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Study of small molecule binding to live cells provides important information on the characterization of ligands pharmacologically. Here we developed and validated a label-free, liquid chromatography-mass spectrometry (LC-MS) based cell binding assay, using centrifugation to separate binders from non-binders. This assay was applied to various target classes, with particular emphasis on those for which protein-based binding assay can be difficult to achieve. In one example, to study a G protein coupled receptor (GPCR), we used one antagonist as probe and multiple other antagonists as competitor ligands. Binding of the probe was confirmed to be specific and saturable, reaching a fast equilibrium. Competition binding analysis by titration of five known ligands suggested a good correlation with their inhibition potency. In another example, this assay was applied to an ion channel target with its agonists, of which the determined binding affinity was consistent with functional assays. This versatile method allows quantitative characterization of ligand binding to cell surface expressed targets in a physiologically relevant environment.

Nucleic acid‐based biosensors for the detection of specific proteins combine the typical programmability of synthetic DNA systems with artificially controlled DNA‐protein communication. The high‐affinity interaction between a target protein and a specific ligand, such as an aptamer sequence, or a double stranded DNA domain, or a small peptide, is paired with a nature‐mimicking molecular mechanism allowing for probing, processing, and translating protein binding activity into a measurable signal. In this Review, two main strategies developed in the context of protein‐responsive nucleic acid‐based biosensors are discussed. One is the design of proximity‐based assays harnessing the spatial colocalization of functional probes within the volume of a multivalent protein. The other is the engineering of dynamic DNA structures that undergo a controlled conformational or structural change upon protein binding. Examples of applications from optical and electrochemical detection of antibodies in biofluids to fluorescence imaging of transcription factors in living cells are reported, and suggestions along with possible future directions in the field are discussed. [ABSTRACT FROM AUTHOR]

The downstream processing of natural active molecules remains the most significant cost in the production pipeline. This considerable cost is largely attributed to rigorous chromatographic purification protocols. In an ongoing effort to abate the dependence on chromatography in downstream processing, alternative affinity matrices in the form of magnetic particles (e.g., iron oxide) have emerged as viable candidates. Nevertheless, biotechnological applications of iron oxide particles are still confined to the research level or for low-throughput clinical applications. Herein, we describe an efficient, quick, and environmentally friendly method for the isolation of astaxanthin and lutein, two carotenoids with very similar chemical structure, from extracts of the microalga Haematococcus pluvialis. The technology proposed, named Selective Magnetic Separation (SMS), is based on the use of magnetic materials carrying affinity ligands that bind carotenoids and is applied as second step of purification. The method, thanks to functionalized magnetic nanoparticles, reduces the use of organic or toxic solvents. In the present work, we examined the most efficient binding conditions such as temperature, magnetic nanoparticles concentration, and elution time, as well as their effects on carotenoids recovery, with the aim to improve the non-covalent binding between the ligand (amines) and astaxanthin/lutein. Our initial results clearly showed that it is possible to use magnetic separation as an alternative to chromatography to isolate important and valuable compounds. [ABSTRACT FROM AUTHOR]

The requirement of high-sensitivity, accurate and rapid determination of glucose in human blood is major challenge from clinical perspective. In this work, boronic acids (BAs) and derivatives were successfully functionalized over graphene nano flakes (GNF) surface as a sensing platform to spot Glucose molecule. The density functional theory-based investigation has been carried out by considering geometrical optimization, density of states, and molecular energy spectrum. Further, molecular properties like molecular orbital investigations, energy gap, chemical hardness, chemical softness, have been evaluated in order to distinguish the rate of reaction and binding energy between GNF-BA and Glucose. Results show that BAs derivatives were physisorbed/chemisorbed over GNF surface. Moreover, the analyte formed the covalent interactions with its diol group towards the trigonal structured B atom of BAs and provided the sensing signal by modifying the DOS peaks of sensing platform. Eventually, the sensitivity of different BA’s derivatives @GNF to Glucose sensing were compared. These findings show the variation at molecular and atomic level behaviour of GNF-BA sensing platform in presence/absences of Glucose. Therefore, our DFT predictions have application in designing sensor in area of biomolecules and bioelectronics for healthcare sector. [ABSTRACT FROM AUTHOR]

Increasing the shelf life of enzymes and making them reusable is a prominent topic in biotechnology. The encapsulation inside hydrogel microparticles (HMPs) can enhance the enzyme’s stability by preserving its native conformation and facilitating continuous biocatalytic processes and enzyme recovery. In this study, we present a method to immobilize β-galactosidase by, first, conjugating the enzyme onto the surface of polymer nanoparticles, and then encapsulating these enzyme-conjugated nanoparticles (ENPs) inside HMPs using microfluidic device paired with UV-LEDs. Polymer nanoparticles act as anchors for enzyme molecules, potentially preventing their leaching through the hydrogel network especially during swelling. The affinity binding (through streptavidin-biotin interaction) was used as an immobilization technique of β-galactosidase on the surface of polymer nanoparticles. The hydrogel microparticles of roughly 400 μm in size (swollen state) containing unbound enzyme and ENPs were produced. The effects of encapsulation and storage in different conditions were evaluated. It was discovered that the encapsulation in acrylamide (AcAm) microparticles caused an almost complete loss of enzymatic activity. Encapsulation in poly(ethylene glycol) (PEG)-diacrylate microparticles, on the other hand, showed a residual activity of 15–25%, presumably due to a protective effect of PEG during polymerization. One of the major factors that affected the enzyme activity was presence of photoinitiator exposed to UV-irradiation. Storage studies were carried out at room temperature, in the fridge and in the freezer throughout 1, 7 and 28 days. The polymer nanoparticles showcased excellent immobilization properties and preserved the activity of the conjugated enzyme at room temperature (115% residual activity after 28 days), while a slight decrease was observed for the unbound enzyme (94% after 28 days). Similar trends were observed for encapsulated ENPs and unbound enzyme. Nevertheless, storage at −26°C resulted in an almost complete loss of enzymatic activity for all samples.

