "receptive field" of the pocket module through a mid-term virtual node fusion. To better integrate sequence-level information from the sequence module and 3D structural information from the pocket module, we incorporate structural information for each amino acid within the sequence module. Besides, to better understand the intrinsic relationship between sequences and 3D atomic information, our SadNet utilizes information stacking from both the early stage and later stage. Experimental results on publicly available benchmark datasets demonstrate the superiority of the proposed dual-stream approach over the state-of-the-art alternatives. The code of this work is available online at https://github.com/wardhong/SadNet.

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]

Cellulose is a biodegradable and renewable material that is one of the most abundant biopolymers with many different applications from low value newsprint products to high value biomedical sensor devices. In the last years, the demand of functionalized cellulose for the development of new packaging materials was constantly rising. In this study, a new two-step method for surface functionalization of cellulose sheets and fibers involving oxidation by 2,2,6,6-tetramethylpiperidinyloxyl (TEMPO) followed by coupling of different proteins was investigated. The cellulose oxidation was monitored via FT-IR at 1610 cm−1, photometrically via toluidine blue staining and via titration for the determination of the –COOH group concentration. TEMPO oxidation increased the amount of –COOH groups from around 0.2 to more than 1.4 mmol g−1 when NaClO2/NaClO regeneration was used. The TEMPO/laccase system instead led to 0.7 mmol g−1 of –COOH groups (determined via HCl titration). The oxidation was monitored over time and showed that 50% of the reaction were completed within the first 60 min of reaction time. Coupling of protein-based hydrophobins or bovine serum albumin using the EDAC/NHS system led to the desired increase in hydrophobicity and detection of protein on cellulose. Coupling was investigated using contact angle measurements and SEM microscopy paired with elemental analysis for oxygen and nitrogen. Hence, the coupled hydrophobins led to a significant increase of the initial contact angle by 33% with water drop stability of over 200 s. In contrary, pure cellulose obtained no visible water drop and for surfaces with uncoupled hydrophobins no stable contact angle, with a soaking time dropping to 55 s was achieved. As a result, the thorough study revealed that the new combinatorial approach of surface functionalization and protein coupling led to the successful increase of hydrophobicity. [ABSTRACT FROM AUTHOR]

Crystallization is an important unit operation in industry, and proteins are widely studied owing to their biological activity and multiple functions. Protein crystals can not only facilitate the elucidation of protein structural information but also have important prospects in biopharmaceuticals. However, owing to their high molecular weight and complex structure, most proteins are difficult to crystallize or the crystallization processes are difficult to control effectively. This paper aims to provide an overview of recent reinforcement techniques for protein crystallization. Several important additive applications and principles in protein crystallization are comprehensively reviewed, including ionic liquids (ILs), deep eutectic solvents (DESs), and molecularly imprinted polymers (MIPs). The mechanism of external fields, including magnetic, electric, ultrasonic, shear, and light fields, enhancing protein crystallization are described and summarized. Finally, the challenges and future research prospects of protein crystallization are proposed. This work is expected to guide the rational design of the protein crystallization process. [ABSTRACT FROM AUTHOR]

In this work, bovine serum albumin (BSA) and Aspergillus sp. laccase (LC) were encapsulated in situ within two lanthanide-based MOFs (TbBTC and GdBTC) through a green one-pot synthesis (almost neutral aqueous solution, T = 25 °C, and atmospheric pressure) in about 1 h. Pristine MOFs and protein-encapsulated MOFs were characterized through wide angle X-ray scattering, scanning electron microscopy, thermogravimetric analysis, Fourier transform infrared and Raman spectroscopies. The location of immobilized BSA molecules, used as a model protein, was investigated through small angle X-ray scattering. BSA occurs both on the inner and on the outer surface of the MOFs. LC@TbBTC, and LC@GdBTC samples were also characterized in terms of specific activity, kinetic parameters, and storage stability both in water and acetate buffer. The specific activity of LC@TbBTC was almost twice that of LC@GdBTC (10.8 μmol min-1 mg-1vs. 6.6 μmol min-1 mg-1). Both biocatalysts showed similar storage stabilities retaining -60% of their initial activity after 7 days and -20% after 21 days. LC@TbBTC dispersed in acetate buffer exhibited a higher storage stability than LC@GdBTC. Additionally, terbium-based MOFs showed interesting luminescent properties. Together, these findings suggest that TbBTC and GdBTC are promising supports for the in situ immobilization of proteins and enzymes. [ABSTRACT FROM AUTHOR]

