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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]

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.

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.

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.

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]

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]

Cysteine-based Michael addition is a widely employed strategy for covalent conjugation of proteins, peptides, and drugs. The covalent reaction is irreversible in most cases, leading to a lack of control over the process. Utilizing spectroscopic analyses along with X-ray crystallographic studies, we demonstrate Michael addition of an engineered cysteine residue in human Cellular Retinol Binding Protein II (hCRBPII) with a coumarin analog that creates a non-fluorescent complex. UV-illumination reverses the conjugation, yielding a fluorescent species, presumably through a retro-Michael process. This series of events can be repeated between a bound and non-bound form of the cysteine reversibly, resulting in the ON–OFF control of fluorescence. The details of the mechanism of photoswitching was illuminated by recapitulation of the process in light irradiated single crystals, confirming the mechanism at atomic resolution. [ABSTRACT FROM AUTHOR]

Immunoassays are frequently used for analysis of protein biomarkers. The specificity of antibodies enables parallel analysis of several target proteins, at the same time. However, the implementation of such multiplexed assays into cost-efficient and mass-producible thermoplastic microfluidic platforms remains difficult due to the lack of suitable immobilization strategies for different capture antibodies. Here, we introduce and characterize a method to functionalize the surfaces of microfluidic devices manufactured in the thermoplastic material cyclic olefin copolymer (COC) by a rapid prototyping process. A laser-induced immobilization process enables the surface patterning of anchor biomolecules at a spatial resolution of 5 μm. We employ the method for the analysis of prostate cancer associated biomarkers by competitive immunoassays in a microchannel with a total volume of 320 nL, and successfully detected the proteins PSA, CRP, CEA and IGF-1 at clinically relevant concentrations. Finally, we also demonstrate the simultaneous analysis of three markers spiked into undiluted human plasma. In conclusion, this method opens the way to transfer multiplexed immunoassays into mass-producible microfluidic platforms that are suitable for point of care applications. [ABSTRACT FROM AUTHOR]

Biothiols play essential roles in balancing the redox state and modulating cellular functions. Fluorescent probes for monitoring/labelling biothiols often suffer from slow reaction rates, strong background fluorescence and cytotoxic byproduct release. Thus, developing facile and versatile probes to overcome the challenges is still in high demand. Here, we report four coumarin–maleimides as fast responding and fluorogenic probes to detect GSH or label peptides/proteins. The probes quantitatively and selectively react with GSH via Michael addition within 1–2 min, achieving an 11–196-fold increase in fluorescence quantum yield via blockage of the photoinduced electron transfer (PET) process. Optimized probe 4 is applied for the detection of GSH in vitro (A549 cells) and in vivo (zebrafish embryos). Taking advantage of the fast Michael addition between the maleimide moiety and the sulfhydryl group, we expand the application of our method for fluorescent labelling of peptides/proteins and for tracking their cellular uptake process. The labelling strategy works for both Cys-bearing and Cys-free proteins after the introduction of a sulfhydryl group using Traut's reagent. Fluorescence assay reveals that the TAT-peptide can efficiently enter cells, but H3 protein, part of nucleosomes, prefers to bind on the cell membrane by electrostatic interactions, shedding light on the cellular uptake activity of nucleosomes and affording a potential membrane staining strategy. Overall, our study illustrates the broad potential of coumarin–maleimide based dual-functional probes for GSH detection and versatile protein labelling in biochemical research. [ABSTRACT FROM AUTHOR]

Herein, we report the construction of a programmable catalytic molecular nanomachine based on a cross-linked catalytic hairpin assembly (CCHA) reaction for the one-step highly sensitive detection of proteins and small molecules. In this system, when the recognition elements attached on split initiators bind to the target proteins, it can trigger the cascade of the CCHA reaction, resulting in the formation of many macromolecular fluorescent products for signaling. This platform couples the advantages of highly efficient DNA-based nanotechnology with specific protein-small molecule interactions. We demonstrated the sensitive detection of streptavidin and anti-digoxigenin antibody with detection limits as low as 48.8 pM and 0.85 nM, respectively. This nanomachine also demonstrated its flexibility in the nanomolar detection of corresponding small molecules, such as biotin and digoxigenin, using a competitive method. In addition, the nanomachine was robust enough to perform well with human serum samples. Overall, this programmable catalytic molecular nanomachine provides a versatile platform for the detection of proteins and small molecules by replacing the recognition elements, which can promote the development of DNA nanotechnology in disease diagnosis and therapeutic drug monitoring. [ABSTRACT FROM AUTHOR]

