Multiple Biomolecules Research Articles

One step 4× and 12× 3D-ExM enables robust super-resolution microscopy of nanoscale cellular structures.

Super-resolution microscopy has become an indispensable tool across diverse research fields, offering unprecedented insights into biological architectures with nanometer scale resolution. Compared with traditional nanometer-scale imaging methods such as electron microscopy, super-resolution microscopy offers several advantages, including the simultaneous labeling of multiple target biomolecules with high specificity and simpler sample preparation, making it accessible to most researchers. In this study, we introduce two optimized methods of super-resolution imaging: 4-fold and 12-fold 3D-isotropic and preserved Expansion Microscopy (4× and 12× 3D-ExM). 3D-ExM is a straightforward expansion microscopy technique featuring a single-step process, providing robust and reproducible 3D isotropic expansion for both 2D and 3D cell culture models. With standard confocal microscopy, 12× 3D-ExM achieves a lateral resolution of <30 nm, enabling the visualization of nanoscale structures, including chromosomes, kinetochores, nuclear pore complexes, and Epstein-Barr virus particles. These results demonstrate that 3D-ExM provides cost-effective and user-friendly super-resolution microscopy, making it highly suitable for a wide range of cell biology research, including studies on cellular and chromatin architectures.

Allosteric stem-loop multicomponent DNAzyme: A versatile stimuli-responsive switch for nanotweezer operations and multifunctional biosensing

DNAzyme, possessing the capabilities of both encoding information and catalyzing chemical reactions, holds significant value in advancing the widespread application of dynamic nanotechnology. Particularly, multicomponent DNAzyme (MNAzyme) has attracted considerable attention due to its remarkable versatility and practicality. However, the inevitable signal leakage of MNAzyme constrains the applications in complex nucleic acid reaction networks. Herein, we present an allosteric stem-loop multicomponent DNAzyme (ASLM) that incorporates a blocking domain to mitigate leakage resulting from secondary structures of MNAzyme while simultaneously stabilizing binding affinity during complex formation. As the sequence of blocking domain serves as an extension independent of the functional region in MNAzyme, this modulation structure can be flexibly assembled with conformation-switchable nanotweezer to form a time-responsive molecular device, enabling dynamic and dissipative operations through nucleic acid and RNase H. More importantly, the ASLM structure can be directly incorporated into DNA catalytic circuits for detecting multiple biomolecules (nucleic acid, protease, and small molecule). With its scalability and versatility, this effector-activated allosteric regulator will serve as a universal stimuli-responsive switch in complex and hierarchical DNA networks, expanding the scope of DNA nanotechnology in molecular device construction and diagnostic biosensing.

Swellable Biopolymer Composite Microneedle Patch for Pain-Free Collection of Interstitial Fluid to Analyze Multiple Biomolecules.

Sampling of interstitial fluid (ISF) using microneedle (MN) patch offers a pain-free minimally invasive alternative to syringe needle-based blood sample collection. However, there is a challenge in the development of MN patch that provides swelling behavior with sufficient mechanical strength for skin penetration. Here, we report fabrication of MN patch made of biopolymer composite containing iota-carrageenan, gelatin, and polyethylene glycol. Calcium chloride was used as a crosslinker to improve mechanical strength. MN patch was characterized for integrity, swelling behavior, mechanical strength, aspiration of fluid from agarose gel, and the excised porcine ear skin. An array of 361 MNs was able to aspirate 36 ± 5 and 14 ± 1 μL fluid after application in agarose gel matrix and the ex vivo porcine skin model, respectively. MN patch applied in vivo rat model for 30 min resulted in the collection of ISF containing 267 ± 128 mg/dL, 24 ± 13 mg/dL, and 0.6 ± 0.4 mIU/mL of glucose, uric acid and thyroid stimulating hormone (TSH), respectively. The concentration of glucose, uric acid, and TSH in rat blood was found to be 199 ± 47 mg/dL, 8.4 ± 6 mg/dL, and 1.1 ± 0.6 mIU/mL at the same time. Furthermore, MN patch applied on the forearm of 10 healthy human volunteers for 30 min was able to aspirate 32 ± 14 μL of ISF. The concentration of glucose, uric acid, and TSH determined from ISF samples of human volunteers was 64 ± 25 mg/dL, 4.2 ± 4.1 mg/dL, and 0.16 ± 0.08 mIU/mL, respectively. The visual analogue scale (VAS) pain score after MN application was lower compared with hypodermic syringe needle insertion. Taken together, biopolymer composite-based swellable MN patch can be developed for collection of ISF for simultaneous determination of multiple biomolecules in a minimally invasive manner.

