Multiple Biomolecules Research Articles

Triple Orthogonal Labeling of Glycans by Applying Photoclick Chemistry.

Bioorthogonal labeling of multiple biomolecules is of current interest in chemical biology. Metabolic glycoengineering (MGE) has been shown to be an appropriate approach to visualizing carbohydrates. Here, we report that the nitrile imine-alkene cycloaddition (photoclick reaction) is a suitable ligation reaction in MGE. Using a mannosamine derivative with an acrylamide reporter group that is efficiently metabolized by cells and that quickly reacts in the photoclick reaction, we labeled sialic acids on the surface of living cells. Screening of several alkenes showed that a previously reported carbamate-linked methylcyclopropene reporter that is well suited for the inverse-electron-demand Diels-Alder (DAinv ) reaction has a surprisingly low reactivity in the photoclick reaction. Thus, for the first time, we were able to triply label glycans by a combination of DAinv , photoclick, and copper-free click chemistry.

Detection of Various Biomarkers and Enzymes via a Nanocluster-Based Fluorescence Turn-on Sensing Platform.

The profiling of disease-related biomarkers is an essential procedure for the accurate diagnosis and intervention of metabolic disorders. Therefore, the development of ultrasensitive and highly selective fluorogenic biosensors for diverse biomarkers is extremely desirable. There is still a considerable challenge to prepare nanocluster-based fluorescence turn-on probes capable of recognizing multiple biomolecules. We herein provide a novel nanocluster-based chemical information processing system (CIPS) for the programmable detection of various metabolites and enzymes. This CIPS consists of biocatalytic reactions between substrates and their respective oxidases to generate H2O2, which was then employed to synthesize bright fluorescent silver nanoclusters (Ag NCs). Utilizing this system, we are able to accurately probe a series of substrates/corresponding oxidases with high sensitivity and specificity, including glucose/glucose oxidase, uric acid/uric acid oxidase, sarcosine/sarcosine oxidase, choline/choline oxidase, xanthine/xanthine oxidase, and lactic acid/lactic acid oxidase. Furthermore, this metabolite profiling CIPS can be integrated with binary logic operations, which create an intelligent tool for the high-throughput screening of various diseases in vitro (e.g., diabetes, gout, prostate cancer, cardiovascular disease, and lactic acidosis).

Coordination-Assisted Bioorthogonal Chemistry: Orthogonal Tetrazine Ligation with Vinylboronic Acid and a Strained Alkene.

Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality that might be present. Recent developments in the field include orthogonal bioorthogonal reactions to modify multiple biomolecules simultaneously. During our research, we observed that the reaction rates for the bioorthogonal inverse-electron-demand Diels-Alder (iEDDA) reactions between nonstrained vinylboronic acids (VBAs) and dipyridyl-s-tetrazines were exceptionally higher than those between VBAs and tetrazines bearing a methyl or phenyl substituent. As VBAs are mild Lewis acids, we hypothesised that coordination of the pyridyl nitrogen atom to the boronic acid promoted tetrazine ligation. Herein, we explore the molecular basis and scope of VBA-tetrazine ligation in more detail and benefit from its unique reactivity in the simultaneous orthogonal tetrazine labelling of two proteins modified with VBA and norbornene, a widely used strained alkene. We further show that the two orthogonal iEDDA reactions can be performed in living cells by labelling the proteasome by using a nonselective probe equipped with a VBA and a subunit-selective VBA bearing a norbornene moiety.