Antibodies are crucial in various biological applications due to their specific binding to target molecules, altering protein function and structure. The advent of single-chain antibodies such as nanobodies has paved the way for broader applicability in both research and therapies due to their small size and efficient tissue penetration. Recently, several approaches have been reported to optically control the antigen-binding affinity of nanobodies. Here, we show an alternative strategy for creating photo-activatable nanobodies. By fusing the photocleavable protein PhoCl with the N-terminus of the nanobody (named optoNb60), we successfully demonstrated light-dependent restoration of the antigen-binding ability and the following modulation of the activity of a target protein, the beta-2 adrenergic receptor. Moreover, the activation of optoNb60 was monitored by the fluorescence changes upon photoconversion. The compatibility of the uncaging design with the previously reported optogenetic molecules using nanobodies will contribute to the further optimization of the response capabilities of existing optogenetic tools, thereby expanding their applicability. [ABSTRACT FROM AUTHOR]

Gold nanoclusters (AuNCs) consist of a few to several hundred gold atoms with a core size of less than 2 nm. Gold nanoclusters are among the most stable metal nanoclusters and have attracted worldwide attention in the biomedical field due to their extraordinary physicochemical properties and excellent biocompatibility. This paper reviews the synthesis and recent research progress of AuNCs using biomolecules as templates. First, we present the synthesis of AuNCs using proteins, peptides, DNA and polysaccharides as templates. Then, recent research advances in AuNCs for bioimaging, disease therapy and drug delivery are discussed. Finally, some research ideas are proposed for future work on gold nanoclusters in biomedical fields. As the research progresses, we expect the bio-template gold nanoclusters to become an essential platform for biomedical applications.

This work has been motivated by the recent paper by the author [M. Toutounji, Phys. Chem. Chem. Phys. , 2021, 23 , 21981] whereby a mixed quantum-classical Liouville equation was used to probe the spectroscopy and dynamics of a spin-boson system. A mixed quantum-classical Liouville equation treats the system of interest quantum mechanically, the bath classically, and the coupling term mixed quantum-classical mechanically. This paper offers a two-fold advantage: correcting the treatment of the electronic transition decay (width in frequency domain) and assessing the local heterogeneous vibrational structure. The homogeneous linear absorption spectrum of a chromophore embedded in a mixed quantum-classical environment at low temperature is composed of a sharp peak called a zero-phonon line (ZPL) and a broad phonon sideband (PSB), whereby the ZPL and the PSB are assimilated by a Lorentzian function and Voigt profiles, respectively. The PSB, in this case, is characterized by a local heterogeneous structure due to a dispersive medium of vibrations, modeled by vibrational Gaussian distributions to represent the arising inhomogeneous broadening and Lorentzians to model the homogeneous vibrations. This description seems to model proteins and amorphous solids exhibiting a local heterogeneous structure as both electronic and vibrational inhomogeneous broadening seems to be large in these media. This work provides a derivation of linear absorption lineshape and vibronic transition dipole moment time correlation functions, both of which account for pure electronic dephasing (ZPL width) the Voigt profile description of the phonon profiles (PSB) in dispersive media.

The development of inhibitors of anti-apoptotic proteins, such as Mcl-1, is currently a very active area in the field of cancer research. One of the very first reported inhibitors of Mcl-1 was the MIM1 molecule, but we have recently demonstrated that the structure of this compound had to be revised from 2 to the derivative 1 (FJ-809). In this paper we first develop a strategy to unambiguously prepare molecules such as 1 with a thiazol-3(2H)-yl)imino core, instead of the [2(3H)-thiazolylidene]hydrazine previously found in MIM1 (2). Next a series of biological studies have been performed on 1, using IGROV1-R10 ovarian cancer cells as models, and they have been complemented by fluorescence polarisation assays. These studies demonstrated that the new compound FJ-809 (1) was devoid of any significant activity on Mcl-1, in contrast to 2. Then molecular modelling and molecular dynamics studies have been performed in order to elucidate the differences between FJ-809 and MIM1 in their interaction with the Mcl-1 protein. [ABSTRACT FROM AUTHOR]