[Display omitted] • Magnetic microcryogels were utilized in an application of the adsorption process. • Magnetic microcryogels were prepared via cryopolymerization. • Polyethylenimine was immobilized on magnetic microcryogels. • The new materials exhibit great potential in the removal of bilirubin. In this study, for the first time, micro-engineered cryogels embedded with Fe 3 O 4 nanoparticles were developed as magnetic adsorbents. Magnetically controllable microcryogels were produced on a microstencil array chip by cryopolymerization and functionalized by polyethylenimine, as an affinity ligand. Macroporous architecture of both magnetic and non-magnetic microcryogels were evaluated via field emission scanning microscopy. Bilirubin adsorption studies were performed to investigate the optimal removal conditions. The results indicated that the adsorption capacity of magnetic microcryogels increased with higher temperature values while decreased with higher salt concentrations. Plasma bilirubin removal assay showed that the removal of MMs-PEI was much higher (i.e., 13-fold) than that of MMs. Magnetic microcryogels exhibited significant reusability, where the adsorption capacity decreased by 7.8 % after 5 adsorption-desorption cycles. The results showed that magnetic micro-engineered cryogels provided good application potential for bilirubin removal and an alternative for the current methods. [ABSTRACT FROM AUTHOR]

The downstream processing of natural active molecules remains the most significant cost in the production pipeline. This considerable cost is largely attributed to rigorous chromatographic purification protocols. In an ongoing effort to abate the dependence on chromatography in downstream processing, alternative affinity matrices in the form of magnetic particles (e.g., iron oxide) have emerged as viable candidates. Nevertheless, biotechnological applications of iron oxide particles are still confined to the research level or for low-throughput clinical applications. Herein, we describe an efficient, quick, and environmentally friendly method for the isolation of astaxanthin and lutein, two carotenoids with very similar chemical structure, from extracts of the microalga Haematococcus pluvialis. The technology proposed, named Selective Magnetic Separation (SMS), is based on the use of magnetic materials carrying affinity ligands that bind carotenoids and is applied as second step of purification. The method, thanks to functionalized magnetic nanoparticles, reduces the use of organic or toxic solvents. In the present work, we examined the most efficient binding conditions such as temperature, magnetic nanoparticles concentration, and elution time, as well as their effects on carotenoids recovery, with the aim to improve the non-covalent binding between the ligand (amines) and astaxanthin/lutein. Our initial results clearly showed that it is possible to use magnetic separation as an alternative to chromatography to isolate important and valuable compounds.

Leishmaniasis considered as the most crucial epidemic-prone diseases according to the World Health Organization. Early diagnoses and therapy of Leishmania infection is a great challenge since, it has no symptom and is resistance to drugs. Therefore, there is an urgent need for sensitive and precise detection of this pathogen. In this study, a new method was developed for optical biosensing of Leishmania spp sequence based on hybridization of Citrate capped Ag nanoparticles bonded to specific single stranded DNA probe of Leishmania spp. Aggregation of the Citrate capped Ag nanoparticles in the existence or lack of a cDNA sequence of Leishmania, cause eye catching and considerable significant alter in the UV–vis. The obtained low limit of quantification (LLOQ) of was achieved as 1ZM. Based on experimental results in optimum conditions, quick bioanalysis of Leishmania spp sequence was performed (2 min). So, this probe can be used for the clinical diagnosis of this pathogen and infection disease.

Growth factors (GFs) are proteins secreted by cells that regulate a variety of biological processes. Although they have long been proposed as potent therapeutic agents, their administration in a soluble form has proven costly and ineffective due to their short half‐lives in biological environments. Biomaterial‐based approaches are increasingly sought as alternatives to improve the efficacy or, ideally, replace the need for exogenous administration of GFs in regenerative medicine strategies. The means by which these systems evolve from biomaterials for conventional controlled release of GFs to the recent extracellular matrix (ECM)–inspired approaches for sequestering these labile molecules and regulating their spatiotemporal activity and presentation are reviewed. Focus is placed on biomaterials functionalized either with ECM components, which show promiscuous GF binding, or with targeted GF ligands (antibodies, aptamers, or peptides). The potential of synthetic platforms with abiotic affinity as cost‐effective alternatives to the current biological ligands is also discussed. Overall, the various GF sequestering systems developed so far have remarkably improved the activity of GFs at reduced doses and, in some cases, completely avoided the need for their exogenous administration to guide cell fates. These bioinspired concepts thus enable the rational exploration of the full therapeutic potential of GFs in regenerative medicine. [ABSTRACT FROM AUTHOR]