Protein acylation is a vital post-translational modification that regulates various protein functions. In particular, protein succinylation has attracted significant attention because of its potential relationship with various biological events and diseases. In this report, we establish a new method for the comprehensive detection and analysis of potentially succinylated proteins using a chemical tagging technology. The newly synthesized alkyne-containing succinyl substrate successfully labeled lysine residues of proteins through intracellular metabolic labeling independent of other acylation pathways such as protein malonylation. Furthermore, reporter molecules such as biotin moieties and fluorescent dyes were conjugated to alkyne-tagged succinylated proteins via Click reactions, permitting enrichment for proteomic analysis and fluorescence imaging of the labeled proteins. We successfully analyzed and identified numerous potential succinylated proteins associated with various biological processes using gel electrophoresis, proteomic and bioinformatic analyses, and their visualization in cells. [ABSTRACT FROM AUTHOR]

Disulfide bonds in proteins have a substantial impact on protein structure, stability, and biological activity. Localizing disulfide bonds is critical for understanding protein folding and higher-order structure. Conventional top-down mass spectrometry (TD-MS), where only terminal fragments are assigned for disulfide-intact proteins, can access disulfide information, but suffers from low fragmentation efficiency, thereby limiting sequence coverage. Here, we show that assigning internal fragments generated from TD-MS enhances the sequence coverage of disulfide-intact proteins by 20–60% by returning information from the interior of the protein sequence, which cannot be obtained by terminal fragments alone. The inclusion of internal fragments can extend the sequence information of disulfide-intact proteins to near complete sequence coverage. Importantly, the enhanced sequence information that arise from the assignment of internal fragments can be used to determine the relative position of disulfide bonds and the exact disulfide connectivity between cysteines. The data presented here demonstrates the benefits of incorporating internal fragment analysis into the TD-MS workflow for analyzing disulfide-intact proteins, which would be valuable for characterizing biotherapeutic proteins such as monoclonal antibodies and antibody–drug conjugates. [ABSTRACT FROM AUTHOR]

In this work, three iridium(III) tetrazolato complexes have been designed and successfully synthesized. Beside photophysical properties, their performances in protein staining have been comprehensively investigated in this work for the first time. Notably, these iridium(III) tetrazolato complexes with high quantum efficiency exhibited much better protein staining properties than the commercial agent Coomassie Brilliant Blue (CBB) under the same experimental conditions, which may pave the way to explore new efficient iridium-based protein staining agents both for commercial markets and academic research in the future. [ABSTRACT FROM AUTHOR]

A novel and highly efficient dual-targeting PtII system was designed to improve the drug delivery capacity and selectivity in cancer treatment. The dual-targeting monofunctional PtII complexes (1–8) having glycosylated pendants as tridentated ligand were achieved by introducing glycosylation modification in the thioaminocarbazone compounds with potential lysosomal targeting ability. The structures and stability of 1–8 were further established by various techniques. Molecular docking studies showed that 2 was efficiently docked into glucose transporters protein 1 (GLUT1) and P-glycoprotein (Pgp) proteins with the optimal CDocker-interaction-energy of −64.84 and −48.85 kcal mol−1. Complex 2 with higher protein binding capacity demonstrated significant and broad-spectrum antitumor efficacy in vitro, even exhibiting a half maximal inhibitory concentration (IC50) value (∼10 μM) than cisplatin (∼17 μM) against human lung adenocarcinoma cells (A549). The inhibitor experiment revealed GLUT-mediated uptake of 2, and the subcellular localization experiment in A549 also proved that 2 could be localized in the lysosome, thereby causing cell apoptosis. Moreover, cellular thermal shift assay (CETSA) confirmed the binding of 2 with the target proteins of GLUT1 and Pgp. The above results indicated that 2 represents a potential anticancer candidate with dual-targeting functions. [ABSTRACT FROM AUTHOR]

