Fusing Peptide Epitopes for Advanced Multiplex Serological Testing for SARS-CoV-2 Antibody Detection.

The tragic COVID-19 pandemic, which has seen a total of 655 million cases worldwide and a death toll of over 6.6 million seems finally tailing off. Even so, new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise, the severity of which cannot be predicted in advance. This is concerning for the maintenance and stability of public health, since immune evasion and increased transmissibility may arise. Therefore, it is crucial to continue monitoring antibody responses to SARS-CoV-2 in the general population. As a complement to polymerase chain reaction tests, multiplex immunoassays are elegant tools that use individual protein or peptide antigens simultaneously to provide a high level of sensitivity and specificity. To further improve these aspects of SARS-CoV-2 antibody detection, as well as accuracy, we have developed an advanced serological peptide-based multiplex assay using antigen-fused peptide epitopes derived from both the spike and the nucleocapsid proteins. The significance of the epitopes selected for antibody detection has been verified by in silico molecular docking simulations between the peptide epitopes and reported SARS-CoV-2 antibodies. Peptides can be more easily and quickly modified and synthesized than full length proteins and can, therefore, be used in a more cost-effective manner. Three different fusion-epitope peptides (FEPs) were synthesized and tested by enzyme-linked immunosorbent assay (ELISA). A total of 145 blood serum samples were used, compromising 110 COVID-19 serum samples from COVID-19 patients and 35 negative control serum samples taken from COVID-19-free individuals before the outbreak. Interestingly, our data demonstrate that the sensitivity, specificity, and accuracy of the results for the FEP antigens are higher than for single peptide epitopes or mixtures of single peptide epitopes. Our FEP concept can be applied to different multiplex immunoassays testing not only for SARS-CoV-2 but also for various other pathogens. A significantly improved peptide-based serological assay may support the development of commercial point-of-care tests, such as lateral-flow-assays.

Peptide Gel Electrolytes for Stabilized Zn Metal Anodes.

The rechargeable aqueous Zn ion battery (AZIB) is considered a promising candidate for future energy storage applications due to its intrinsic safety features and low cost. However, Zn dendrites and side reactions (e.g., corrosion, hydrogen evolution reaction, and inactive side product (Zn hydroxide sulfate) formation) at the Zn metal anode have been serious obstacles to realizing a satisfactory AZIB performance. The application of gel electrolytes is a common strategy for suppressing these problems, but the normally used highly cross-linked polymer matrix (e.g., polyacrylamide (PAM)) brings additional difficulties for battery assembly and recycling. Herein, we have developed a gel electrolyte for Zn metal anode stabilization, where a peptide matrix, a highly biocompatible material, is used for gel construction. Various experiments and simulations elucidate the sulfate anion-assisted self-assembly gel formation and its effect in stabilizing Zn metal anodes. Unlike polymer gel electrolytes, the peptide gel electrolyte can reversibly transform between gel and liquid states, thus facilitating the gel-involved battery assembly and recycling. Furthermore, the peptide gel electrolyte provides fast Zn ion diffusion (comparable to conventional liquid electrolyte) while suppressing side reactions and dendrite growth, thus achieving highly stable Zn metal anodes as validated in various cell configurations. We believe that our concept of gel electrolyte design will inspire more future directions for Zn metal anode protection based on gel electrolyte design.

Selectively Positioned Catechol Moiety Supports Ultrashort Self-Assembling Peptide Hydrogel Adhesion for Coral Restoration.

Coral reef survival is threatened globally. One way to restore this delicate ecosystem is to enhance coral growth by the controlled propagation of coral fragments. To be sustainable, this technique requires the use of biocompatible underwater adhesives. Hydrogels based on rationally designed ultrashort self-assembling peptides (USP) are of great interest for various biological and environmental applications, due to their biocompatibility and tunable mechanical properties. Implementing superior adhesion properties to the USP hydrogel compounds is crucial in both water and high ionic strength solutions and is relevant in medical and marine environmental applications such as coral regeneration. Some marine animals secrete large quantities of the aminoacids dopa and lysine to enhance their adhesion to wet surfaces. Therefore, the addition of catechol moieties to the USP sequence containing lysine (IIZK) should improve the adhesive properties of USP hydrogels. However, it is challenging to place the catechol moiety (Do) within the USP sequence at an optimal position without compromising the hydrogel self-assembly process and mechanical properties. Here, we demonstrate that, among three USP hydrogels, DoIIZK is the least adhesive and that the adhesiveness of the IIZDoK hydrogel is compromised by its poor mechanical properties. The best adhesion outcome was achieved using the IIZKDo hydrogel, the only one to show equally sound adhesive and mechanical properties. A mechanistic understanding of this outcome is presented here. This property was confirmed by the successful gluing of coral fragments by means of IIZKDo hydrogel that are still thriving after more than three years since the deployment. The validated biocompatibility of this underwater hydrogel glue suggests that it could be advantageously implemented for other applications, such as surgical interventions.