Development of multi-layered three-dimensional printed scaffold for sequential delivery of biomolecules

Spatiotemporal control of exogenous growth factors plays a crucial role in the sequential repair of damaged tissues. However, traditional therapeutic trials have mostly relied on the delivery of a single molecule because of the limitations of rapid diffusion and the instability of biomolecules. In this study, we developed a novel strategy using a composite of gelatin and silica as an alternative for the stable and sequential release of biomolecules. Two biomolecules, basic fibroblast growth factor and bone morphogenetic protein-2, were incorporated into two separate composites and sequentially layered onto the activated polymer surface. Each molecule was sequentially released from each layer of the scaffold and the silica composite prevented rapid diffusion owing to its nano-porous structure. The adhesion, proliferation, and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells were significantly enhanced in the double-layered group containing separately delivered dual molecules compared with the control or single groups. Our developed gelatin&amp;ndash;silica composite was successfully placed in double layers on polymer scaffolds, and cellular responses were promoted in this manner. These results demonstrated that our scaffolding system has potential as a therapeutic strategy for the delivery of dual or multiple biomolecules in regenerative medicine. &amp;nbsp;

Synthesis of colicin Ia neoglycoproteins: tools towards glyco-engineering of bacterial cell surfaces.

Colicins are antimicrobial proteins produced by certain strains of Escherichia coli that function as offensive weapons against closely-related competitor strains. Their bactericidal properties and narrow bacterial targeting range has made them of therapeutic interest. Furthermore, the applications of engineered non-bactericidal colicins are of interest as a cell surface-directed protein anchor for decorating E. coli with biomolecules. We previously demonstrated that an engineered non-bacteriocidal colicin E9 could be used to label bacterial cells with multiple biomolecules including glycans. Herein we extend our approach to colicin Ia, constructing mannose-presenting colicin la neoglycoproteins, through N-terminal organocatalyst-mediated protein aldol ligation (OPAL), or maleimide ligation targeting an internal cysteine. This work further highlights the potential utility of engineered colicins for non-genetic glyco-engineering of the E. coli cell surface.

Broadband multiple resonant metasurface for mixture surface-enhanced infrared absorption based on polarization-sensitive folded nanoantennas

Multiple resonant metasurfaces for mixture component detection by surface-enhanced infrared absorption in a broadband spectrum region are still highly desirable. This paper presents a polarization-sensitive multi-resonant broadband metasurface in the mid-infrared (MIR) region based on an odd symmetric folded antenna array. Six major resonant modes are effectively excited in 6–15 μm covering the absorption peak of multiple gases and biomolecules. In addition, the calculation indicates the maximum electric field intensity enhancement reaches 3607 and the average electric enhancement factors are dominantly amplified to a maximum of 75.08. The excited multiple hotspots provide more spatial overlap with analytes and would be beneficial for label-free detection. Moreover, owing to the contributions of the intrinsic phonons and polaritons in the ENZ material, the spectroscopy features are relatively stable when changing the geometry parameters and the incident angles, allowing more fabrication tolerance and experiment flexibility. Furthermore, the multiple resonant metasurface is also effectively enabled under orthogonal circular polarizations. Finally, the sensing abilities of the designed metasurface for polyethylene terephthalate and isopropanol molecules have been computationally validated. These results indicate the potential for nanophotonic detection and mixture spectroscopy analysis.

One step 4x and 12x 3D-ExM: robust super-resolution microscopy in cell biology.