Immobilization of enzymes provides many benefits, including facile separation and recovery of enzymes from reaction mixtures, enhanced stability, and co‐localization of multiple enzymes. Calcium‐phosphate‐protein supraparticles imbued with a leucine zipper binding domain (ZR) serve as a modular immobilization platform for enzymes fused to the complementary leucine zipper domain (ZE). The zippers provide high‐affinity, specific binding, separating enzymatic activity from the binding event. Using fluorescent model proteins (mCherryZE and eGFPZE), an amine dehydrogenase (AmDHZE), and a formate dehydrogenase (FDHZE), the efficacy of supraparticles as a biocatalytic solid support was assessed. Supraparticles demonstrated several benefits as an immobilization support, including predictable loading of multiple proteins, structural integrity in a panel of solvents, and the ability to elute and reload proteins without damaging the support. The dual‐enzyme reaction successfully converted ketone to amine on supraparticles, highlighting the efficacy of this system. [ABSTRACT FROM AUTHOR]

Cadmium ion (Cd2+) is a highly toxic heavy metal ion that threatens the environment and human health. To achieve rapid and sensitive detection of Cd2+, here we developed a reagent-less aptamer electrochemical sensor by immobilizing an engineered high-affinity DNA aptamer with a redox tag of methylene blue (MB) on the gold electrode. After testing a series of engineered aptamer sequences, we employed an optimal and new 15-mer aptamer with a short 3-bp stem for sensor fabrication, which underwent large conformation change upon Cd2+ binding. This aptamer retained high affinity with a K d about 360 nM, verified by isothermal titration calorimetry (ITC) analysis. In the presence of Cd2+, this aptamer folded into a stem-loop structure, drawing the MB into a close proximity to the electrode surface and generating enhanced current in square wave voltammetry (SWV). Under the optimized conditions, this aptamer sensor enabled us to sensitively detect Cd2+ in a wide concentration range from 0.5 nM to 4 μM, and the detection limit was 90 pM. The developed electrochemical aptasensor has the advantages in easy preparation, rapid response, high stability, high selectivity and easy regeneration and reuse, showing the potential for Cd2+ detection in broad applications. [Display omitted] • Sensitive electrochemical sensor for Cd2+ using new high-affinity 15-mer DNA aptamer. • Cd2+ binding induced large aptamer conformation change and great current increase. • Single base pair mutation in stem greatly improves electrochemical responses. • Showing rapid response, high stability, high selectivity and easy regeneration. [ABSTRACT FROM AUTHOR]

Spatio-temporal presentation of growth factors is one of the key attributes of the cell's microenvironment. The design of macroporous alginate scaffolds, wherein TGF-β1 or BMP-4 is electrostatically bound to affinity binding sites of alginate sulfate, mimicking their presentation by the extracellular matrix (ECM), was previously shown to enable sustained presentation and release of each factor, thus increasing their biological activity. Specifically, TGF-β1/affinity-bound scaffolds induced the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded within these scaffolds. The prolonged activity of the affinity-bound TGF-β1 enabled efficient induction of signaling pathways leading to chondrogenesis, up to the appearance of committed chondrocytes. Similarly, BMP-4 affinity-bound to the macroporous alginate scaffold enabled efficient induction of osteogenic differentiation in hMSC constructs. Subsequent construction of a multicompartment inductive system, spatially-presenting TGF-β1 and BMP-4 in two distinct layers, enabled complete differentiation of hMSC to chondrocytes and osteoblasts, depending on the type of factor in use in the respective layer. This paper describes in detail the preparation method of the TGF-β1 or BMP4/ affinity-bound alginate scaffolds, and the set of analyses performed to characterize the resultant scaffolds, including release profile study, released factor bioactivity, and functionality of the scaffolds as hMSC-inductive scaffolds.

Fusion tags – amino acid sequences that are genetically coded to be expressed as attached moieties to a protein – have the potential to enhance the activity of native enzyme, enable specific purification of the enzyme, and promote simple and efficient immobilization of enzymes onto material supports. In this work, we demonstrate the effect of a Strep-tag II fusion tag on the properties of free and immobilized lipase B from Candida antarctica (CALB). The gene encoding the mature portion of CALB was codon-optimized and cloned in pASG-IBA2 plasmid for expression in E. coli. Purified recombinant Strep-tag II CALB was immobilized to Strep-Tactin based support through affinity binding, and the immobilized and free Strep-tag II CALB were compared to a commercial CALB. Following modification, the enzyme could be selectively purified from culture media with no observable non-specific binding. The catalytic efficiency of the purified fusion-tagged enzyme was significantly greater than that of the commercial CALB in its free form. Immobilization of the fusion-tagged enzyme to Strep-Tactin modified crosslinked agarose support yielded a catalytically active enzyme; however, the kcat of the immobilized enzyme was significantly reduced compared to the free tagged enzyme. This work indicates that a C-terminus Strep-tag II fusion tag may be employed to improve the catalytic efficiency of free CALB, but may not be suitable for immobilized applications that employ binding of the enzyme to a Strep-Tactin-modified support.