Anhydrous guanine (AG) crystals are widely applied in organisms due to their excellent optical properties. However, the influence of biomolecules on the biomineralization process of guanine crystals in organisms remains unexplored. In this study, mixed proteins from the silver film of carp fish scales were extracted and were used as a model system to elucidate the effect of biomolecules on guanine crystallization for the first time. The mixed proteins were enriched with amino acids such as Asp, Glu, Leu and Lys. Elongated hexagonal microplates of α-form AG (α-AG) mixed with a small amount of β-form AG (β-AG) mainly exposing the (100) plane were obtained in the presence of the mixed proteins under mild conditions while irregular particles were formed in the absence of proteins, indicating that the extracted mixed proteins have a strong influence on the formation of AG microplates exposing the (100) plane. It is found that the proteins not only adsorb on the surface but also occluded into the AG crystals. We also find that guanine analogues have profound synergetic effects on the morphology and polymorphs of guanine in the in vitro synthetic system. The obtained α-AG exhibit superior optical properties such as high reflectivity, strong depolarization and pearlescent luster. This in vitro synthesis work shows clearly the strong influence of biomolecules for the biomineralization process of guanine crystals in organisms. [ABSTRACT FROM AUTHOR]

Experimentally measured transition path time distributions are usually analyzed theoretically in terms of a diffusion equation over a free energy barrier. It is though well understood that the free energy profile separating the folded and unfolded states of a protein is characterized as a transition through many stable micro-states which exist between the folded and unfolded states. Why is it then justified to model the transition path dynamics in terms of a diffusion equation, namely the Smoluchowski equation (SE)? In principle, van Kampen has shown that a nearest neighbor Markov chain of thermal jumps between neighboring microstates will lead in a continuum limit to the SE, such that the friction coefficient is proportional to the mean residence time in each micro-state. However, the practical question of how many microstates are needed to justify modeling the transition path dynamics in terms of an SE has not been addressed. This is a central topic of this paper where we compare numerical results for transition paths based on the diffusion equation on the one hand and the nearest neighbor Markov jump model on the other. Comparison of the transition path time distributions shows that one needs at least a few dozen microstates to obtain reasonable agreement between the two approaches. Using the Markov nearest neighbor model one also obtains good agreement with the experimentally measured transition path time distributions for a DNA hairpin and PrP protein. As found previously when using the diffusion equation, the Markov chain model used here also reproduces the experimentally measured long time tail and confirms that the transition path barrier height is ∼3 k B T . This study indicates that in the future, when attempting to model experimentally measured transition path time distributions, one should perhaps prefer a nearest neighbor Markov model which is well defined also for rough energy landscapes. Such studies can also shed light on the minimal number of microstates needed to unravel the experimental data.

This paper reviews methods for detecting proteins based on molecular digitization, i.e., the isolation and detection of single protein molecules or singulated ensembles of protein molecules. The single molecule resolution of these methods has resulted in significant improvements in the sensitivity of immunoassays beyond what was possible using traditional "analog" methods: the sensitivity of some digital immunoassays approach those of methods for measuring nucleic acids, such as the polymerase chain reaction (PCR). The greater sensitivity of digital protein detection has resulted in immuno-diagnostics with high potential societal impact, e.g., the early diagnosis and therapeutic intervention of Alzheimer's Disease. In this review, we will first provide the motivation for developing digital protein detection methods given the limitations in the sensitivity of analog methods. We will describe the paradigm shift catalyzed by single molecule detection, and will describe in detail one digital approach – which we call digital bead assays (DBA) – based on the capture and labeling of proteins on beads, identifying "on" and "off" beads, and quantification using Poisson statistics. DBA based on the single molecule array (Simoa) technology have sensitivities down to attomolar concentrations, equating to ∼10 proteins in a 200 μL sample. We will describe the concept behind DBA, the different single molecule labels used, the ways of analyzing beads (imaging of arrays and flow), the binding reagents and substrates used, and integration of these technologies into fully automated and miniaturized systems. We provide an overview of emerging approaches to digital protein detection, including those based on digital detection of nucleic acids labels, single nanoparticle detection, measurements using nanopores, and methods that exploit the kinetics of single molecule binding. We outline the initial impact of digital protein detection on clinical measurements, highlighting the importance of customized assay development and translational clinical research. We highlight the use of DBA in the measurement of neurological protein biomarkers in blood, and how these higher sensitivity methods are changing the diagnosis and treatment of neurological diseases. We conclude by summarizing the status of digital protein detection and suggest how the lab-on-a-chip community might drive future innovations in this field. [ABSTRACT FROM AUTHOR]