Dipeptide-Based Photoreactive Instant Glue for Environmental and Biomedical Applications.

Nature-inspired smart materials offer numerous advantages over environmental friendliness and efficiency. Emulating the excellent adhesive properties of mussels foot proteins, where the lysine is in close proximity with the 3,4-dihydroxy-l-phenylalanine (DOPA), we report the synthesis of a novel photocurable peptide-based adhesive consisting exclusively of these two amino acids. Our adhesive is a highly concentrated aqueous solution of a monomer, a cross-linker, and a photoinitiator. Lap-shear adhesion measurements on plastic and glass surfaces and comparison with different types of commercial adhesives showed that the adhesive strength of our glue is comparable when applied in air and superior when used underwater. No toxicity of our adhesive was observed when the cytocompatibility on human dermal fibroblast cells was assessed. Preliminary experiments with various tissues and coral fragments showed that our adhesive could be applied to wound healing and coral reef restoration. Given the convenience of the facile synthesis, biocompatibility, ease of application underwater, and high adhesive strength, we expect that our adhesive may find application, but not limited, to the biomedical and environmental field.

The Impact of Mechanical Cues on the Metabolomic and Transcriptomic Profiles of Human Dermal Fibroblasts Cultured in Ultrashort Self-Assembling Peptide 3D Scaffolds.

Cells' interactions with their microenvironment influence their morphological features and regulate crucial cellular functions including proliferation, differentiation, metabolism, and gene expression. Most biological data available are based on in vitro two-dimensional (2D) cellular models, which fail to recapitulate the three-dimensional (3D) in vivo systems. This can be attributed to the lack of cell-matrix interaction and the limitless access to nutrients and oxygen, in contrast to in vivo systems. Despite the emergence of a plethora of 3D matrices to address this challenge, there are few reports offering a proper characterization of these matrices or studying how the cell-matrix interaction influences cellular metabolism in correlation with gene expression. In this study, two tetrameric ultrashort self-assembling peptide sequences, FFIK and FIIK, were used to create in vitro 3D models using well-described human dermal fibroblast cells. The peptide sequences are derived from naturally occurring amino acids that are capable of self-assembling into stable hydrogels without UV or chemical cross-linking. Our results showed that 2D cultured fibroblasts exhibited distinct metabolic and transcriptomic profiles compared to 3D cultured cells. The observed changes in the metabolomic and transcriptomic profiles were closely interconnected and influenced several important metabolic pathways including the TCA cycle, glycolysis, MAPK signaling cascades, and hemostasis. Data provided here may lead to clearer insights into the influence of the surrounding microenvironment on human dermal fibroblast metabolic patterns and molecular mechanisms, underscoring the importance of utilizing efficient 3D in vitro models to study such complex mechanisms.

Fabrication of lumen-forming colorectal cancer organoids using a newly designed laminin-derived bioink.

160Three-dimensional (3D) bioprinting systems, which are the prominent tools for biofabrication, should evolve around the cutting-edge technologies of tissue engineering. This is the case with organoid technology, which requires a plethora of new materials to evolve, including extracellular matrices with specific mechanical and biochemical properties. For a bioprinting system to facilitate organoid growth, it must be able to recreate an organ-like environment within the 3D construct. In this study, a well-established, self-assembling peptide system was employed to generate a laminin-like bioink to provide signals of cell adhesion and lumen formation in cancer stem cells. One bioink formulation led to the formation of lumen with outperforming characteristics, which showed good stability of the printed construct.

A Parkinson's disease model composed of 3D bioprinted dopaminergic neurons within a biomimetic peptide scaffold.