Super-resolution microscopy has become an indispensable tool across diverse research fields, offering unprecedented insights into biological architectures with nanometer scale resolution. Compared to traditional nanometer-scale imaging methods such as electron microscopy, super-resolution microscopy offers several advantages, including the simultaneous labeling of multiple target biomolecules with high specificity and simpler sample preparation, making it accessible to most researchers. In this study, we introduce two optimized methods of super-resolution imaging: 4-fold and 12-fold 3D-isotropic and preserved Expansion Microscopy (4x and 12x 3D-ExM). 3D-ExM is a straightforward expansion microscopy method featuring a single-step process, providing robust and reproducible 3D isotropic expansion for both 2D and 3D cell culture models. With standard confocal microscopy, 12x 3D-ExM achieves a lateral resolution of under 30 nm, enabling the visualization of nanoscale structures, including chromosomes, kinetochores, nuclear pore complexes, and Epstein-Barr virus particles. These results demonstrate that 3D-ExM provides cost-effective and user-friendly super-resolution microscopy, making it highly suitable for a wide range of cell biology research, including studies on cellular and chromatin architectures.

Diffusion-driven height readout analytical device based on gold nanobipyramid-doped supramolecular hydrogel for point-of-care bioanalysis

Point-of-care (POC) diagnostics is crucial for rapid on-site detection in resource-limited settings. However, there are still some challenges in the development of quantitative POC bioassays. Herein, we propose a simple, visual, and instrument-free height readout analytical device injected with target-responsive gold nanobipyramid (AuNBP)-doped guanosine-quartet (G4) supramolecular hydrogel for the quantitative detection of glucose. This sensing platform relies on H2O2 generated by glucose oxidation to induce iodide-mediated etching of AuNBPs, resulting in visualized color-changed hydrogel bar-chart driven by molecular diffusion in a polycarbonate tube. The visual height of the color-changed hydrogel is correlated with the concentration of glucose, and the quantitative determination was achieved simply by using a household ruler or mobile software as a readout. More importantly, this method has been successfully used to detect glucose in human serum samples with high accuracy and reliability. In addition, the AuNBPs are utilized as plasmonic probes to construct two-input logic gates, providing an intelligent strategy to simultaneously detect multiple biomolecules. This height-readout sensing platform demonstrates great promise for applications in POC and intelligent bioanalysis.

Recent Advances in Hydrogel-Based 3D Bioprinting and Its Potential Application in the Treatment of Congenital Heart Disease.

Congenital heart disease (CHD) is the most common birth defect, requiring invasive surgery often before a child's first birthday. Current materials used during CHD surgery lack the ability to grow, remodel, and regenerate. To solve those limitations, 3D bioprinting is an emerging tool with the capability to create tailored constructs based on patients' own imaging data with the ability to grow and remodel once implanted in children with CHD. It has the potential to integrate multiple bioinks with several cell types and biomolecules within 3D-bioprinted constructs that exhibit good structural fidelity, stability, and mechanical integrity. This review gives an overview of CHD and recent advancements in 3D bioprinting technologies with potential use in the treatment of CHD. Moreover, the selection of appropriate biomaterials based on their chemical, physical, and biological properties that are further manipulated to suit their application are also discussed. An introduction to bioink formulations composed of various biomaterials with emphasis on multiple cell types and biomolecules is briefly overviewed. Vasculogenesis and angiogenesis of prefabricated 3D-bioprinted structures and novel 4D printing technology are also summarized. Finally, we discuss several restrictions and our perspective on future directions in 3D bioprinting technologies in the treatment of CHD.

Multi-modal mass spectrometry imaging of a single tissue section.