Aiming to reduce the computational cost in the current explicit solvent molecular dynamics (MD) simulation, this paper proposes a fast-slow method for the fast MD simulation of biomolecules in explicit solvent. This fast-slow method divides the entire system into two parts: a core layer (typically solute or biomolecule) and a peripheral layer (typically solvent molecules). The core layer is treated using standard MD method but the peripheral layer is treated by a slower dynamics method to reduce the computational cost. We compared four different simulation models in testing calculations for several small proteins. These include gas-phase, implicit solvent, fast-slow explicit solvent and standard explicit solvent MD simulations. Our study shows that gas-phase and implicit solvent models do not provide a realistic solvent environment and fail to correctly produce reliable dynamic structures of proteins. On the other hand, the fast-slow method can essentially reproduce the same solvent effect as the standard explicit solvent model while gaining an order of magnitude in efficiency. This fast-slow method thus provides an efficient approach for accelerating the MD simulation of biomolecules in explicit solvent.

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]

The demand for proteins with high purity, high activity and high stability is increasing, especially in the biopharmaceutical industry. Traditional industrial protein production technologies include chromatography, which is expensive, and salting out and traditional crystallization methods, which require large amounts of salt and have an adverse effect on the environment. In recent years, a new method—solvent freeze-out (SFO) technology for protein crystallization—has been developed. Compared with traditional methods, SFO requires less salt, and can be carried out at low temperatures, resulting in more stable and active proteins. In addition, SFO can control the nucleation and crystal growth process under different supersaturation levels by setting appropriate operating temperatures, which can ensure industrial application of this technology. This paper reviews important advances in SFO technology for the crystallization of proteins. [ABSTRACT FROM AUTHOR]

Protein S-acylation is an important lipid modification characteristic for heterogeneity in the acyl chain and dynamicity in the acylation/deacylation cycle. Most S-acylproteomic research has been limited by indirect identification of modified proteins/peptides without attached fatty acids, resulting in the failure to precisely characterize S-acylated sites with attached fatty acids. The study of S-acylation turnover is still limited at the protein level. Herein, aiming to site-specifically profile both the heterogeneity and the turnover of S-acylation, we first developed a site-specific strategy for intact S-acylated peptide analysis by introducing an acid cleavable bioorthogonal tag into a metabolic labelling method (ssMLCC). The cleavable bioorthogonal tag allowed for the selective enrichment and efficient MS analysis of intact S-acylated peptides so that S-acylated sites and their attached fatty acids could be directly analysed, enabling the precise mapping of S-acylated sites, as well as circumventing false positives from previous studies. Moreover, 606 S-palmitoylated (C16:0) sites of 441 proteins in HeLa cells were identified. All types of S-acylated peptides were further characterized by an open search, providing site-specific profiling of acyl chain heterogeneity, including S-myristoylation, S-palmitoylation, S-palmitoleylation, and S-oleylation. Furthermore, site-specific monitoring of S-palmitoylation turnover was achieved by coupling with pulse-chase methods, facilitating the detailed observation of the dynamic event at each site in multi-palmitoylated proteins, and 85 rapidly cycling palmitoylated sites in 79 proteins were identified. This study provided a strategy for the precise and comprehensive analysis of protein S-acylation based on intact S-acylated peptide analysis, contributing to the further understanding of its complexity and biological functions. [ABSTRACT FROM AUTHOR]

To tackle the obstacles related to tumor targeting and overcome the limitations of single treatment models, we have developed a nanoplatform that is both tumor-targeted and enzyme-responsive. This nanoplatform integrates photothermal gold nanorods (AuNRs) and protein drugs into a single system. This nanosystem, known as AuNRs@HA-mPEG-Deta-LA, was fabricated by modifying gold nanorods (AuNRs) with a polymeric ligand called hyaluronic acid-grafted-(mPEG/diethylenetriamine-conjugated-lipoic acid). The purpose of this fabrication was to load cytochrome c (CC) and utilize it for the synergetic protein–photothermal therapy of cancer. The resulting nanoplatform exhibited a high efficiency in loading proteins and demonstrated excellent stability in different biological environments. Additionally, CC-loaded AuNRs@HA-mPEG-Deta-LA not only enabled localized hyperthermia for photothermal therapy (PTT) with laser irradiation but also facilitated the release of CC under the action of hyaluronidase, an enzyme known to be overexpressed in tumor cells. The confocal imaging results demonstrated that the presence of a specific polymeric ligand on this nanoparticle enhances the internalization of CD44-positive cancer cells, accelerates endo/lysosomal escape, and facilitates the controlled release of CC within the cells. Furthermore, the results of the MTT assay also showed that AuNRs@HA-mPEG-Deta-LA as a protein nanocarrier demonstrated excellent biocompatibility. Importantly, this synergistic therapeutic strategy effectively induced apoptosis in A549 cancer cells by increasing the intracellular concentration of CC and utilizing the photothermal conversion of AuNRs, which was observed to be more effective compared to using only protein therapy or PTT. Therefore, this study showcased a nanoplatform based on AuNRs that has great potential for tumor-targeted protein delivery in combination with PTT in cancer treatment. [ABSTRACT FROM AUTHOR]