Simultaneous biosynthesis of (R)-acetoin and ethylene glycol from D-xylose through in vitro metabolic engineering

(R)-acetoin is a four-carbon platform compound used as the precursor for synthesizing novel optically active materials. Ethylene glycol (EG) is a large-volume two-carbon commodity chemical used as the anti-freezing agent and building-block molecule for various polymers. Currently established microbial fermentation processes for converting monosaccharides to either (R)-acetoin or EG are plagued by the formation of undesirable by-products. We show here that a cell-free bioreaction scheme can generate enantiomerically pure acetoin and EG as co-products from biomass-derived D-xylose. The seven-step, ATP-free system included in situ cofactor regeneration and recruited enzymes from Escherichia coli W3110, Bacillus subtilis shaijiu 32 and Caulobacter crescentus CB 2. Optimized in vitro biocatalytic conditions generated 3.2 mM (R)-acetoin with stereoisomeric purity of 99.5% from 10 mM D-xylose at 30 °C and pH 7.5 after 24 h, with an initial (R)-acetoin productivity of 1.0 mM/h. Concomitantly, EG was produced at 5.5 mM, with an initial productivity of 1.7 mM/h. This in vitro biocatalytic platform illustrates the potential for production of multiple value-added biomolecules from biomass-based sugars with no ATP requirement.

Incorporating β-cyclodextrin into collagen scaffolds to sequester growth factors and modulate mesenchymal stem cell activity

The development of biomaterials for a range of tissue engineering applications increasingly requires control over the bioavailability of biomolecular cues such as growth factors in order to promote desired cell responses. While efforts have predominantly concentrated on covalently-bound or freely-diffusible incorporation of biomolecules in porous, three-dimensional biomaterials, opportunities exist to exploit transient interactions to concentrate growth factor activity over desired time frames. Here, we report the incorporation of β-cyclodextrin into a model collagen-GAG scaffold as a means to exploit the passive sequestration and release of growth factors via guest-host interactions to control mesenchymal stem cell differentiation. Collagen-GAG scaffolds that incorporate β-cyclodextrin show improved sequestration as well as extended retention and release of TGF-β1. We further show extended retention and release of TGF-β1 and BMP-2 from β-cyclodextrin modified scaffolds was sufficient to influence the metabolic activity and proliferation of mesenchymal stem cells as well as differential activation of Smad 2/3 and Smad 1/5/8 pathways associated with differential osteo-chondral differentiation. Further, gene expression analysis showed TGF-β1 release from β-cyclodextrin CG scaffolds promoted early chondrogenic-specific differentiation. Ultimately, this work establishes a novel method for the incorporation and display of growth factors within CG scaffolds via supramolecular interactions. Such a design framework offers opportunities to selectively alter the bioavailability of multiple biomolecules within a three-dimensional collagen-GAG scaffold to enhance cell activity for a range of musculoskeletal regenerative medicine applications. Statement of SignificanceWe describe the incorporation of β-cyclodextrin into a model CG-scaffold under development for musculoskeletal tissue engineering applications. We show β-cyclodextrin modified scaffolds promote the sequestration of soluble TGF-β1 and BMP-2 via guest-host interactions, leading to extended retention and release. Further, β-cyclodextrin modified CG scaffolds promote TGF-β1 or BMP-2 specific Smad signaling pathway activation associated with divergent osseous versus chondrogenic differentiation pathways in mesenchymal stem cells.

Resolving molecule-specific information in dynamic lipid membrane processes with multi-resonant infrared metasurfaces

A multitude of biological processes are enabled by complex interactions between lipid membranes and proteins. To understand such dynamic processes, it is crucial to differentiate the constituent biomolecular species and track their individual time evolution without invasive labels. Here, we present a label-free mid-infrared biosensor capable of distinguishing multiple analytes in heterogeneous biological samples with high sensitivity. Our technology leverages a multi-resonant metasurface to simultaneously enhance the different vibrational fingerprints of multiple biomolecules. By providing up to 1000-fold near-field intensity enhancement over both amide and methylene bands, our sensor resolves the interactions of lipid membranes with different polypeptides in real time. Significantly, we demonstrate that our label-free chemically specific sensor can analyze peptide-induced neurotransmitter cargo release from synaptic vesicle mimics. Our sensor opens up exciting possibilities for gaining new insights into biological processes such as signaling or transport in basic research as well as provides a valuable toolkit for bioanalytical and pharmaceutical applications.