Mass spectrometry imaging (MSI) was developed to visualize spatial chemical information within tissues, thereby facilitating spatial multi-omic analysis. However, due to the limited spatial information provided by individual modal MSI, correlating various chemical data within tissues remains a significant challenge. In recent years, multimodal MSI has garnered considerable attention due to its ability to visualize the spatial distributions of multiple biomolecules within tissues. Among the strategies employed in this field, multimodal imaging on a single tissue section circumvents multiple issues introduced by integration of images of consecutive tissue sections. In this minireview, we provide an overview of multimodal MSI on a single tissue section, with a particular focus on the use of Matrix-Assisted Laser Desorption/Ionization-MSI for spatial multi-omic investigations that offer a comprehensive and in-depth elucidation of the biological state and activities, aiming to inspire the development of new approaches in this field.

Efficient colorimetric point-of-care detection and imaging of multiple biomolecules utilizing photonic liquid crystal composite on gold nanoisland thin films for label-free sensing

Efficient colorimetric point-of-care detection and imaging of multiple biomolecules utilizing photonic liquid crystal composite on gold nanoisland thin films for label-free sensing

GENESIS 2.1: High-Performance Molecular Dynamics Software for Enhanced Sampling and Free-Energy Calculations for Atomistic, Coarse-Grained, and Quantum Mechanics/Molecular Mechanics Models.

GENeralized-Ensemble SImulation System (GENESIS) is a molecular dynamics (MD) software developed to simulate the conformational dynamics of a single biomolecule, as well as molecular interactions in large biomolecular assemblies and between multiple biomolecules in cellular environments. To achieve the latter purpose, the earlier versions of GENESIS emphasized high performance in atomistic MD simulations on massively parallel supercomputers, with or without graphics processing units (GPUs). Here, we implemented multiscale MD simulations that include atomistic, coarse-grained, and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. They demonstrate high performance and are integrated with enhanced conformational sampling algorithms and free-energy calculations without using external programs except for the QM programs. In this article, we review new functions, molecular models, and other essential features in GENESIS version 2.1 and discuss ongoing developments for future releases.

Ti3C2Tx Coated with TiO2 Nanosheets for the Simultaneous Detection of Ascorbic Acid, Dopamine and Uric Acid.

Two-dimensional MXenes have become an important material for electrochemical sensing of biomolecules due to their excellent electric properties, large surface area and hydrophilicity. However, the simultaneous detection of multiple biomolecules using MXene-based electrodes is still a challenge. Here, a simple solvothermal process was used to synthesis the Ti3C2Tx coated with TiO2 nanosheets (Ti3C2Tx@TiO2 NSs). The surface modification of TiO2 NSs on Ti3C2Tx can effectively reduce the self-accumulation of Ti3C2Tx and improve stability. Glassy carbon electrode was modified by Ti3C2Tx@TiO2 NSs (Ti3C2Tx@TiO2 NSs/GCE) and was able simultaneously to detect dopamine (DA), ascorbic acid (AA) and uric acid (UA). Under concentrations ranging from 200 to 1000 μM, 40 to 300 μM and 50 to 400 μM, the limit of detection (LOD) is 2.91 μM, 0.19 μM and 0.25 μM for AA, DA and UA, respectively. Furthermore, Ti3C2Tx@TiO2 NSs/GCE demonstrated remarkable stability and reliable reproducibility for the detection of AA/DA/UA.

In situ single-cell spontaneous Raman spectroscopy differentiates tumor-associated macrophages

The analysis of in situ single cells in tissue is crucial for the study of tumors. However, simultaneous analysis of multiple biomolecules for single cells by fluorescence immunohistochemistry is challenging. Spontaneous Raman spectroscopy provides biochemical fingerprints of individual cells in a label-free and nondestructive manner, yet it is not well applied to single cells in tissue. Here, we develop an in situ single-cell spontaneous Raman spectroscopy (SRS) system that can localize a single cell in tissue with fluorescence imaging, while obtaining the single-cell Raman spectra for molecular analysis. Our system is verified by measuring the standard polystyrene beads of different sizes. Measurements of in situ single macrophages in clinical cancerous and para-cancerous tissues are then performed. By using discriminant analysis, it is shown that CD68+ macrophages in cancerous and para-cancerous tissues are classified with an accuracy of 98.3 %. The molecular content of single macrophages in tissue is analyzed by using multivariate curve resolution-alternating least squares method. We find that the lipid content of tumor-associated macrophages in cancerous tissues is higher while both the contents of protein and nucleic acid are lower than those in para-cancerous tissues, which is challenging to achieve by conventional immunohistochemistry. Our in situ single-cell SRS system has the capability for the obtaining of molecular information of a localized single cell in tissue, which may find potential applications for clinical cancer diagnosis.