Recent advances in experimental measurements of transition path time distributions have raised intriguing theoretical questions. The present interpretation of the experimental data indicates a small value of the fitted transition path barrier height as compared to the barrier height of the unfolded to folded transition. Secondly, as shown in this paper, it is essential to analyse the experimental data using absorbing boundary conditions at the end points used to determine the transition paths. Such an analysis reveals long time tails that have thus far eluded quantitative theoretical interpretation. Is this due to uncertainty in the experimental data or does it call for a rethinking of the theoretical interpretation? A detailed study of the transition path time distribution using a diffusive model leads to the following conclusions. a. The present experimental data is not accurate enough to discern between functional forms of bell shaped free energy barriers. b. Long time tails indicate the possible existence of a "trap" in the transition path region. c. The "trap" may be considered as a well in the free energy surface. d. The long time tail is quite sensitive to the form of the trap so that future measurements of the long time tail as a function of the location of the end points of the transition path may make it possible to not only determine the well depth but also to distinguish between different functional forms for the free energy surface. e. Introduction of a well along the transition path leads to good fits with the experimental data provided that the transition path barrier height is ∼3 k B T , substantially higher than the estimates of ∼1 k B T based on bell shaped functions. The results presented here negate the need of introducing multi-dimensional effects, free energy barrier asymmetry, sub-diffusive memory kernels or systematic ruggedness to explain the experimentally measured data.

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]

Self-assembly of proteins on lipid membranes underlies many important processes in cell biology, such as, exo- and endo-cytosis, assembly of viruses, etc. An attractive force that can cause self-assembly is mediated by membrane thickness interactions between proteins. The free energy profile associated with this attractive force is a result of the overlap of thickness deformation fields around the proteins which can be calculated from the solution of a boundary value problem. Yet, the time scales over which two inclusions coalesce has not been explored, even though the evolution of particle concentrations on membranes has been modeled using phase-field approaches. In this paper we compute this time scale as a function of the initial distance between two inclusions by viewing their coalescence as a first passage time problem. The mean first passage time is computed using Langevin dynamics and a partial differential equation, and both methods are found to be in excellent agreement. Inclusions of three different shapes are studied and it is found that for two inclusions separated by about hundred nanometers the time to coalescence is hundreds of milliseconds irrespective of shape. An efficient computation of the interaction energy of inclusions is central to our work. We compute it using a finite difference technique and show that our results are in excellent agreement with those from a previously proposed semi-analytical method based on Fourier-Bessel series. The computational strategies described in this paper could potentially lead to efficient methods to explore the kinetics of self-assembly of proteins on lipid membranes.

Dilute acid (DA) pretreatment of biomass generates multiple inhibitory compounds within the pretreated hydrolysates. These compounds subsequently contribute to the formation of pseudo-lignin on the surface of the substrate, consequently impeding the efficiency of enzymatic digestibility. To detoxify the DA pretreated hydrolysates, post-incubation with non-catalytic proteins (amaranth protein, AP; soy protein, SP; bovine serum albumin, BSA) was performed in the present study. The enzymatic digestibility of DA-pretreated substrates was increased from 40.0% (without non-catalytic proteins) to 64.9, 53.8, and 56.4%, respectively, in the presence of AP (50 mg g−1), SP (65 mg g−1), and BSA (50 mg g−1). The post-incubation of pretreated substrates with non-catalytic proteins led to high hydrophobicity, contact angle, and accessibility, likely due to less formation of pseudo-lignin. Furthermore, gas chromatography/mass spectrometry analysis revealed that AP, SP, and BSA could lower the inhibitor concentrations in the pretreated hydrolysates by 39–100%, 5–100%, and 3–100%, respectively. The detoxification of the pretreated hydrolysates by AP demonstrated superior effectiveness compared to SP and BSA. To assess the affinity between inhibitors and non-catalytic proteins, surface plasmon resonance analysis was conducted, revealing the following affinity rates: AP (18.65 nM) > SP (17.04 nM) > BSA (16.87 nM). Additionally, molecular docking analysis revealed numerous molecular binding sites (i.e., hydrogen, polar, acidic, basic, and greasy contacts) with strong binding affinity ranging from −36.17 to −76.98 kcal mol−1 between the inhibitors and the amino acids of AP. Thus, this study highlights the potential application of AP as a cost-effective strategy for achieving a viable biorefinery. Also, the findings provide valuable insights that can be utilized to advance the development of (hemi)cellulases that exhibit enhanced resistance to lignin and inhibitors. [ABSTRACT FROM AUTHOR]