Autonomous Magnetic Microrobots by Navigating Gates for Multiple Biomolecules Delivery.

The precise delivery of biofunctionalized matters is of great interest from the fundamental and applied viewpoints. In spite of significant progress achieved during the last decade, a parallel and automated isolation and manipulation of rare analyte, and their simultaneous on-chip separation and trapping, still remain challenging. Here, a universal micromagnet junction for self-navigating gates of microrobotic particles to deliver the biomolecules to specific sites using a remote magnetic field is described. In the proposed concept, the nonmagnetic gap between the lithographically defined donor and acceptor micromagnets creates a crucial energy barrier to restrict particle gating. It is shown that by carefully designing the geometry of the junctions, it becomes possible to deliver multiple protein-functionalized carriers in high resolution, as well as MCF-7 and THP-1 cells from the mixture, with high fidelity and trap them in individual apartments. Integration of such junctions with magnetophoretic circuitry elements could lead to novel platforms without retrieving for the synchronous digital manipulation of particles/biomolecules in microfluidic multiplex arrays for next-generation biochips.

Valency-Controlled Framework Nucleic Acid Signal Amplifiers.

Weak ligand-receptor recognition events are often amplified by recruiting multiple regulatory biomolecules to the action site in biological systems. However, signal amplification in in vitro biomimetic systems generally lack the spatiotemporal regulation in vivo. Herein we report a framework nucleic acid (FNA)-programmed strategy to develop valence-controlled signal amplifiers with high modularity for ultrasensitive biosensing. We demonstrated that the FNA-programmed signal amplifiers could recruit nucleic acids, proteins, and inorganic nanoparticles in a stoichiometric manner. The valence-controlled signal amplifier enhanced the quantification ability of electrochemical biosensors, and enabled ultrasensitive detection of tumor-relevant circulating free DNA (cfDNA) with sensitivity enhancement of 3-5 orders of magnitude and improved dynamic range.

Valency‐Controlled Framework Nucleic Acid Signal Amplifiers

AbstractWeak ligand–receptor recognition events are often amplified by recruiting multiple regulatory biomolecules to the action site in biological systems. However, signal amplification in in vitro biomimetic systems generally lack the spatiotemporal regulation in vivo. Herein we report a framework nucleic acid (FNA)‐programmed strategy to develop valence‐controlled signal amplifiers with high modularity for ultrasensitive biosensing. We demonstrated that the FNA‐programmed signal amplifiers could recruit nucleic acids, proteins, and inorganic nanoparticles in a stoichiometric manner. The valence‐controlled signal amplifier enhanced the quantification ability of electrochemical biosensors, and enabled ultrasensitive detection of tumor‐relevant circulating free DNA (cfDNA) with sensitivity enhancement of 3–5 orders of magnitude and improved dynamic range.

Identifying apparent local stable isotope equilibrium in a complex non-equilibrium system.

Although being out of equilibrium, biomolecules in organisms have the potential to approach isotope equilibrium locally because enzymatic reactions are intrinsically reversible. A rigorous approach that can describe isotope distribution among biomolecules and their apparent deviation from equilibrium state is lacking, however. Applying the concept of distance matrix in graph theory, we propose that apparent local isotope equilibrium among a subset of biomolecules can be assessed using an apparent fractionation difference (|Δα|) matrix, in which the differences between the observed isotope composition (δ') and the calculated equilibrium fractionation factor (1000lnβ) can be more rigorously evaluated than by using a previous approach for multiple biomolecules. We tested our |Δα| matrix approach by re-analyzing published data of different amino acids (AAs) in potato and in green alga. Our re-analysis shows that biosynthesis pathways could be the reason for an apparently close-to-equilibrium relationship inside AA families in potato leaves. Different biosynthesis/degradation pathways in tubers may have led to the observed isotope distribution difference between potato leaves and tubers. The analysis of data from green algae does not support the conclusion that AAs are further from equilibrium in glucose-cultured green algae than in the autotrophic ones. Application of the |Δα| matrix can help us to locate potential reversible reactions or reaction networks in a complex system such as a metabolic system. The same approach can be broadly applied to all complex systems that have multiple components, e.g. geochemical or atmospheric systems of early Earth or other planets.