The existence of life beyond Earth has long captivated humanity, and the study of extremophiles—organisms surviving and thriving in extreme environments—provides crucial insights into this possibility. Extremophiles overcome severe challenges such as enzyme inactivity, protein denaturation, and damage of the cell membrane by adopting several strategies. This feature article focuses on the molecular strategies extremophiles use to maintain the cell membrane's structure and fluidity under external stress. Key strategies include homeoviscous adaptation (HVA), involving the regulation of lipid composition, and osmolyte-mediated adaptation (OMA), where small organic molecules protect the lipid membrane under stress. Proteins also have direct and indirect roles in protecting the lipid membrane. Examining the survival strategies of extremophiles provides scientists with crucial insights into how life can adapt and persist in harsh conditions, shedding light on the origins of life. This article examines HVA and OMA and their mechanisms in maintaining membrane stability, emphasizing our contributions to this field. It also provides a brief overview of the roles of proteins and concludes with recommendations for future research directions. [ABSTRACT FROM AUTHOR]

Utilizing a novel approach known as aptamer-assisted phage display (APD), we identified an anti-PD-L1 peptide, NV Pep, capable of simultaneous binding to PD-L1 alongside the DNA aptamer MJ5C. Combined inhibition using NV Pep and MJ5C demonstrated significant enhancement compared to individual ligands against the PD-1/PD-L1 interaction. [ABSTRACT FROM AUTHOR]

Noncanonical G-quadruplex nucleic acid structures can be used as probes in biosensors for the detection of metal ions, proteins and nucleic acids. Angiotensinogen (AGT) is a glycosylated globulin found in serum, which can regulate blood pressure and body fluid homeostasis. AGT is an important part of the renin–angiotensin system (RAS) and can be potentially used as a biomarker of diseases with RAS alterations. G-quadruplex based biosensors can detect targets with high accuracy and speed and low cost. Employing the magnetic bead enrichment method we constructed a reliable and efficient fluorescent biosensor platform for G-quadruplex based detection of the human AGT protein. The primary antibody in our biosensor is recognized by fluorescently labeled secondary antibodies, which leads to the detection of AGT captured by the G-quadruplex aptamer coupled magnetic beads. This G-quadruplex based fluorescent biosensor designed with a detection limit of 5 × 10–5 mg mL−1 was used for the successful detection of AGT at the cellular level. Our G-quadruplex based fluorescent biosensor will contribute to the more reliable and efficient detection of AGT. [ABSTRACT FROM AUTHOR]

Carbon-supported bimetallic NiCo nanoparticles (NPs) have emerged as attractive catalysts and adsorbents for the reduction of 4-nitrophenol (4-NP) and separation of histidine-rich (His-rich) protein recently due to their low cost, high catalytic activity and good affinity for His-rich protein. In this study, new strongly coupled nickel–cobalt alloy/N,P,S co-doped carbon (NPSC) nanocages are rationally designed via chemical etching of the ZIF-67 dodecahedron with Ni2+ under sonication at room temperature, followed by poly(cyclotriphosphazene-co-4,4′-sulfonyldiphenol) (PZS) coating and subsequent carbonization treatment in a nitrogen atmosphere. When evaluated as a catalyst for 4-NP or an adsorbent for His-rich protein, the as-prepared NiCo@NPSC nanocages obtained at 700 °C show better performance than those obtained at other temperatures (500 and 900 °C). This improved catalytic effect is attributed to the controllable size and fine distribution of the NiCo NPs together with the effective contact between the catalysts and the N,P,S co-doped carbon matrix, leading to a superior catalytic effect on the reduction of 4-NP and the adsorption of His-rich protein. This catalyst design principle can be easily extended to other catalysis research fields. [ABSTRACT FROM AUTHOR]