We described an aptamer based and Mg2+ mediated free zone capillary electrophoresis-laser induced fluorescence (CE-LIF) assay for aflatoxin B1 (AFB1) detection. This CE-LIF assay applied an anti-AFB1 aptamer with a single fluorescein (FAM) label at 5′ end and a short complementary DNA (cDNA). In the absence of AFB1, the cDNA hybridized with the aptamer probe and formed a duplex DNA. The use of running buffer containing MgCl 2 allowed good isolation of the duplex DNA from the single stranded DNA in CE. We found introducing a biotin label on the cDNA further improved the isolation. When AFB1 existed in sample solution, the aptamer probe bound with AFB1, dissociating from the duplex DNA. Thus, the duplex DNA peak decreased, while the aptamer probe peak increased during CE-LIF analysis. We achieved detection of AFB1 by measuring the aptamer probe peak. The length of cDNA, the ratio of aptamer to cDNA, and the concentration of MgCl 2 in sample buffer and separation buffer had great effect on the aptamer based CE-LIF assay. Under optimized conditions, the detection limit of AFB1 was 0.2 nM, and the dynamic range was from 0.2 nM to 500 nM. Limit of quantitation was 0.5 nM. This CE-LIF assay enabled detection of AFB1 spiked in diluted human serum, diluted human urine, and corn flour samples. This assay exhibits potential for wide application as it integrates the rapidity, high sensitivity, low sample consumption of CE-LIF analysis and the strengths of aptamer. An aptamer free zone capillary electrophoresis coupled with laser induced fluorescence assay (CE-LIF) was developed for detection of aflatoxin B1 (AFB1). AFB1 competes with a short complementary DNA (cDNA) in the binding to a fluorescein (FAM)-labeled aptamer probe. The duplex DNA is well isolated from the FAM-labeled aptamer in CE with separation buffer containing MgCl 2. The presence of AFB1 caused decrease of the aptamer-cDNA duplex peak and increase of aptamer probe peak during CE-LIF analysis. Image 1 • Aptamer free zone capillary electrophoresis (CE) assay for aflatoxin B1 (AFB1). • Complementary DNA (cDNA) and fluorescein-labeled aptamer probe are used. • MgCl 2 mediated CE allows separation of aptamer probe and aptamer-cDNA duplex. • AFB1 binding causes decrease of duplex peak and increase of aptamer probe peak. • AFB1 is detected by peaks changes in CE coupled with laser induced fluorescence. [ABSTRACT FROM AUTHOR]

Highlights • Novel macroporous polymer supports were developed and tested for protein microarray. • Effect of porous characteristics of matrix on analytical efficiency was established. • The developed biochips are suitable for the quantitative study of binding properties. • Novel method for the detection of acetylcholinesterase was suggested. • Developed biochips can be used for the determination of enzyme activity on a chip. Abstract A series of rigid macroporous polymer layers differed by hydrophobic–hydrophilic properties was synthesized in situ in preliminary fabricated wells and applied as the platforms for protein biochips. Scanning electron microscopy, etalon porosimetry and BET analysis were used for materials characterization. The comparison of analytical efficiency of the developed platforms allowed for the choice of the most optimal polymer, as well as the evaluation of impact of material porous properties. The quantitative parameters of affinity interaction between two different protein pairs were calculated depending on biochip characteristics using the developed analytical protocol. Moreover, the described biochips were successfully tested to detect acetylcholinesterase via catalytic reaction followed by the formation of fluoresceine as a product. Different parameters of enzymatic reaction were calculated for the reaction on a chip and compared to those established for in solution process. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • E. coli O157:H7 biofilm affinity peptide SGVYKVAYDWQH was obtained by biopanning. • Designed affinity surfactant peptide SGVYKVAYDWQHKKK formed micelle effectively. • Biofilm removal efficiency reached 75% at 125 mg/L affinity surfactant peptide. • Designed peptide adsorbed on biofilm before removing and surrounding the bacteria. • Different constituents of biofilm associated with binding and removal efficiency. Abstract Conventional chemical surfactants attach on blots randomly, accompanied with health and environmental issues. To address this, a surfactant peptide was designed to mimic chemical surfactants with an affinity binding peptide as a hydrophobic tail for the cleanup of biofilm contaminations. The micelle forming and structural changes of the peptide in aqueous solution were systematically investigated. More importantly, the biofilm removal efficiency toward Escherichia coli O157:H7 biofilm reached 75% in neutral aqueous solutions at the concentration of 125 mg/L (critical micelle concentration 91 mg/L), a significant improvement in comparison to conventional surfactants and random surfactant peptide. The dynamic removal process reported by confocal laser scanning microscope (CLSM) also displayed the different constituents of biofilm blots, which associated with surfactant peptide binding efficiency. Hopefully, this surfactant strategy will eventually provide new scopes in the design of surface active biological agents. [ABSTRACT FROM AUTHOR]

Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins and draws great concern in health and food safety. A DNA aptamer against AFB1 having a stem-loop structure shows high binding affinity to AFB1 and promise in assay development for AFB1 detection. Based on the structure-switching property of the aptamer, we report an aptamer fluorescence assay for AFB1 detection. Aptamer with fluorescein (FAM) label at 5′ end was used as affinity ligand, while its short complementary DNA (cDNA) with BHQ1 (black hole quencher 1) label at 3′ end was used as a quencher. In the absence of AFB1, FAM-labeled aptamer hybridized with BHQ1-labeled cDNA, forming a duplex of cDNA and aptamer, resulting in fluorescence quenching of FAM. When AFB1 bound with aptamer, the BHQ1-labeled cDNA was displaced from aptamer, causing fluorescence restoration of FAM. We tested a series of FAM-labeled aptamers and BHQ1-labeled cDNAs with different lengths. The lengths of the aptamer stem and the cDNA, Mg2+ in binding buffer, and temperature had significant influence on the performance of the assay. Under optimized conditions, we achieved sensitive detection of AFB1 by using a 29-mer FAM-labeled aptamer and a 14-mer BHQ1-labeled cDNA, and the detection limit of AFB1 reached 0.2 nM. The maximum fluorescence recovery rate of FAM-labeled aptamer caused by AFB1 was about 69-fold. This method enabled the detection of AFB1 in complex sample matrix, e.g., diluted wine samples and maize flour samples. This aptamer-based fluorescent assay for AFB1 determination shows potential for broad applications.ᅟ [ABSTRACT FROM AUTHOR]

The introduction of aptameric ligands onto disk-monolithic adsorbent, representing a unique strategy for convective isolation of target molecules with high specificity and selectivity, is investigated for the first time. Experimental results showed that the disk monolith possessed a good permeability of 1.67 ± 0.05 × 10-14 m2 (RSD = 3.2%). The aptameric ligand density for the aptamer-modified disk monolith was 480 pmol/uL. Chromatographic analysis of the aptamer disk-monolith efficiency showed an optimum linear velocity of 126 cm/min (≈0.25 mL/min) at room temperatures 25 ± 2°C. The theoretical number of plates corresponding to the optimum linear velocity was 128.2 with an height equivalent to the theoretical plate of 0.022 mm. The disk aptamer-immobilised monolithic system demonstrated good selectivity and isolation of thrombin from non-targets. [ABSTRACT FROM AUTHOR]

In this study, poly- l -lysine (PLL) immobilized PHEMA cryogel was designed for the specific bilirubin removal from human plasma. The surface of PHEMA cryogels is surrounded by PLL chains to enhance specific binding of bilirubin molecules via specific complementary electrostatic interactions. The functionalization of the PHEMA cryogel was carried out by direct coupling of PLL to pre-activated OH-group of the HEMA alcohol units via carbodiimide activation. Prior to removal of bilirubin from human plasma, the optimization parameters including, initial bilirubin concentration, flow rate, temperature, ionic strength, and adsorption rates on adsorption of PLL-PHEMA cryogel were investigated from the aqueous medium. According to the elemental analyses results, the incorporation of PLL was 690.0 μmol/g cryogel. The cryogel has highly interconnected supermacroporous structure sized between 20 and 100 μm with a positive surface charge value. The maximum bilirubin adsorption capacity was found as 59.9 mg/g of the dry weight of PLL-PHEMA cryogel. Reusability study explored that PLL-PHEMA could be used with a negligible loss in the bilirubin adsorption capacity after successive ten adsorption-desorption cycles using the same adsorbent. The results of the study demonstrated that the PLL-PHEMA cryogel exhibited high specificity that can be used as an efficient column for removing the bilirubin from the human plasma. [ABSTRACT FROM AUTHOR]

Antibody-coated surfaces (Ab surfaces) play a key role in bioanalytical tools developed for biosensors and diagnostics. Therefore, a high and well-defined functional activity of the Ab surface is required. The functional activity of the Ab surface depends on its available binding sites i.e. “the active sites” that are able to capture antigen (Ag). The number of active binding sites strongly depends on the immobilization strategy used to fix the Ab on the solid surface. Determination of layer thickness or surface topology are often used to characterize the Ab surfaces but there is no gold standard method for the study of the functionality of the Ab surfaces. For that purpose, we aim at developing an assay allowing to determine the performances of Ab surfaces. In the present study, anti-horseradish peroxidase antibody (anti-HRP Ab) is used as capture Ab covalently bound to the surface and enzyme HRP as Ag. This direct assay permits, in one-step, to generate a signal utilizing the catalytic properties of HRP. The signal is directly proportional to the amount of HRP bound on the Ab surface, and therefore to the active binding sites of immobilized Ab. The HRP/anti-HRP Ab interactions may be a useful indicator to construct accurate and reproducible active Ab surfaces and also to improve their performances in term of stability and sensitivity. Optimization of the assay parameters and quality of the results are presented. A good repeatability and an acceptable inter-day precision (RSD < 10%) are reported. [ABSTRACT FROM AUTHOR]

Fusion tags – amino acid sequences that are genetically coded to be expressed as attached moieties to a protein – have the potential to enhance the activity of native enzyme, enable specific purification of the enzyme, and promote simple and efficient immobilization of enzymes onto material supports. In this work, we demonstrate the effect of a Strep-tag II fusion tag on the properties of free and immobilized lipase B from Candida antarctica (CALB). The gene encoding the mature portion of CALB was codon-optimized and cloned in pASG-IBA2 plasmid for expression in E. coli . Purified recombinant Strep-tag II CALB was immobilized to Strep-Tactin based support through affinity binding, and the immobilized and free Strep-tag II CALB were compared to a commercial CALB. Following modification, the enzyme could be selectively purified from culture media with no observable non-specific binding. The catalytic efficiency of the purified fusion-tagged enzyme was significantly greater than that of the commercial CALB in its free form. Immobilization of the fusion-tagged enzyme to Strep-Tactin modified crosslinked agarose support yielded a catalytically active enzyme; however, the k cat of the immobilized enzyme was significantly reduced compared to the free tagged enzyme. This work indicates that a C-terminus Strep-tag II fusion tag may be employed to improve the catalytic efficiency of free CALB, but may not be suitable for immobilized applications that employ binding of the enzyme to a Strep-Tactin-modified support. [ABSTRACT FROM AUTHOR]