Design and numerical simulation demonstration of multi-functional holographic phase plate for large depth of field single molecular localization microscopy

The development of nanoscale single-molecule localization and tracking technology for multiple bio-molecules in intact cells has important significance for studying the dynamic process in life process. Since most of cells are several microns in depth, but the focal depth of traditional optical microscopes are less than one micron, the limited depth of field is the main drawback of conventional single molecular localization microscopy that prevents observation and tracking of multiple molecules in intact cells. In this paper, based on the wavefront coding technique, a new type of holographic phase plate with high efficiency is proposed and designed to extend the depth of field of single molecular localization microscopy, which combines the distorted multi-value pure-phase grating (DMVPPG) with the double-helix point spread function (DH-PSF). The DMVPPG can be used to realize multiplane imaging of several tens of layers of a sample in a single detection plane. And the DH-PSF is an engineered point spread function which encodes the lateral and axial position with high precision of a molecule in the center of its two lobes and the angle between them respectively. Using the combined holographic phase plate, the molecules in dozens layers of a whole cell can be simultaneously imaged on the same detection plane with DH-PSF. Not only can the axial resolving power be improved, but the imaging depth can also be extended without scanning. Adding such a holographic phase plate to the imaging path, the limited imaging depth problem in single-molecule-localization microscopy can be solved without sacrificing the localization accuracy. The proposed new type of holographic phase plate can also be implemented with a spatial light modulator. In the following numerical simulation experiments, the designed holographic phase plate is composed of 600×600 pixels with a pixel size of 10 μm. The distance between two adjacent focal planes is designed to be 0.5 μm. Such a holographic phase plate is placed on the Fourier transform plane of the detection light path. When an emitter is located on the focal plane, it can be imaged as two lobes without rotation in a center area of the field of view. If an emitter is -6 μm away from the focal plane, the DH-PSF appears in the upper-left area of the field of view. Simulation results demonstrate that a total of 25 sample layers can be simultaneously imaged on the single detection plane and the 12 μm detection range can be achieved, thus proving the feasibility of this method.

Blood derivatives awaken in regenerative medicine strategies to modulate wound healing.

Blood components play key roles in the modulation of the wound healing process and, together with the provisional fibrin matrix ability to selectively bind bioactive molecules and control its spatial-temporal presentation, define the complex microenvironment that characterize this biological process. As a biomimetic approach, the use of blood derivatives in regenerative strategies has awakened as a source of multiple therapeutic biomolecules. Nevertheless, and despite their clinical relevance, blood derivatives have been showing inconsistent therapeutic results due to several factors, including proper control over their delivery mechanisms. Herein, we highlight recent trends on the use biomaterials to protect, sequester and deliver these pools of biomolecules in tissue engineering and regenerative medicine approaches. Particular emphasis is given to strategies that enable to control their spatiotemporal delivery and improve the selectivity of presentation profiles of the biomolecules derived from blood derivatives rich in platelets. Finally, we discussed possible directions for biomaterials design to potentiate the aimed regenerative effects of blood derivatives and achieve efficient therapies.

Controlled Synthesis of Gold Nanoparticles on Fluorescent Nanodiamond via Electron-Beam-Induced Reduction Method for Dual-Modal Optical and Electron Bioimaging

Hybrid nanoparticles are emergent nanomaterials that combine particles with different characteristic properties to enhance their original functions or modulate their original physical or chemical properties for application in catalysis, sensing, and imaging. Fluorescent nanodiamonds (fNDs) have recently become more attractive for bioimaging because of their characteristic physicochemical properties and biocompatibility. Their wide applicability in bioimaging has been utilized in the single-particle tracking of biomolecules, local environmental sensors in cells, and stem cell tracking in tissues. However, the use of fNDs as multiscale spatial mapping probes for multiple biomolecules and cells in optical and electron microscopy techniques has been limited because of their broad fluorescence spectrum and composition of mainly light elements (C, O, H, N, etc.). On the other hand, metal nanoparticles (metal NPs) with unique photonic properties have been employed as functional labeling probes in bioimaging. The...