We have developed a point-of-care (POC) lateral flow biosensor (LFB) for universal protein detection based on a proximity hybridization-mediated protein-to-DNA signal transducer, isothermal exponential amplification (EXPAR) and catalytic hairpin assembly (CHA) with high sensitivity and specificity. In this assay, a protein signal transducer was employed to convert the input protein to the output DNA signal. Antibody conjugated DNA1 was firstly hybridized with the output DNA (DNA3). The binding of antibody conjugated DNA1 and DNA2 to the same protein was able to increase the local concentrations, resulting in strand displacement between DNA3 and DNA2. DNA3 with nicking endonuclease recognition sequences at the 5′ end then hybridized with hairpin probe 1 to mediate EXPAR in the presence of nicking endonuclease and polymerase. A large number of single strand DNA were produced in the circle of nicking, polymerization and strand displacement. The resulting ssDNA products were further amplified by CHA to generate double-stranded DNA products. The double-stranded DNA products were detected with a lateral flow biosensor within 5 min. This proposed assay has very high sensitivity and selectivity with a dynamic response ranging from 1 fM to 100 nM, and the detection limit was 0.74 fM. This work provides a universal and simple method for protein detection. [ABSTRACT FROM AUTHOR]

The identification of molecular targets for achieving beneficial effects from small-molecule drugs is a crucial and currently unsolved challenge, which leads to high costs and long development cycles. Therefore, it is urgent to develop methods for easily and quickly acquiring information about protein–drug interaction at a molecular level. In this study, we propose a novel method for the study of protein–drug interaction by fluorescence correlation spectroscopy (FCS) based on organic solvent-induced protein aggregation. We used β-secretase (BACE-1) and dihydrofolate reductase (DHFR) as model proteins. Fluorescence-labelled proteins aggregated in aqueous solutions containing organic solvents. In the presence of drugs, the aggregation of proteins was inhibited greatly, and FCS was used to characterize protein aggregates. The decrease in the characteristic diffusion time (τD) of protein aggregates demonstrated a strong interaction between proteins and drug molecules. We presented a new parameter IC50 to assess the inhibitory effects of drugs on the basis of the changes in the τD of fluorescence-labelled proteins under different concentrations of the drugs in the presence of organic solvents. We acquired a remarkable difference in the IC50 values for different drugs and in terms of the trend, our results were consistent with those reported by other methods. Compared with current methods, our approach is simple, low-cost, and time-saving, and has the potential to become a promising and universal tool for drug screening at the molecular level. [ABSTRACT FROM AUTHOR]

COVID-19 has caused millions of cases and deaths all over the world since late 2019. Rapid detection of the virus is crucial for controlling its spread through a population. COVID-19 is currently detected by nucleic acid-based tests and serological tests. However, these methods have limitations such as the requirement of high-cost reagents, false negative results and being time consuming. Surface-enhanced Raman scattering (SERS), which is a powerful technique that enhances the Raman signals of molecules using plasmonic nanostructures, can overcome these disadvantages. In this study, we developed a virus-infected cell model and analyzed this model by SERS combined with Principal Component Analysis (PCA). HEK293 cells were transfected with plasmids encoding the nucleocapsid (N), membrane (M) and envelope (E) proteins of SARS-CoV-2 via polyethyleneimine (PEI). Non-plasmid transfected HEK293 cells were used as the control group. Cellular uptake was optimized with green fluorescence protein (GFP) plasmids and evaluated by fluorescence microscopy and flow cytometry. The transfection efficiency was found to be around 60%. The expression of M, N, and E proteins was demonstrated by western blotting. The SERS spectra of the total proteins of transfected cells were obtained using a gold nanoparticle-based SERS substrate. Proteins of the transfected cells have peak positions at 646, 680, 713, 768, 780, 953, 1014, 1046, 1213, 1243, 1424, 2102, and 2124 cm−1. To reveal spectral differences between plasmid transfected cells and non-transfected control cells, PCA was applied to the spectra. The results demonstrated that SERS coupled with PCA might be a favorable and reliable way to develop a rapid, low-cost, and promising technique for the detection of COVID-19. [ABSTRACT FROM AUTHOR]