Smart Biointerfaces via Click Chemistry-Enabled Nanopatterning of Multiple Bioligands and DNA Force Sensors.

Nanoscale biomolecular placement is crucial for advancing cellular signaling, sensor technology, and molecular interaction studies. Despite this, current methods fall short in enabling large-area nanopatterning of multiple biomolecules while minimizing nonspecific interactions. Using bioorthogonal tags at a submicron scale, we introduce a novel hole-mask colloidal lithography method for arranging up to three distinct proteins, DNA, or peptides on large, fully passivated surfaces. The surfaces are compatible with single-molecule fluorescence microscopy and microplate formats, facilitating versatile applications in cellular and single-molecule assays. We utilize fully passivated and transparent substrates devoid of metals and nanotopographical features to ensure accurate patterning and minimize nonspecific interactions. Surface patterning is achieved using bioorthogonal TCO-tetrazine (inverse electron-demand Diels-Alder, IEDDA) ligation, DBCO-azide (strain-promoted azide-alkyne cycloaddition, SPAAC) click chemistry, and biotin-avidin interactions. These are arranged on surfaces passivated with dense poly(ethylene glycol) PEG brushes crafted through the selective and stepwise removal of sacrificial metallic and polymeric layers, enabling the directed attachment of biospecific tags with nanometric precision. In a proof-of-concept experiment, DNA tension gauge tether (TGT) force sensors, conjugated to cRGD (arginylglycylaspartic acid) in nanoclusters, measured fibroblast integrin tension. This novel application enables the quantification of forces in the piconewton range, which is restricted within the nanopatterned clusters. A second demonstration of the platform to study integrin and epidermal growth factor (EGF) proximal signaling reveals clear mechanotransduction and changes in the cellular morphology. The findings illustrate the platform's potential as a powerful tool for probing complex biochemical pathways involving several molecules arranged with nanometer precision and cellular interactions at the nanoscale.

Design and analysis of hetero-dielectric Junctionless-TFET(JL-TFET) with N+ pocket as label free biosensors

This paper includes sensitivity assessment of label-free biosensors using hetero dielectric Junctionless-TFET (HD-JL-TFET) thorough TCAD simulator. The fundamental structure, operation and design of a Junctionless-TFET (HD-JL-TFET) as biosensor are investigated in this paper. For the purpose of detecting the biomolecule, a nano-gap is added close to the source end between the gate and channel. To test the sensing potential, we adjusted the charge density and material dielectric constant (K) by comprehensive calibrated device simulation. For several biomolecules, the device’s sensitivity was examined as surface potential, electron tunnelling rate, and conduction-valence band edge fluctuation. Additionally, the Id versus VGS features, the sensitivity to the drain current, and the ION/IOFF fluctuation are also examined. By contrasting neutral or charged biomolecules using various dielectric constants, the sensitivity characteristics of positive, negative, and neutral biomolecules are examined. The development of biosensors, which enable the rapid and precise detection of multiple biomolecules, has revolutionized the field of bioanalysis.