Electrochemicolor Imaging Using an LSI-Based Device for Multiplexed Cell Assays.

Multiplexed bioimaging systems have triggered the development of effective assays, contributing new biological information. Although electrochemical imaging is beneficial for quantitative analysis in real time, monitoring multiple cell functions is difficult. We have developed a novel electrochemical imaging system, herein, using a large-scale integration (LSI)-based amperometric device for detecting multiple biomolecules simultaneously. This system is designated as an electrochemicolor imaging system in which the current signals from two different types of biomolecules are depicted as a multicolor electrochemical image. The mode-selectable function of the 400-electrode device enables the imaging system and two different potentials can be independently applied to the selected electrodes. The imaging system is successfully applied for detecting multiple cell functions of the embryonic stem (ES) cell and the rat pheochromocytoma (PC12) cell aggregates. To the best of our knowledge, this is the first time that a real-time electrochemical mapping technique for multiple electroactive species, simultaneously, has been reported. The imaging system is a promising bioanalytical method for exploring complex biological phenomena.

Toward Universal SERS Detection of Disease Signaling Bioanalytes Using 3D Self-Assembled Nonplasmonic near-Quantum-Scale Silicon Probe

Currently, the quantum-scale surface-enhanced Raman scattering (SERS) properties of Si materials have yet to be discovered for universal biosensing applications. In this study, a potential universal biosensing probe is generated by activating the SERS functionality of Si nanostructures through near quantum-scale (nQS) engineering. We introduce herein 3D nonplasmonic Si nanomesh structure with nQS defects for SERS biosensing applications. Through ionization of a single-crystal defect-free Si wafer, highly defect-rich Si subnano-orbs (sNOs) are fabricated and self-assemble as connective 3D Si nanomesh structures with enhanced SERS biosensing activity. By amending the laser ionization and ion-ion interactions, we observe the controlled synthesis of engineered nQS defects in the form of nQS-grain boundary disorder or surface nQS voids within the interconnected Si sNOs. To our knowledge, it is shown here for the first time that defect-rich Si nanomesh structures exhibit enhanced Raman activity, with the nQS morphological and crystallographic defects acting as the prime SERS contributors without a plasmonic contribution. The SERS biosensing sensitivity with the synthesized defect-rich Si nanomesh structures without an additional plasmonic material was evaluated using of a tripeptide biomarker l-glutathione (GSH); we observe an enhancement factor value of ∼102 for the GSH biomolecules with 10-9 M sensitivity, a phenomena to our knowledge that has yet to be reported. Additionally, the SERS detection of multiple disease-signaling biomolecules (cysteine, tryptophan, and methionine) is achieved at very low analyte concentration (10-9 M). These results indicate a potential new dimension to universal SERS biosensing applications with these unique nonplasmonic defect-rich 3D nQS-Si nanostructures.

ComplexViewer: visualization of curated macromolecular complexes.

SummaryProteins frequently function as parts of complexes, assemblages of multiple proteins and other biomolecules, yet network visualizations usually only show proteins as parts of binary interactions. ComplexViewer visualizes interactions with more than two participants and thereby avoids the need to first expand these into multiple binary interactions. Furthermore, if binding regions between molecules are known then these can be displayed in the context of the larger complex.Availability and implementationfreely available under Apache version 2 license; EMBL-EBI Complex Portal: http://www.ebi.ac.uk/complexportal; Source code: https://github.com/MICommunity/ComplexViewer; Package: https://www.npmjs.com/package/complexviewer; http://biojs.io/d/complexviewer. Language: JavaScript; Web technology: Scalable Vector Graphics; Libraries: D3.js.