We report a novel hydrogel for pre-concentration, fluorescent labelling, and light-triggered release of proteins for detection of low abundance biomarkers. The hydrogel was a co-polymer of acrylamide/bisacrylamide and methacrylamide attached to fluorescein isothiocyanate via a light cleavable bond and a poly(ethylene glycol) spacer arm of molecular weight of 3400 g mol−1. Unlike previous work, proteins were captured by an irreversible chemical reaction rather than by non-covalent affinity binding or physical entrapment. Because the protein-reactive group was attached to fluorescein, which in turn was coupled to the hydrogel by a photocleavable bond, on release the protein was labelled with fluorescein. Our hydrogel offered a pre-concentration factor of up to 236 for a model protein, streptavidin. Each protein molecule was labelled with 85 fluorescein molecules, and 50% of the proteins in the hydrogel were released after UV exposure for ∼100 s. The proteins released from the hydrogel were captured in biotinylated microtitre plates and detected by fluorescence, allowing measurement of at least 0.01 ppm (or ∼166 pM) of protein in sample solutions. The reported hydrogel is promising for detection of low abundance proteins while being less laborious than enzyme-linked immunosorbent assay and less affected by changes in environmental conditions than label-free biosensors. [ABSTRACT FROM AUTHOR]

Proteomics studies generate tables with thousands of entries. A significant component of being a proteomics scientist is the ability to process these tables to identify regulated proteins. Many bioinformatics tools are freely available for the community, some of which within reach for scientists with limited or no background in programming and statistics. However, proteomics has become popular in most other biological and biomedical disciplines, resulting in more and more studies where data processing is delegated to specialists that are not lead authors of the scientific project. This creates a risk or at least a limiting factor, as the biological interpretation of a dataset is contingent of a third-party specialist transforming data without the input of the project leader. We acknowledge in advance that dedicated scripts and software have a higher level of sophistication; but we hereby claim that the approach we describe makes proteomics data processing immediately accessible to every scientist. In this paper, we describe key steps of the typical data transformation, normalization and statistics in proteomics data analysis using a simple spreadsheet. This manuscript aims to demonstrate to those who are not familiar with the math and statistics behind these workflows that a proteomics dataset can be processed, simplified and interpreted in software like Microsoft Excel. With this, we aim to reach the community of non-specialists in proteomics to find a common language and illustrate the basic steps of -omics data processing.

Methylation, which is one of the most prominent post-translational modifications on proteins, regulates many important cellular functions. Though several model-based methylation site predictors have been reported, all existing methods employ machine learning strategies, such as support vector machines and random forest, to predict sites of methylation based on a set of "hand-selected" features. As a consequence, the subsequent models may be biased toward one set of features. Moreover, due to the large number of features, model development can often be computationally expensive. In this paper, we propose an alternative approach based on deep learning to predict arginine methylation sites. Our model, which we termed DeepRMethylSite, is computationally less expensive than traditional feature-based methods while eliminating potential biases that can arise through features selection. Based on independent testing on our dataset, DeepRMethylSite achieved efficiency scores of 68%, 82% and 0.51 with respect to sensitivity (SN), specificity (SP) and Matthew's correlation coefficient (MCC), respectively. Importantly, in side-by-side comparisons with other state-of-the-art methylation site predictors, our method performs on par or better in all scoring metrics tested.