A novel universal fluorescence detection platform based on target binding-mediated primer exchange reaction strategy for sensitive and accurate detection of multiple biomolecules

This study introduces a universal fluorescent detection platform based on the binding-mediated primer exchange reaction strategy, designed for highly sensitive and accurate detection of multiple biomolecules. This method uses an intact DNA hybridization probe as the initial target recognition site, forming a stable probe-target complex upon binding to the target. This complex drives the primer exchange reaction to amplify the signal, and then to achieve signal output through fluorescence resonance energy transfer (FRET). The reaction process is initiated directly by the catalytic target binding medium, ensuring method specificity. The versatile platform, equipped with various recognition elements such as antisense nucleic acids, biotin, and split aptamers, demonstrates exceptional universality in detecting a range of biomolecules, including virus nucleic acid fragments, streptavidin, and ATP. The method exhibits strong linear relationships with detection limits of 1.02 pM for virus nucleic acid fragments, 1.08 nM for streptavidin, and 15.7 μM for ATP, respectively. Moreover, the method displays excellent selectivity and outstanding performance in complex biological samples. This promising biosensing platform is believed to find numerous applications spanning bioanalysis, disease diagnosis, and biomedicine.

Advances in the co-production of biosurfactant and other biomolecules: statistical approaches for process optimization.

An efficient microbial conversion for simultaneous synthesis of multiple high-value compounds, such as biosurfactants and enzymes, is one of the most promising aspects for an economical bioprocess leading to a marked reduction in production cost. Although biosurfactant and enzyme production separately have been much explored, there are limited reports on the predictions and optimization studies on simultaneous production of biosurfactants and other industrially important enzymes, including lipase, protease, and amylase. Enzymes are suited for an integrated production process with biosurfactants as multiple common industrial processes and applications are catalysed by these molecules. However, the complexity in microbial metabolism complicates the production process. This study details the work done on biosurfactant and enzyme co-production and explores the application and scope of various statistical tools and methodologies in this area of research. The use of advanced computational tools is yet to be explored for the optimization of downstream strategies in the co-production process. Given the complexity of the co-production process and with various new methodologies based on artificial intelligence (AI) being invented, the scope of AI in shaping the biosurfactant-enzyme co-production process is immense and would lead to not only efficient and rapid optimization, but economical extraction of multiple biomolecules as well.

In Situ Electrospinning MOF-Derived Highly Dispersed α-Cobalt Confined in Nitrogen-Doped Carbon Nanofibers Nanozyme for Biomolecule Monitoring.

Designing a metal-organic framework (MOF)-derived nanozyme with highly dispersed active sites and high catalytic activity as well as robust structure for colorimetric biosensing of diverse biomolecules remains a substantial challenge. Here, an MOF-derived highly dispersed and pure α-cobalt confined in a nitrogen-doped carbon nanofiber (α-Co@NCNF) nanozyme with superior glucose oxidase (GOD)- and peroxidase (POD)-like activities was constructed for colorimetric assay of multiple biomolecules. Specifically, the α-Co@NCNF nanozyme was synthesized, utilizing in situ electrospinning Co-MOFs into polyacrylonitrile nanofiber (PAN) followed by a pyrolysis process. Taking advantage of the in situ electrospinning strategy, the α-Co nanoparticles were confined in continuous porous NCNF to restrict the growth and prevent the aggregation and oxidation during the pyrolysis process. The resulting special structure considerably improved the enzyme-like performance. A series of experiments validate that the enzyme-like activity of the α-Co@NCNF nanozyme was superior to that of Co@CoO@NCNF (derivatives from Co-MOFs grown on the surface of PAN nanofiber) and nature enzymes. Furthermore, α-Co@NCNF nanozyme-based colorimetric biosensing was developed for monitoring glucose, hydrogen peroxide (H2O2), and glutathione (GSH) and the corresponding linear ranges are 0.1-50 and 50-900 μM and 5-55 and 0.1-20 μM accompanied by the corresponding low detection of 0.03, 1.66, and 0.03 μM. The proposed method for the construction of α-Co@NCNF nanozyme with dual enzyme-like properties provides a new insight for designing novel nanozymes and has prospects for application in colorimetric biosensing.

A detection system using sensing motif-tethered oligodeoxynucleotides for multiplex biomolecular analysis.

We developed a system to detect multiple target biomolecules through sensing motif-tethered oligodeoxynucleotides. DNA-based molecular probes gave the primary amine motif upon reaction with the target biomolecules, glutathione (GSH) and H2O2. After labelling with biotin, the product DNAs were selectively collected to be quantified by qPCR.