As a multifunctional serine protease, human neutrophil elastase (HNE) plays critical roles in a variety of physiopathological processes, such as acute lung injury, emphysema, atherosclerosis, and arthritis. The quantification of HNE is important in many applications. In this paper, we report an aptamer affinity capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) assay for detection of HNE using a tetramethylrhodamine (TMR)-labeled DNA aptamer probe. The affinity complex of HNE and DNA aptamer probe was well separated from the unbound aptamer probe in CE separation based on the difference of electrophoretic mobility. Broad complex peaks appeared due to possible multiple binding. The 45-mer aptamer having TMR labeling on the 40th T base was used as affinity probe, as larger complex peaks were obtained. We investigated the effects of various metal cations (Na, K, and Mg) in sample buffer on the binding of HNE and the aptamer in CE-LIF analysis. The presence of Na, K, or Mg in sample buffer caused a decrease of complex peaks, and Mg showed a larger effect. Under optimized conditions, this aptamer CE-LIF assay enabled the detection of HNE at 0.5 nM. This assay showed good specificity and allowed for detection of HNE spiked in diluted human serum sample. [ABSTRACT FROM AUTHOR]

The authors describe a surface plasmon resonance (SPR) based aptasensor for the carcinogenic mycotoxin aflatoxin B1 (AFB1) in a direct assay format. The aptamer is immobilized on the surface of a commercial sensor chip, and the SPR signal increases on binding of AFB1. The sensor chip can be fully regenerated by passing a flow of buffer over it upon which bound AFB1 dissociates from the aptamer. The biosensor works in the 0.4 nM to 200 nM AFB1 concentration range and has a 0.4 nM detection limit. It allows AFB1 to be determined in complex samples such as diluted red wine and beer. The assay is sensitive, and the chip is easily regenerated and stable. The method therefore overcomes certain limitations of antibody-based SPR assays and of competitive SPR assays for AFB1. [ABSTRACT FROM AUTHOR]

Integration of gallic acid (GA) and its derivative epigallocatechin gallate (EGCG; 20, 120, and 240 μmol/g, protein basis) into whey protein isolate (WPI) at room temperature and pH 3.0 and pH 7.0 was investigated. At pH 7.0, both phenolics caused significant structural changes and EGCG induced greater digestibility of WPI. Total sulfhydryl in WPI decreased from 28.6 to 7.6 μmol/g and surface hydrophobicity declined by nearly 50% with 240 μmol/g EGCG at pH 7.0. Similar but less appreciable changes were produced by GA and at pH 3.0. Isothermal titration and fluorescence quenching tests showed moderately weak binding of WPI with GA but strong binding with EGCG at pH 7.0. Both phenolics at high concentrations lowered the thermal transition temperature of β-lactoglobulin by 0.5 °C to 1.4 °C and promoted its digestion. The phenolics also displayed a remarkable synergism with peptides in the WPI digest exerting radical scavenging activity. [ABSTRACT FROM AUTHOR]

Aptamer affinity capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) combines the advantages of affinity aptamer, rapid CE separation, and high sensitivity detection. Here we reported an affinity CE-LIF assay for thrombin by using a fluorophore-labeled RNA aptamer containing 2′-fluoro modification in sugar rings of pyrimidine nucleotides (C and U) as affinity ligand. This RNA aptamer has high binding affinity, specificity and biostability. Thrombin at 0.2 nM was successfully detected. This RNA aptamer allowed for the detection of thrombin spiked in diluted human serum sample due to the nuclease resistance. The RNA aptamer has comparable binding affinity to a 29-mer DNA aptamer for thrombin, and the binding site of the RNA aptamer on thrombin partially overlaps with the binding site of the 29-mer DNA aptamer on thrombin. It shows the nuclease-resistant RNA aptamers are promising in assays for thrombin. [ABSTRACT FROM AUTHOR]

The authors describe a competitive aptamer based assay for detection of the platelet-derived growth factor BB (PDGF-BB; used as a model protein). The assay is making use of thrombin (a serine protease) as an enzyme label for reporting signals. It is taking advantage of a highly selective aptamer and of the fairly specific enzymatic activity of thrombin in terms of cleaving artificial fluorogenic peptide substrates. In a first step, the surface of wells of microplates is coated with PDGF-BB. On addition of a sample containing PDGF-BB, free and bound PDGF-BB compete with each other for binding to a DNA probe that consists of an aptamer sequence for PDGF-BB and a 29-mer aptamer sequence for thrombin. After washing, thrombin is added and will attach to the DNA probe that bound to the PDGF-BB on the microplates. Following addition of a fluorogenic peptide substrate, the bound thrombin will catalyze the cleavage of the substrate to generate a fluorescent product whose fluorescence intensity is measured at excitation/emission wavelengths of 370/440 nm. Fluorescence intensity decreases with increasing PDGF-BB concentration in the sample because less thrombin will bind to the PDGF-BB coated surface of the microplate. Under optimal conditions, PDGF-BB can be quantified in the 0.125 to 3 nM concentration range. This assay was successfully applied to the determination of PDGF-BB in spiked 100-fold diluted human serum. [ABSTRACT FROM AUTHOR]