Nanostructured raspberry-like gelatin microspheres for local delivery of multiple biomolecules.

Multicompartment particles have gained considerable interest during the past decade to facilitate simultaneous and differential delivery of multiple biomolecules in various biomedical applications. Nevertheless, common methods employed for the production of such particles are often complex and only offer small-scale production. Here, we report the formation of raspberry-like gelatin (RLG) microparticles composed of gelatin nanoparticles as colloidal building blocks through supercritical CO2 processing. We show that these microparticles are cytocompatible and facilitate differential release of two model compounds with different molecular sizes, promising successful applications in various biomedical areas. Summarizing, this paper presents a novel strategy that can be utilized as a cost-effective route for production of various types of multicompartment particles using a wide range of colloidal building blocks.

Clinical Study of NeuroRegen Scaffold Combined with Human Mesenchymal Stem Cells for the Repair of Chronic Complete Spinal Cord Injury

Regeneration of damaged neurons and recovery of sensation and motor function after complete spinal cord injury (SCI) are challenging. We previously developed a collagen scaffold, NeuroRegen, to promote axonal growth along collagen fibers and inhibit glial scar formation after SCI. When functionalized with multiple biomolecules, this scaffold promoted neurological regeneration and functional recovery in animals with SCI. In this study, eight patients with chronic complete SCI were enrolled to examine the safety and efficacy of implanting NeuroRegen scaffold with human umbilical cord mesenchymal stem cells (hUCB-MSCs). Using intraoperative neurophysiological monitoring, we identified and surgically resected scar tissues to eliminate the inhibitory effect of glial scarring on nerve regeneration. We then implanted NeuroRegen scaffold loaded with hUCB-MSCs into the resection sites. No adverse events (infection, fever, headache, allergic reaction, shock, perioperative complications, aggravation of neurological status, or cancer) were observed during 1 year of follow-up. Primary efficacy outcomes, including expansion of sensation level and motor-evoked potential (MEP)-responsive area, increased finger activity, enhanced trunk stability, defecation sensation, and autonomic neural function recovery, were observed in some patients. Our findings suggest that combined application of NeuroRegen scaffold and hUCB-MSCs is safe and feasible for clinical therapy in patients with chronic SCI. Our study suggests that construction of a regenerative microenvironment using a scaffold-based strategy may be a possible future approach to SCI repair.

Quantitative volumetric Raman imaging of three dimensional cell cultures

The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell–material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy.

Biomimetic calcium phosphate/polyelectrolyte multilayer coatings for sequential delivery of multiple biological factors.

Combinations of growth factors synergistically enhance tissue regeneration, but typically require sequential, rather than co-delivery from biomaterials for maximum efficacy. Polyelectrolyte multilayer (PEM) coatings can deliver multiple factors without loss of activity; however, sequential delivery from PEM has been limited due to interlayer diffusion that results in co-delivery of the factors. This study shows that addition of a biomimetic calcium phosphate (bCaP) barrier layer to a PEM coating effectively prevents interlayer diffusion and enables sequential delivery of two different biomolecules via direct cell access. A simulated body fluid method was used to deposit a layer of bCaP followed by 30 bilayers of PEM made with poly-l-Lysine (+) and poly l-Glutamic acid (-) (bCaP-PEM). Measurements of MC3T3-E1 proliferation and viability over time on bCaP-PEM were used to demonstrate the sequential delivery kinetics of a proliferative factor [fibroblast growth factor-2 (FGF-2)] followed by a cytotoxic factor (antimycin A, AntiA). FGF-2 and AntiA both retained their bioactivity within bCaP-PEM, yet no release of FGF-2 or AntiA from bCaP-PEM was observed when cells were absent indicating a cell-mediated, local delivery process. This coating technique is useful for a variety of applications that would benefit from highly localized, sequential delivery of multiple biomolecules governed by cell initiated degradation that avoids off-target effects associated with diffusion-based release. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1500-1509, 2017.