Parkinson's disease (PD) is a progressive neurological disorder that affects movement. It is associated with lost dopaminergic (DA) neurons in thesubstantia nigra, a process that is not yet fully understood. To understand this deleterious disorder, there is an immense need to develop efficientin vitrothree-dimensional (3D) models that can recapitulate complex organs such as the brain. However, due to the complexity of neurons, selecting suitable biomaterials to accommodate them is challenging. Here, we report on the fabrication of functional DA neuronal 3D models using ultrashort self-assembling tetrapeptide scaffolds. Our peptide-based models demonstrate biocompatibility both for primary mouse embryonic DA neurons and for human DA neurons derived from human embryonic stem cells. DA neurons encapsulated in these scaffolds responded to 6-hydroxydopamine, a neurotoxin that selectively induces loss of DA neurons. Using multi-electrode arrays, we recorded spontaneous activity in DA neurons encapsulated within these 3D peptide scaffolds for more than 1 month without decrease of signal intensity. Additionally, vascularization of our 3D models in a co-culture with endothelial cells greatly promoted neurite outgrowth, leading to denser network formation. This increase of neuronal networks through vascularization was observed for both primary mouse DA and cortical neurons. Furthermore, we present a 3D bioprinted model of DA neurons inspired by the mouse brain and created with an extrusion-based 3D robotic bioprinting system that was developed during previous studies and is optimized with time-dependent pulsing by microfluidic pumps. We employed a hybrid fabrication strategy that relies on an external mold of the mouse brain construct that complements the shape and size of the desired bioprinted model to offer better support during printing. We hope that our 3D model provides a platform for studies of the pathogenesis of PD and other neurodegenerative disorders that may lead to better understanding and more efficient treatment strategies.

Direct Visualization and Identification of Membrane Voltage-Gated Sodium Channels from Human iPSC-Derived Neurons by Multiple Imaging and Light Enhanced Spectroscopy.

In this study, transmission electron microscopy atomic force microscopy, and surface enhanced Raman spectroscopy are combined through a direct imaging approach, to gather structural and chemical information of complex molecular systems such as ion channels in their original plasma membrane. Customized microfabricated sample holder allows to characterize Nav channels embedded in the original plasma membrane extracted from neuronal cells that are derived from healthy human induced pluripotent stem cells. The identification of the channels is accomplished by using two different approaches, one of them widely used in cryo-EM (the particle analysis method) and the other based on a novel Zernike Polynomial expansion of the images bitmap. This approach allows to carry out a whole series of investigations, one complementary to the other, on the same sample, preserving its state as close as possible to the original membrane configuration.

Micro/Nanopatterned Superhydrophobic Surfaces Fabrication for Biomolecules and Biomaterials Manipulation and Analysis.

Superhydrophobic surfaces display an extraordinary repulsion to water and water-based solutions. This effect emerges from the interplay of intrinsic hydrophobicity of the surface and its morphology. These surfaces have been established for a long time and have been studied for decades. The increasing interest in recent years has been focused towards applications in many different fields and, in particular, biomedical applications. In this paper, we review the progress achieved in the last years in the fabrication of regularly patterned superhydrophobic surfaces in many different materials and their exploitation for the manipulation and characterization of biomaterial, with particular emphasis on the issues affecting the yields of the fabrication processes and the quality of the manufactured devices.

Fabrication of a Lateral Flow Assay for Rapid In-Field Detection of COVID-19 Antibodies Using Additive Manufacturing Printing Technologies.

The development of lateral flow immunoassay (LFIA) using three-dimensional (3D) printing and bioprinting technologies can enhance and accelerate the optimization process of the fabrication. Therefore, the main goal of this study is to investigate methods to speed up the developing process of a LFIA as a tool for community screening. To achieve this goal, an in-house developed robotic arm and microfluidic pumps were used to print the proteins during the development of the test. 3D printing technologies were used to design and print the housing unit for the testing strip. The proposed design was made by taking into consideration the environmental impact of this disposable medical device.

Clustering of Major Histocompatibility Complex-Class I Molecules in Healthy and Cancer Colon Cells Revealed from Their Nanomechanical Properties.