Herein, we present a fast and sensitive biosensor for detection of Ochratoxin A (OTA) in a red wine that utilizes gold nanoparticle-enhanced surface plasmon resonance (SPR). By combining an indirect competitive inhibition immunoassay and signal enhancement by secondary antibodies conjugated with gold nanoparticles (AuNPs), highly sensitive detection of low molecular weight compounds (such as OTA) was achieved. The reported biosensor allowed for OTA detection at concentrations as low as 0.75 ng mL −1 and its limit of detection was improved by more than one order of magnitude to 0.068 ng mL −1 by applying AuNPs as a signal enhancer. The study investigates the interplay of size of AuNPs and affinity of recognition elements affecting the efficiency of the signal amplification strategy based on AuNP. Furthermore, we observed that the presence of polyphenolic compounds in wine samples strongly interferes with the affinity binding on the surface. To overcome this limitation, a simple pre-treatment of the wine sample with the binding agent poly(vinylpyrrolidone) (PVP) was successfully applied. [ABSTRACT FROM AUTHOR]

Here we describe a thrombin-linked aptamer assay (TLAA) for protein by using thrombin as an enzyme label, harnessing enzyme activity of thrombin and aptamer affinity binding. TLAA converts detection of specific target proteins to the detection of thrombin by using a DNA sequence that consists of two aptamers with the first aptamer binding to the specific target protein and the second aptamer binding to thrombin. Through the affinity binding, the thrombin enzyme is labeled on the protein target, and thrombin catalyzes the hydrolysis of small peptide substrate into product, generating signals for quantification. As a proof of principle, we show a sandwich TLAA for platelet derived growth factor BB (PDGF-BB) by using anti-PDGF-BB antibody coated on magnetic beads and an oligonucleotide containing the aptamer for PDGF-BB and the aptamer for thrombin. The binding of PDGF-BB to both the antibody and the aptamer results in labeling the complex with thrombin. We achieved detection of PDGF-BB at 16 pM. This TLAA contributes a new application of thrombin and its aptamer in bioanalysis, and shows potentials in assay developments. Graphical abstractfx1Highlights· Thrombin is used as an enzyme label in aptamer-based assay for protein detection. · A DNA probe contains dual aptamer sequences for binding with thrombin and target. · Thrombin is labeled on protein target through aptamer affinity binding. · Thrombin catalyzes cleavage of a substrate to a product for signal generation. · PDGF-BB is detected by the thrombin-linked aptamer assay on magnetic beads. [ABSTRACT FROM AUTHOR]

The synergistic effects of the polysaccharides chitosan (CS) and hyaluronic acid (HA) formulated into hybrid nanoparticles are promising for drug delivery. In the present work, we performed a detailed analysis of the molecular interactions involved in the TPP-assisted ionotropic gelation of CS hybrid nanoparticles with the objective of investigating the impact of HA inclusion on the particle formulation and on the in vitro release of insulin (INS) as a protein cargo. To do that, an in-depth thermodynamic study was carried out by isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC) techniques. Such analysis allowed us to elucidate the type and extent of interactions established by INS within the hybrid nanoparticles and to get further knowledge on the nature of its release mechanism in vitro. Overall, INS release from the CS nanoparticles was thermodynamically driven, and when including HA a weaker INS binding to the nanoparticles, hence, a faster release rate in vitro were observed. As a negative polyelectrolyte, HA might have sterically blocked the activated sites of CS, such as the amino groups, through chain entanglement, thereby, attenuating the competitive binding interactions of INS. As a consequence, INS might have experienced a spatial exclusion onto the surface of the hybrid nanoparticles to a greater extent which, in turn, would explain its initial abrupt release. [ABSTRACT FROM AUTHOR]

Controlled delivery of heparin-binding (HB) proteins represents a challenge in regenerative medicine strategies. Here, we describe the features of novel nanoparticles (NPs), spontaneously coassembled due to affinity interactions between HB proteins and the semisynthetic anionic polysaccharide, alginate-sulfate. The NPs efficiently encapsulated and protected the proteins from proteolysis. Injection of a combination of NPs encapsulating multiple therapeutic growth factors promoted effective and long-term tissue repair in animal models of severe ischemia (murine model of hindlimb ischemia and acute myocardial infarction in rats). This simple yet efficient NP fabrication method is amenable for clinical use. [ABSTRACT FROM AUTHOR]