Almost 60% of commercialized pharmaceutical proteins are glycosylated. Glycosylation is considered a critical quality attribute, as it affects the stability, bioactivity and safety of proteins. Hence, the development of analytical methods to characterise the composition and structure of glycoproteins is crucial. Currently, existing methods are time-consuming, expensive, and require significant sample preparation steps, which can alter the robustness of the analyses. In this work, we suggest the use of a fast, direct, and simple Fourier transform infrared spectroscopy (FT-IR) combined with a chemometric strategy to address this challenge. In this context, a database of FT-IR spectra of glycoproteins was built, and the glycoproteins were characterised by reference methods (MALDI-TOF, LC-ESI-QTOF and LC-FLR-MS) to estimate the mass ratio between carbohydrates and proteins and determine the composition in monosaccharides. The FT-IR spectra were processed first by Partial Least Squares Regression (PLSR), one of the most used regression algorithms in spectroscopy and secondly by Support Vector Regression (SVR). SVR has emerged in recent years and is now considered a powerful alternative to PLSR, thanks to its ability to flexibly model nonlinear relationships. The results provide clear evidence of the efficiency of the combination of FT-IR spectroscopy, and SVR modelling to characterise glycosylation in therapeutic proteins. The SVR models showed better predictive performances than the PLSR models in terms of RMSECV, RMSEP, R 2CV , R 2Pred and RPD. This tool offers several potential applications, such as comparing the glycosylation of a biosimilar and the original molecule, monitoring batch-to-batch homogeneity, and in-process control. [ABSTRACT FROM AUTHOR]

Dirhodium complexes of general formula [Rh2(O2CR)4]L2 are a well-known class of bimetallic compounds that are used as efficient catalysts for a variety of reactions and have been shown to be potent antibacterial and anticancer agents. The catalytic and biological properties of these complexes largely depend on the nature of the bridging carboxylate ligands. Trifluoroacetate (tfa)-containing dirhodium compounds have been used to build artificial metalloenzymes upon reaction with peptides and have been shown to be more cytotoxic than dirhodium tetraacetate. However, there is no structural information on the interaction between these compounds and proteins. Here, cis-Rh2(μ-O2CCH3)2(μ-O2CCF3)2 ([cis-Rh2(OAc)2(tfa)2]) has been synthesized and its reaction with bovine pancreatic ribonuclease (RNase A) and hen egg white lysozyme (HEWL) was analyzed using a combination of different techniques, including Fluorine-19 nuclear magnetic resonance spectroscopy and macromolecular X-ray crystallography, with the aim to unveil the differences in the reactivity of tfa-containing dihrodium complexes with proteins when compared to [Rh2(OAc)4]. [cis-Rh2(OAc)2(tfa)2] and [Rh2(OAc)4] bind the N atoms of His side chains of RNase A at the axial position; however the fluorine-containing compound rapidly loses its tfa ligands, while [Rh2(OAc)4] can retain the acetate ligands upon protein binding. The reactivity of [cis-Rh2(OAc)2(tfa)2] with HEWL is slightly distinct when compared to that of [Rh2(OAc)4] under the same experimental conditions; however, both [cis-Rh2(OAc)2(tfa)2] and [Rh2(OAc)4] degrade when soaked within HEWL crystals. These results provide a structural-based guide for the design of new heterogenous chiral dirhodium/peptide and dirhodium/protein adducts with application in the fields of organic synthesis and asymmetric catalysis. [ABSTRACT FROM AUTHOR]

It is widely believed that high-purity protein is critical to crystallization. Impurities can induce lattice strain, internal stress, and rotation disorder, thus decreasing protein crystallization success rate and crystal quality. However, some studies showed that protein could be crystallized from impure solutions; thus, the effect of impurities on protein crystallization remains unclear. Here, we systemically studied the effect of impurity type and concentration on protein crystallization. Results showed that protein crystallization was different when using different impurities. Protein crystallization could be promoted with a low concentration of impurities and inhibited with a high concentration of impurities, and this inhibition can be weakened by an audible sound. The effect of impurities on protein crystallization can be explained by the phase separation theory. In summary, we suggest that proper impurities might be beneficial to enhance protein crystallization. [ABSTRACT FROM AUTHOR]

