Adapting arrays and lab-on-a-chip technology for proteomics

The impact of proteomics as a discovery engine in life science and in drug discovery has increased tremendously over the last seven years. At the same time, proteomics has expanded from the initial trust as a two-dimensional gel based approach to cover more functional and structural properties of proteins. The development of lab-on-a-chip and protein arrays for proteomics will have to evolve with the changes in proteomics to stay relevant. Here, we review the changes in the field of proteomics and their impact on the development in protein arrays and lab-on-a-chip.     

Self-assembly of repeat proteins: Concepts and design of new interfaces

In nature, assembled protein structures offer the most complex functional structures. The understanding of the mechanisms ruling protein–protein interactions opens the door to manipulate protein assemblies in a rational way. Proteins are versatile scaffolds with great potential as tools in nanotechnology and biomedicine because of their chemical, structural, and functional versatility. Currently, bottom-up self-assembly based on biomolecular interactions of small and well-defined components, is an attractive approach to biomolecular engineering and biomaterial design. Specifically, repeat proteins are simplified systems for this purpose.In this work, we provide an overview of fundamental concepts of the design of new protein interfaces. We describe an experimental approach to form higher order architectures by a bottom-up assembly of repeated building blocks. For this purpose, we use designed consensus tetratricopeptide repeat proteins (CTPRs). CTPR arrays contain multiple identical repeats that interact through a single inter-repeat interface to form elongated superhelices. Introducing a novel interface along the CTPR superhelix allows two CTPR molecules to assemble into protein nanotubes. We apply three approaches to form protein nanotubes: electrostatic interactions, hydrophobic interactions, and π-π interactions. We isolate and characterize the stability and shape of the formed dimers and analyze the nanotube formation considering the energy of the interaction and the structure in the three different models. These studies provide insights into the design of novel protein interfaces for the control of the assembly into more complex structures, which will open the door to the rational design of nanostructures and ordered materials for many potential applications in nanotechnology.     

Peptoids at the 7th Summit: toward macromolecular systems engineering

Methods for facile synthesis of extraordinarily diverse peptide-like oligomers have placed peptoids at the center of a broad and vibrant area of foldamer science and technology. The 7th Peptoid Summit offered a perspective on the current state of peptoid science and technology and on prospects for engineering supramolecular assemblies that rival the complexity of biomolecular systems. Methods for engineering biomolecular systems based on DNA and protein are advancing rapidly, building a technology platform for engineering increasingly large and complex self-assembled nanosystems. A comparative review of the physical basis for DNA, protein, and peptoid engineering indicates that the characteristics of peptoids suit them for a strong role in developing self-assembled nanosystems. Physical parallels between peptoids and proteins indicate that peptoid engineering, like protein engineering, will require specialized software to support design. Access to novel side-chain functionality will enable peptoid designers to exploit novel binding interactions, including many that have been discovered and exploited in crystal engineering, a field that has extensively explored the self-assembly of small organic molecules to form well-ordered structures. Developments in DNA, protein, and inorganic nanotechnologies are converging to provide a technology platform for the design and fabrication of complex, functional, atomically precise nanosystems. Peptoid-based foldamer technologies, can contribute to this convergence, expanding the scope of the emerging field of atomically precise macromolecular nanosystems.     

合成生物学技术在材料科学中的应用

材料是人类赖以生存与发展的物质基础,科技和社会的进步都离不开材料技术的发展,未来先进材料的合成和制备必然朝着绿色可持续、低耗高产出、精细可调控、高效多功能的方向发展。以"基因调控·工程设计"为核心的合成生物学技术从分子、细胞层面极大地推动了生命科学的发展,也已经并继续为材料科学的发展注入新的思路和活力。本文将围绕合成生物学技术在材料科学中的应用,以基因回路设计为核心,概念应用为线索,重点介绍合成生物学技术在高分子生物材料和无机纳米材料领域的开发和生产,细胞展示和蛋白定向进化战略对分子材料的筛选和优化,"活体"功能材料、工程菌调节的人工光合系统功能材料体系以及基因回路在材料科学中的应用。     

Molecular and functional aspects of latent transforming growth factor-β binding protein: just a masking protein?

Latent transforming growth factor-beta (TGFbeta) binding protein (LTBP), a component of the high-molecular-weight latent TGFbeta complex, is found in various cell and tissue types. Originally described as a TGFbeta-masking protein, recent detections of four isoforms and numerous splice variants provide new aspects of its putative functional role. Regulation and sequestration of TGFbeta activity and structural remodeling of the extracellular matrix (ECM) seem to be the main tasks, but other possible functions might exist. The mechanism by which LTBP interacts with cell surface molecules or cellular receptors and ECM components remains unclear. Cellular, molecular and functional aspects will be discussed.     