Circular dichroism (CD) spectroscopy is a widely-used method in biochemistry, structural biology and pharmaceutical chemistry. More than 24 000 papers published in the past decade have included CD characterisations of proteins; many of those studies have also included other complementary chemical, biophysical, and computational chemistry methods. This tutorial review describes the background to the technique of CD spectroscopy and good practice methods for high quality data collection. It specifically focuses on both established and new methods and tools available for experimental design and interpretation, data processing, visualisation, analysis, validation, archiving, and accession, including tools developed to enhance the complementarity of this method with other structural and chemical biology studies. [ABSTRACT FROM AUTHOR]

The inherent in vivo instability of oligonucleotides presents one of many challenges in the development of RNAi-based therapeutics. Chemical modification to the 5′-terminus serves as an existing paradigm which can make phosphorylated antisense strands less prone to degradation by endogenous enzymes. It has been recently shown that installation of 5′-cyclopropyl phosphonate on the terminus of an oligonucleotide results in greater knockdown of a targeted protein when compared to its unmodified phosphate derivative. In this paper we report the synthesis of a 5′-modified uridine. [ABSTRACT FROM AUTHOR]

This paper discusses a novel, simple, and inexpensive micro-volume ultrafiltration technique for protein concentration, desalting, buffer exchange, and size-based protein purification. The technique is suitable for processing protein samples in a high-throughput mode. It utilizes a combination of capillary action, and osmosis for drawing water and other permeable species from a micro-volume sample droplet applied on the surface of an ultrafiltration membrane. A macromolecule coated on the permeate side of the membrane functions as the osmolyte. The action of the osmolyte could, if required, be augmented by adding a supersorbent polymer layer over the osmolyte. The mildly hydrophobic surface of the polymeric ultrafiltration membrane used in this study minimized sample droplet spreading, thus making it easy to recover the retained material after separation, without sample interference and cross-contamination. High protein recoveries were observed in the micro-volume ultrafiltration experiments described in the paper. [ABSTRACT FROM AUTHOR]

We report a method for embedding cell-free protein synthesis reactions in macro-scale hydrogel materials without a free liquid phase. This paper focuses on methods of preparation for a variety of hydrogels and an investigation of the impact that the hydrogel material has on cell-free protein synthesis.

The ability to efficiently detect low-abundance protein biomarkers in tiny blood samples is a significant challenge in clinical and laboratory settings. Currently, high-sensitivity approaches require specialized instrumentation, involve multiple washing steps, and lack the ability to parallelize, preventing their widespread implementation. Herein, we developed a parallelized, wash-free, and ultrasensitive centrifugal droplet digital protein detection (CDPro) technology that achieves a femtomolar limit of detection (LoD) of target proteins with sub-microliters of plasma. The CDPro combines two techniques, namely a centrifugal microdroplet generation device and a digital immuno-PCR assay. Miniaturized centrifugal devices enable emulsification of hundreds of samples within 3 minutes using a common centrifuge. The bead-free digital immuno-PCR assay not only eliminates the need for multistep washing, but also possesses ultra-high detection sensitivity and accuracy. We characterized the performance of CDPro using recombinant interleukins (IL-3 and IL-6) as example targets and reported a LoD of 0.0128 pg mL−1. We also quantified IL-6 from 7 human clinical blood samples using the CDPro with only 0.5 μL plasma, which showed excellent agreement with an existing clinical protein diagnostic system with 25 μL plasma from those samples (R2 = 0.98). [ABSTRACT FROM AUTHOR]

The evolution of the SARS-CoV2 coronavirus spike S-protein is studied using a mass spectrometry based protein phylogenetic approach. A study of a large dataset comprising sets of peptide masses derived from over 3000 proteins of the SARS-CoV2 virus shows that the approach is capable of resolving and correctly displaying the evolution of the major variants of concern. Using these numerical datasets, through a pairwise comparison of sets of proteolytic peptide masses for each protein, the tree is built without the need for the sequence data itself or any sequence alignment. In the same analysis, single point mutations are calculated from peptide mass differences of different protein sets and these are displayed at the branch nodes on the tree. The tree topology is found to be consistent with that generated using conventional sequence-based phylogenetics by a manual visualisation and using a tree comparison algorithm. The mass tree resolves major variants of the virus and displays non-synonymous mutations, calculated based on the mass data alone, on the tree that enable protein evolution to be charted and tracked along interconnected branches. Tracking the evolution of the SARS-CoV2 coronavirus S-protein is of particular importance given its role in the attachment of the virus to host cells ahead of viral replication. [ABSTRACT FROM AUTHOR]