A series of highly charged nanoscale chelators were fabricated by grafting of poly(glycidyl methacrylate-iminodiacetic acid) (pGI) chains with iminodiacetic acid (IDA) chelating group on silica nanoparticles (SNPs) via atom transfer radical polymerization (ATRP). The nanoscale chelators, denoted as SNPs-pGI, possessed a nickel ion chelating capacity as high as 2800 μmol/g, 50 times higher than the IDA-modified Sepharose FF (IDA-Sepharose) resin reported in literature and offered a high affinity binding capacity for hexahistidine-tagged enhanced green fluorescence protein (6 × His-EGFP) after nickel ion loading. More importantly, the anionic SNPs-pGI of high charge densities displayed much better performance than IDA-Sepharose in facilitating the refolding of like-charged 6 × His-EGFP from inclusion bodies (IBs). For example, for 0.2 mg/mL 6 × His-EGFP IB refolding, addition of 6.2 μL/mL SNPs-pGI with the highest charge density led to a refolding yield of 90%, over 43% higher than that obtained with 460 μL/mL IDA-Sepharose. It is notable that the much higher efficiency of the nanoscale chelator was obtained with a chelator consumption corresponding to only 1.4% of IDA-Sepharose. Moreover, the highly charged SNPs-pGI could efficiently facilitate the refolding of 6 × His-EGFP at higher IB concentrations (0.4 and 0.8 mg/mL). After refolding, nickel ions addition led to the recovery of the refolded 6 × His-EGFP with high yield (80%), purity (96%) and enrichment ratio (1.8). All the results suggest that the SNPs-pGI of high charge densities were promising for cost-effective recovery of His-tagged proteins expressed as IBs with the integrative like-charge facilitated refolding and metal-chelate affinity purification strategy. [ABSTRACT FROM AUTHOR]

Alginate biomaterial is widely utilized for tissue engineering and regeneration due to its biocompatibility, non-thrombogenic nature, mild and physical gelation process, and the resemblance of its hydrogel matrix texture and stiffness to that of the extracellular matrix. In this review, we describe the versatile biomedical applications of alginate, from its use as a supporting cardiac implant in patients after acute myocardial infarction (MI) to its employment as a vehicle for stem cell delivery and for the controlled delivery and presentation of multiple combinations of bioactive molecules and regenerative factors into the heart. Preclinical and first-in-man clinical trials are described in details, showing the therapeutic potential of injectable acellular alginate implants to inhibit the damaging processes after MI, leading to myocardial repair and tissue reconstruction. [ABSTRACT FROM AUTHOR]

Biomolecule-nanoparticle hybrid bioconjugates based on bioscaffolds such as protein cages and virus capsules have been widely studied. Highly stable and durable biotemplates are a vital pillar in constructing bio-inorganic functional hybrid composites. Here, we introduce a highly heat-resistant coat protein (CP) of Sulfolobus tengchongensis spindle-shaped virus 1 (STSV1) isolated from the hyperthermophilic archaeon as a prospective biological matrix. Our experiments showed that STSV1 CP was successfully cloned and solubly expressed in the Escherichia coli Rosetta-(DE3) host strain. Protein expression was verified by SDS-PAGE and western blot analysis of the reference C-terminally six-histidine (His6) tagged STSV1 CP (HT-CP). Thermal stability experiments showed that the STSV1 coat protein remained fairly stable at 80 °C. The proteins can be purified facilely by heat treatment followed by size exclusion chromatography (SEC). Transmission electron microscopy (TEM) analysis of the purified STSV1 CP protein aggregates demonstrated that the protein could self-assemble into rotavirus-like nanostructures devoid of genetic materials under our experimental conditions. Similar results were obtained for the HT-CP purified by heat treatment followed by Ni-NTA and SEC, indicating that moderately engineered STSV1 CP can retain its self-assembly property. In addition, the STSV1 CP has a high binding affinity for TiO nanoparticles. This illustrates that the STSV1 CP can be used as a bioscaffold in nanobiotechnological applications. [ABSTRACT FROM AUTHOR]

Abstract: Boronic acids are important for effective separation of biological active cis-diols. For the purpose of constructing a new type of saccharide-sensitive material which can not only provide convenient separation but also improve the access of boronic acid to guest molecules, the fluorogenic boronic acid terminated, thermo-sensitive polymers (BA-polyNIPAm) were grafted to an alkyne modified silica gel through the exploitation of click chemistry. The BA-polyNIPAm grafted silica gel (BA-polyNIPAm-SG) was characterized by FT-IR, fluorescence spectra, fluorescence microscopy, elemental analysis (EA), thermal gravimetric analysis (TGA), scanning electron microscope (SEM) and so on. BA-polyNIPAm-SG displayed affinity binding ability for saccharides under physiological pH value and allowed saccharides to be conveniently separated from solution. The maximum binding capacities for fructose and glucose are 83.2μmol/g and 70.4μmol/g polymer, respectively. The intensity of fluorescence emission of BA-polyNIPAm-SG increased with the increasing of fructose concentration. The present study provides a new kind of composite material which contains moveable and flexible grippers for recognizing and binding guest molecules. [Copyright &y& Elsevier]

The commonly used trypan blue dye exclusion method and other modified cell viability methods, such as fluorescein dye and tetrazolium dye exclusion, artificially introduce toxic chemicals to cells and, thus, alter cellular organelles when measuring cell viability. Therefore, cell viability could be affected by the processes currently used to observe viability. In this study, the cell viability of Chinese hamster ovary (CHO) cells was measured by simply counting attached cells after the cultured CHO cells were attached on a Concanavalin A (Con A) substrate. The efficiency of cell attachment to Con A surfaces was different for live and dead cells allowing the cell viability of CHO cells to be measured without any chemical modifications to the cells. [ABSTRACT FROM AUTHOR]