Arraying proteins by cell-free synthesis

Recent advances in life science have led to great motivation for the development of protein arrays to study functions of genome-encoded proteins. While traditional cell-based methods have been commonly used for generating protein arrays, they are usually a time-consuming process with a number of technical challenges. Cell-free protein synthesis offers an attractive system for making protein arrays, not only does it rapidly converts the genetic information into functional proteins without the need for DNA cloning, but also presents a flexible environment amenable to production of folded proteins or proteins with defined modifications. Recent advancements have made it possible to rapidly generate protein arrays from PCR DNA templates through parallel on-chip protein synthesis. This article reviews current cell-free protein array technologies and their proteomic applications.     

Synthetic biology advancing clinical applications

The 'omics' era, with its identification of genetic and protein components, has combined with systems biology, which provided insights into network structures, to set the stage for synthetic biology, an emerging interdisciplinary life science that uses engineering principles. By capitalizing on an iterative design cycle that involves molecular and computational biology tools to assemble functional designer devices from a comprehensive catalogue of standardized biological components with predictable functions, synthetic biology has significantly advanced our understanding of complex control dynamics that program living systems. Such insights, collected over the past decade, are priming a variety of synthetic biology-inspired biomedical applications that have the potential to revolutionize drug discovery and production technologies, as well as treatment strategies for infectious diseases and metabolic disorders.     

Prospects of nanoparticle–DNA binding and its implications in medical biotechnology

Bio-nanotechnology is a new interdisciplinary R&D area that integrates engineering and physical science with biology through the development of multifunctional devices and systems, focusing biology inspired processes or their applications, in particular in medical biotechnology. DNA based nanotechnology, in many ways, has been one of the most intensively studied fields in recent years that involves the use and the creation of bio-inspired materials and their technologies for highly selective biosensing, nanoarchitecture engineering and nanoelectronics. Increasing researches have been offered to a fundamental understanding how the interactions between the nanoparticles and DNA molecules could alter DNA molecular structure and its biochemical activities. This minor review describes the mechanisms of the nanoparticle–DNA binding and molecular interactions. We present recent discoveries and research progresses how the nanoparticle–DNA binding could vary DNA molecular structure, DNA detection, and gene therapy. We report a few case studies associated with the application of the nanoparticle–DNA binding devices in medical detection and biotechnology. The potential impacts of the nanoparticles via DNA binding on toxicity of the microorganisms are briefly discussed. The nanoparticle–DNA interactions and their impact on molecular and microbial functionalities have only drown attention in recent a few years. The information presented in this review can provide useful references for further studies on biomedical science and technology.     

S-layers as a tool kit for nanobiotechnological applications

Crystalline bacterial cell surface layers (S-layers) have been identified in a great number of different species of bacteria and represent an almost universal feature of archaea. Isolated native S-layer proteins and S-layer fusion proteins incorporating functional sequences self-assemble into monomolecular crystalline arrays in suspension, on a great variety of solid substrates and on various lipid structures including planar membranes and liposomes. S-layers have proven to be particularly suited as building blocks and patterning elements in a biomolecular construction kit involving all major classes of biological molecules (proteins, lipids, glycans, nucleic acids and combinations of them) enabling innovative approaches for the controlled 'bottom-up' assembly of functional supramolecular structures and devices. Here, we review the basic principles of S-layer proteins and the application potential of S-layers in nanobiotechnology and biomimetics including life and nonlife sciences.     

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

Protein stability and ligand‐binding affinity measurements are widely required for the formulation of biopharmaceutical proteins, protein engineering and drug screening within life science research. Current techniques either consume too much of often precious biological or compound materials, in large sample volumes, or alternatively require chemical labeling with fluorescent tags to achieve measurements at submicrolitre volumes with less sample. Here we present a quantitative and accurate method for the determination of protein stability and the affinity for small molecules, at only 1.5–20 nL optical sample volumes without the need for fluorescent labeling, and that takes advantage of the intrinsic tryptophan fluorescence of most proteins. Coupled to appropriate microfluidic sample preparation methods, the sample requirements could thus be reduced 85,000‐fold to just 10⁸ molecules. The stability of wild‐type FKBP‐12 and a destabilizing binding‐pocket mutant are studied in the presence and absence of rapamycin, to demonstrate the potential of the technique to both drug screening and protein engineering. The results show that 75% of the interaction energy between FKBP‐12 and rapamycin originates from residue Phe99 in the binding site.     