The activation of the T cell mediated immune response relies on the fine interaction between the T cell receptor on the immune cell and the antigen-presenting major histocompatibility complex (MHC) molecules on the membrane surface of antigen-presenting cells. Both the distribution and quantity of MHC/peptide complexes and their adequate morphological presentation affect the activation of the immune cells. In several types of cancer the immune response is down-regulated due to the low expression of MHC-class I (MHC-I) molecules on the cell's surface, and in addition, the mechanical properties of the membrane seem to play a role. Herein, we investigate the distribution of MHC-I molecules and the related nanoscale mechanical environment on the cell surface of two cell lines derived from colon adenocarcinoma and a healthy epithelial colon reference cell line. Atomic force microscopy (AFM) force spectroscopy analysis using an antibody-tagged pyramidal probe specific for MHC-I molecules and a formula that relates the elasticity of the cell to the energy of adhesion revealed the different population distributions of MHC-I molecules in healthy cells compared to cancer cells. We found that MHC-I molecules are significantly less expressed in cancer cells. Moreover, the local elastic modulus is significantly reduced in cancer cells. We speculate that these results might be related to the proven ability of cancer cells to evade the immune system, not only by reducing MHC-I cell surface expression but also by modifying the local mechanical properties affecting the overall morphology of MHC-I synapse presentation to immune cells.

Ultrashort Peptide Bioinks Support Automated Printing of Large-Scale Constructs Assuring Long-Term Survival of Printed Tissue Constructs.

We report about rationally designed ultrashort peptide bioinks, overcoming severe limitations in current bioprinting procedures. Bioprinting is increasingly relevant in tissue engineering, regenerative and personalized medicine due to its ability to fabricate complex tissue scaffolds through an automated deposition process. Printing stable large-scale constructs with high shape fidelity and enabling long-term cell survival are major challenges that most existing bioinks are unable to solve. Additionally, they require chemical or UV-cross-linking for the structure-solidifying process which compromises the encapsulated cells, resulting in restricted structure complexity and low cell viability. Using ultrashort peptide bioinks as ideal bodylike but synthetic material, we demonstrate an instant solidifying cell-embedding printing process via a sophisticated extrusion procedure under true physiological conditions and at cost-effective low bioink concentrations. Our printed large-scale cell constructs and the chondrogenic differentiation of printed mesenchymal stem cells point to the strong potential of the peptide bioinks for automated complex tissue fabrication.

Engineered Microgels-Their Manufacturing and Biomedical Applications.

Microgels are hydrogel particles with diameters in the micrometer scale that can be fabricated in different shapes and sizes. Microgels are increasingly used for biomedical applications and for biofabrication due to their interesting features, such as injectability, modularity, porosity and tunability in respect to size, shape and mechanical properties. Fabrication methods of microgels are divided into two categories, following a top-down or bottom-up approach. Each approach has its own advantages and disadvantages and requires certain sets of materials and equipments. In this review, we discuss fabrication methods of both top-down and bottom-up approaches and point to their advantages as well as their limitations, with more focus on the bottom-up approaches. In addition, the use of microgels for a variety of biomedical applications will be discussed, including microgels for the delivery of therapeutic agents and microgels as cell carriers for the fabrication of 3D bioprinted cell-laden constructs. Microgels made from well-defined synthetic materials with a focus on rationally designed ultrashort peptides are also discussed, because they have been demonstrated to serve as an attractive alternative to much less defined naturally derived materials. Here, we will emphasize the potential and properties of ultrashort self-assembling peptides related to microgels.

A droplet reactor on a super-hydrophobic surface allows control and characterization of amyloid fibril growth.

Methods to produce protein amyloid fibrils, in vitro, and in situ structure characterization, are of primary importance in biology, medicine, and pharmacology. We first demonstrated the droplet on a super-hydrophobic substrate as the reactor to produce protein amyloid fibrils with real-time monitoring of the growth process by using combined light-sheet microscopy and thermal imaging. The molecular structures were characterized by Raman spectroscopy, X-ray diffraction and X-ray scattering. We demonstrated that the convective flow induced by the temperature gradient of the sample is the main driving force in the growth of well-ordered protein fibrils. Particular attention was devoted to PHF6 peptide and full-length Tau441 protein to form amyloid fibrils. By a combined experimental with the molecular dynamics simulations, the conformational polymorphism of these amyloid fibrils were characterized. The study provided a feasible procedure to optimize the amyloid fibrils formation and characterizations of other types of proteins in future studies.