C-reactive proteins (CRPs; an acute-phase protein) in patients with initial acute cerebral infarction neurological rehabilitation prediction have a significant correlation. In this work, a simple and sensitive anodic-stripping voltammetric (ASV) immunosensing system was innovatively designed for the quantitative screening of target CRPs using biomimetic-mineralized bifunctional antibody-Cu3(PO4)2 nanoflowers as molecular tags. In this system, a monoclonal anti-CRP antibody-anchored microtiter plate was utilized to specifically capture target CRPs from the sample. For detection, a sandwiched immunoreaction mode was employed with the antibody-Cu3(PO4)2 nanoflowers in the presence of analytes. Subsequent ASV measurement of copper ions (Cu2+) released under acidic conditions from the bifunctional nanoflowers was conducted at an in situ prepared mercury film electrode. The introduction of hybrid nanoflowers greatly increased the loading amount of copper ions on the molecular tag, thereby amplifying the detectable signal of electrochemical immunoassay. Meanwhile, factors influencing the analytical properties of the electrochemical immunoassay were investigated in detail. By combining the high-efficiency nanohybrids with signal amplification, the dynamic concentration range of electrochemical immunoassay spanned from 0.01 ng mL−1 to 100 ng mL−1 toward the target CRP. The limit of detection was calculated to be 0.0079 ng mL−1 at 3Sblank criterion. Intra- and interassay imprecisions (relative standard deviations: RSDs) were less than or equal to 6.72%. Good anti-interference ability, long-term storage stability, and acceptable accuracy for the evaluation of human serum specimens were observed during a series of procedures to determine the target protein. In addition, the bifunctional nanoflower-based immunosensing system offers promise for the simple, cost-effective analysis of disease-related proteins. [ABSTRACT FROM AUTHOR]

Chemical labeling methods for proteins are highly researched. Herein, we introduced β-carbonyl sulfonium compounds for selective cysteine modification in proteins within biological systems. Structural tuning led to sulfonium-based probes with high reactivity and selectivity. These probes show excellent biocompatibility, cell uptake, and specificity towards cysteine profiling in live cells. [ABSTRACT FROM AUTHOR]

Attaching proteins to surfaces while maintaining bioactivity is a promising avenue for developing new functional materials. This work describes a method to rapidly apply bioactive coatings by conjugating protein into an adhesive network using an ortho-phthaldialdehyde (oPA)-based linker. The polymer matrix was generated using a copper-catalyzed azide–alkyne cycloaddition (CuAAC) polymerization on a commercial, antifouling copper-based paint that had not been previously examined as a catalytic material. After undergoing bioconjugation with the oPA linker, protein was retained at a higher rate on the surface when compared either with the control, which lacked a linker, or with a commercially-available maleimide linker. The ability to apply in situ, polymerize, and adhere biomacromolecules to surfaces while retaining their efficacy enables the creation of novel functional materials with properties usually reserved for the biological domains. [ABSTRACT FROM AUTHOR]

In this study, silica particles were used to improve target protein batch crystallisation from a binary protein mixture at a 5 mL scale. Lysozyme (40 mg mL−1) was used as the target protein and thaumatin (0.1–8 mg mL−1) was regarded as a protein impurity. It was demonstrated that even an impurity at the concentration as low as 0.1 mg mL−1 (0.25 w/w% of the target protein) would delay target protein crystallisation, predominantly by extending the induction time. When the silica particles were employed in the system to facilitate crystallisation, target protein crystallisation was significantly improved with a much shorter induction time and higher yield at the end of the experiment. It was also shown that the effectiveness of silica on target protein crystallisation depended on the impurity concentration and silica loading amount. [ABSTRACT FROM AUTHOR]