Exosomes are considered as promising biomarkers for early cancer diagnosis and prognosis. However, the majority of the research studies focused on a single type of exosomal biomarkers, which cannot comprehensively reflect the state of cancer for accurate diagnosis. To address this problem, we presented a ship-shaped microfluidic device containing a microcolumn array for simultaneous in situ detection of exosomal surface proteins and miRNAs. Exosomes were first captured in the microchannels modified with CD63 protein aptamer. Exosomal surface proteins and miRNAs were simultaneously detected in four parallel channels to avoid the interference of fluorescent signals using specific aptamers labeled by Cy5 and catalytic hairpin assembly (CHA) based signal amplification strategy. The limit of detection for multiplexed markers in exosomes was 83 exosomes per μL, which is comparable to previously reported methods. Through quantitative analysis of two disease-specific surface proteins and miRNAs derived from different cancer cells and clinical serum samples, different cancer subtypes as well as cancer patients and healthy people could be significantly distinguished. These results suggest that this simple, highly sensitive, and more accurate analytical strategy by simultaneous in situ profiling of different types of exosomal biomarkers has potential applications in cancer diagnosis and stage monitoring. [ABSTRACT FROM AUTHOR]

Enzymes are used to amplify signals for detection of antigen proteins in biological samples. However, the enzymes conventionally used for this purpose have limitations, such as the presence of the same (i.e., endogenous) activity in human cells and difficulty in simultaneous use of multiple enzymes because of differences in their required reaction conditions. In this report, we identify an enzyme that can overcome these problems: β- D -galacturonidase (GalUAase) from Eisenbergiella tayi. GalUAase activity was confirmed to be absent from human cells. The substrate of GalUAase, galacturonic acid, is highly hydrophilic because of its anionic carboxylate group; high substrate hydrophilicity is an ideal characteristic for the substrate of an enzyme used for detection because it decreases nonspecific adsorption to biological samples. We show that E. tayi GalUAase could be used in the detection of antigen proteins on live human cells with lower background signal than the conventionally used enzyme β- D -galactosidase. The combinatorial use of GalUAase with β- D -galactosidase enabled simultaneous detection of two antigens on live cells. [ABSTRACT FROM AUTHOR]

C-Reaction protein (CRP) is a marker of nonspecific immunity for vital signs and wound assessment, and it can be used to diagnose infections in clinical medicine. However, measuring CRP level currently requires hospital-based instruments, high-cost reagents, and a complex process, all of which have limited its full capabilities for self-detection, a growing trend in modern medicine. In this study, we developed a novel smartphone-based device using advanced methods of magnetoelastic immunosensing to mitigate these limitations. We combined a system-on-chip (SoC) hardware architecture with smartphone apps to realize the sampling of resonance frequency shift on magnetoelastic chips, which can determine the ultra-sensitivity to mass change caused by the binding of anti-CRP antibody and CRP. Through detecting a multi-group of samples, we found that the resonance frequency shift was linearly proportional to the CRP concentration in the range from 0.1 to 100 μg mL−1, with a sensitivity of 12.90 Hz μg−1 mL−1 and a detection limit of 2.349 × 10−4 μg mL−1. Meanwhile, compared with the large-scale instrument used in clinical settings, the performance of our device was stable and significantly more portable, rapid and cost-effective, offering excellent potential for modern home-based diagnosis. [ABSTRACT FROM AUTHOR]

2-(Bis(pyridin-2-ylmethyl)amino) acetic acid (PIC) and ethylenediaminetetraacetic acid (EDTA) vehicle-based acetamidobenzoxazolone probes have been evaluated for the assessment of a 18 kDa translocator protein (TSPO). Here, PIC and EDTA work as a vehicle for possible binding of requisite metal and acetamidobenzoxazolone pharmacophore (MBP) has been used as a specific binding probe for TSPO. In silico interaction analysis was carried out to assess MBP's binding efficacy towards the TSPO. TSPO is a protein that is known to be overexpressed during inflammatory conditions, therefore, during in vivo experiments, an LPS-induced lung inflammation mouse model was developed for the administration of 99mTc-PIC-MBP and 99mTc-EDTA-MBP to understand absorption and distribution in several peripheral organs over time through SPECT imaging. In addition, photophysical analysis with serum protein, i.e., bovine serum albumin (BSA) was also performed using different spectroscopic techniques to understand the in vivo transportation/excretion of these two probes PIC-MPB and EDTA-MPB to better understand the nature of these probes as specific diagnostic agents. [ABSTRACT FROM AUTHOR]