The structure of DNA by direct imaging.

The structure of DNA was determined in 1953 by x-ray fiber diffraction. Several attempts have been made to obtain a direct image of DNA with alternative techniques. The direct image is intended to allow a quantitative evaluation of all relevant characteristic lengths present in a molecule. A direct image of DNA, which is different from diffraction in the reciprocal space, is difficult to obtain for two main reasons: the intrinsic very low contrast of the elements that form the molecule and the difficulty of preparing the sample while preserving its pristine shape and size. We show that through a preparation procedure compatible with the DNA physiological conditions, a direct image of a single suspended DNA molecule can be obtained. In the image, all relevant lengths of A-form DNA are measurable. A high-resolution transmission electron microscope that operates at 80 keV with an ultimate resolution of 1.5 Å was used for this experiment. Direct imaging of a single molecule can be used as a method to address biological problems that require knowledge at the single-molecule level, given that the average information obtained by x-ray diffraction of crystals or fibers is not sufficient for detailed structure determination, or when crystals cannot be obtained from biological molecules or are not sufficient in understanding multiple protein configurations.

Visualization of single proteins from stripped native cell membranes: a protocol for high-resolution atomic force microscopy.

Atomic force microscopy (AFM) proved to be able to obtain high-resolution three-dimensional images of single-membrane proteins, isolated, crystallized, or included in reconstructed model membranes. The extension of this technique to native systems, such as the protein immersed in a cell membrane, needs a careful manipulation of the biological sample to meet the experimental constraints for high-resolution AFM imaging. In this article, a general protocol for sample preparation is presented, based on the mechanical stretch of the cell membrane. The effectiveness for AFM imaging has been tested on the basis of an integrated optical and AFM approach and the proposed method has been applied to cells expressing cystic fibrosis transmembrane conductance regulator, a channel involved in cystic fibrosis, showing the possibility to identify and analyze single proteins in the plasma membrane.

Reflection-mode TERS on Insulin Amyloid Fibrils with Top-Visual AFM Probes.

Tip-enhanced Raman spectroscopy provides chemical information while raster scanning samples with topographical detail. The coupling of atomic force microscopy and Raman spectroscopy in top illumination optical setup is a powerful configuration to resolve nanometer structures while collecting reflection mode backscattered signal. Here, we theoretically calculate the field enhancement generated by TER spectroscopy with top illumination geometry and we apply the technique to the characterization of insulin amyloid fibrils. We experimentally confirm that this technique is able to enhance the Raman signal of the polypeptide chain by a factor of 105, thus revealing details down to few molecules resolution.

Direct imaging of DNA fibers: the visage of double helix.

Direct imaging becomes important when the knowledge at few/single molecule level is requested and where the diffraction does not allow to get structural and functional information. Here we report on the direct imaging of double stranded (ds) λ-DNA in the A conformation, obtained by combining a novel sample preparation method based on super hydrophobic DNA molecules self-aggregation process with transmission electron microscopy (TEM). The experimental breakthrough is the production of robust and highly ordered paired DNA nanofibers that allowed its direct TEM imaging and the double helix structure revealing.

An ON/OFF biosensor based on blockade of ionic current passing through a solid-state nanopore.

Single nanopores have attracted interest for their use as biosensing devices. In general, methods involve measuring ionic current blockades associated with translocation of analytes through the nanopore, but the detection of such short time lasting events requires complex equipment and setup that are critical for convenient routine biosensing. Here we present a novel biosensing concept based on a single nanopore in a silicon nitride membrane and two anchor-linked DNA species that forms trans-pore hybrids, realizing a stable blockade of ionic current through the pore. Molecular recognition events affecting the DNA hybrids cause a pore opening and the consequent establishment of an ionic current. In the present implementation of the device, we constructed a magnetic bead/streptavidin/biotin-DNA1/DNA2-biotin/streptavidin/Quantumdot-cluster complex (where DNA1 is a mismatched reverse complement of DNA2) through a sub-micrometric pore and monitored DNA strand displacement events occurring after addition of an oligonucleotide complementary to DNA2. The electric and mechanical aspects of the novel device, as well as its potential in biosensing are discussed.