Heterogeneous surfaces, such as solid particles, are known to efficiently increase nucleation rates during crystallisations from solution. In this work, air bubbles have been used to act as heterogeneous surfaces, to facilitate the critical nuclei formation of large protein molecules. Protein crystallisation experiments were performed using the hanging-drop method, to investigate the effects of concentrations of lysozyme and sodium chloride, and air bubbles on the nucleation of crystals. The introduction of an air bubble template resulted in an overall reduction in the nucleation induction time, over the majority of the experimental conditions. With air bubbles in the hanging droplets, the population density of the lysozyme crystals was up to 1.5 times higher than that in the droplets that contained no bubbles. For the studied experimental conditions, the mass yield was also found to increase by adding air bubbles into the droplet. [ABSTRACT FROM AUTHOR]

In this work, we designed and developed a method to detect S1 spike protein of SARS-CoV-2. The portable Localized Surface Plasmon Resonance instrument equipped with a two-channel system was combined with the biotin–streptavidin platform on a nanogold surface to immobilize biotinylated aptamers. The proposed assay does not utilize antibodies or enzyme-based reagents, further simplifying the detection method. Using aptamer–protein bioaffinity interactions, the aptasensor selectively and specifically detected in real-time S1 spike protein, rather than S2 spike protein, RBD spike protein, or bovine serum albumin. The dynamic range and limit of detection of the aptasensor was determined to be 1 nM–100 nM and 0.26 nM, respectively. Notably, aptasensor detected preferentially S1 protein of SARS-CoV-2 compared to SARS-CoV and detected S1 protein with >95% recovery in artificial saliva, and serum albumin, excellent repeatability and shelf-life stability. The method may provide a low-cost, rapid, and real-time detection and monitoring of viruses in the general public. [ABSTRACT FROM AUTHOR]

Herein, copper adhesive tape attached to the reverse side of a glass slide was developed as a new method to achieve protein in situ detection and imaging in a formalin fixed paraffin-embedded (FFPE) tissue section on a non-conductive glass slide by MALDI-MSI. The use of this new method led to 223 protein ions being imaged from a rat brain FFPE-tissue section on a non-conductive glass slide by MALDI-MS, compared to only 145 and 163 protein ions detected on an ITO glass slide and an AnchorChip target plate, respectively. This new method has great potential to become standard practice for protein MALDI-imaging in FFPE-tissue sections on non-conductive glass slides. [ABSTRACT FROM AUTHOR]

The affinity between functional nanoparticles (NPs) and proteins could determine the efficacy of nanoprobes, nanosensors, nanocarriers, and many other devices for biomedical applications. Therefore, it is necessary to develop analytical strategies to accurately evaluate the magnitude of these protein corona interactions in physiological media. In this work, different electrokinetic strategies were implemented to accurately determine the interactions between PEGylated ZnGa1.995Cr0.005O4 persistent luminescent NPs (ZGO-PEG) and two important serum proteins: human serum albumin (HSA), the most abundant serum protein, and apolipoprotein-E (ApoE), associated with the active transport of NPs through the blood--brain barrier. Firstly, the injection of ZGO-PEG in a background electrolyte (BGE) containing individual proteins allowed an affinity study to separately characterize each NP--protein system. Then, the same procedure was applied in a buffer containing a mixture of the two proteins at different molar ratios. Finally, the NPs were pre-incubated with one protein and thereafter electrokinetically separated in a BGE containing the second protein. These analytical strategies revealed the mechanisms (comparative, cooperative or competitive systems) and the magnitude of their interactions, resulting in all cases in notably higher affinity and stability between ZGO-PEG and ApoE (Ka = 1.96 ± 0.25 x 1010 M-M) compared to HSA (Ka = 4.60 ± 0.41 x 106 M-M). For the first time, the inter-protein ApoE/HSA interactions with ZGO-PEG were also demonstrated, highlighting the formation of a ternary ZGO-PEG/ApoE/HSA nanocomplex. These results open the way for a deeper understanding of the protein corona formation, and the development of versatile optical imaging applications for ZGO-PEG and other systemically delivered nanoprobes ideally vectorized to the brain. [ABSTRACT FROM AUTHOR]