同步PCR技术及其在植物核酸分子定量中的应用

同步PCR是一种集生化、光电和计算机技术于一体的封闭式DNA扩增系统,采用荧光染料将扩增与检测过程结合在一起,实现了在PCR过程中在线显示PCR反应,通过检测荧光强度来绝对定量起始模板的拷贝数。该技术大大简化和加速了核酸分子的定量过程,不仅快速、灵敏、准确、重复性好,而且很容易计算出待测样品中核酸分子的绝对起始拷贝数。同微阵列等分子生物技术一起,同步PcR技术将会在功能基因解析和病害分子诊断等方面发挥重要作用。本综述除了介绍同步．PCR技术的原理和应用外,还介绍了定量拟南芥,Aux／正4,4基因的转录水平的实验,并就同步PCR操作过程中的问题进行了讨论。     

Composite scaffolds for cartilage tissue engineering

Tissue engineering remains a promising therapeutic strategy for the repair or regeneration of diseased or damaged tissues. Previous approaches have typically focused on combining cells and bioactive molecules (e.g., growth factors, cytokines and DNA fragments) with a biomaterial scaffold that functions as a template to control the geometry of the newly formed tissue, while facilitating the attachment, proliferation, and differentiation of embedded cells. Biomaterial scaffolds also play a crucial role in determining the functional properties of engineered tissues, including biomechanical characteristics such as inhomogeneity, anisotropy, nonlinearity or viscoelasticity. While single-phase, homogeneous materials have been used extensively to create numerous types of tissue constructs, there continue to be significant challenges in the development of scaffolds that can provide the functional properties of load-bearing tissues such as articular cartilage. In an attempt to create more complex scaffolds that promote the regeneration of functional engineered tissues, composite scaffolds comprising two or more distinct materials have been developed. This paper reviews various studies on the development and testing of composite scaffolds for the tissue engineering of articular cartilage, using techniques such as embedded fibers and textiles for reinforcement, embedded solid structures, multi-layered designs, or three-dimensionally woven composite materials. In many cases, the use of composite scaffolds can provide unique biomechanical and biological properties for the development of functional tissue engineering scaffolds.     

同步PCR技术及其在植物核酸分子定量中的应用

同步PCR是一种集生化、光电和计算机技术于一体的封闭式DNA扩增系统,采用荧光染料将扩增与检测过程结合在一起,实现了在PCR过程中在线显示PCR反应,通过检测荧光强度来绝对定量起始模板的拷贝数.该技术大大简化和加速了核酸分子的定量过程,不仅快速、灵敏、准确、重复性好,而且很容易计算出待测样品中核酸分子的绝对起始拷贝数.同微阵列等分子生物技术一起,同步PCR技术将会在功能基因解析和病害分子诊断等方面发挥重要作用.本综述除了介绍同步PCR技术的原理和应用外,还介绍了定量拟南芥Aux/IAA基因的转录水平的实验,并就同步PCR操作过程中的问题进行了讨论.     

Rational Design of Metal‐Organic Framework Derived Hollow NiCo2O4 Arrays for Flexible Supercapacitor and Electrocatalysis

Metal‐organic frameworks (MOFs) are promising porous precursors for the construction of various functional materials for high‐performance electrochemical energy storage and conversion. Herein, a facile two‐step solution method to rational design of a novel electrode of hollow NiCo₂O₄ nanowall arrays on flexible carbon cloth substrate is reported. Uniform 2D cobalt‐based wall‐like MOFs are first synthesized via a solution reaction, and then the 2D solid nanowall arrays are converted into hollow and porous NiCo₂O₄ nanostructures through an ion‐exchange and etching process with an additional annealing treatment. The as‐obtained NiCo₂O₄ nanostructure arrays can provide rich reaction sites and short ion diffusion path. When evaluated as a flexible electrode material for supercapacitor, the as‐fabricated NiCo₂O₄ nanowall electrode shows remarkable electrochemical performance with excellent rate capability and long cycle life. In addition, the hollow NiCo₂O₄ nanowall electrode exhibits promising electrocatalytic activity for oxygen evolution reaction. This work provides an example of rational design of hollow nanostructured metal oxide arrays with high electrochemical performance and mechanical flexibility, holding great potential for future flexible multifunctional electronic devices.     

Polypeptoid polymers: Synthesis,characterization, and properties

Polypeptoids, a class of peptidomimetic polymers, have emerged at the forefront of macromolecular and supramolecular science and engineering as the technological relevance of these polymers continues to be demonstrated. The chemical and structural diversity of polypeptoids have enabled access to and adjustment of a variety of physicochemical and biological properties (eg, solubility, charge characteristics, chain conformation, HLB, thermal processability, degradability, cytotoxicity and immunogenicity). These attributes have made this synthetic polymer platform a potential candidate for various biomedical and biotechnological applications. This review will provide an overview of recent development in synthetic methods to access polypeptoid polymers with well‐defined structures and highlight some of the fundamental physicochemical and biological properties of polypeptoids that are pertinent to the future development of functional materials based on polypeptoids.     

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays¹. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration^2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein ⁴, protein-RNA⁵ or protein-DNA⁶ interactions.The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins⁷, DNA⁸, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.     

Nanotechnology in regenerative medicine: the materials side

Regenerative medicine is an emerging multidisciplinary field that aims to restore, maintain or enhance tissues and hence organ functions. Regeneration of tissues can be achieved by the combination of living cells, which will provide biological functionality, and materials, which act as scaffolds to support cell proliferation. Mammalian cells behave in vivo in response to the biological signals they receive from the surrounding environment, which is structured by nanometre-scaled components. Therefore, materials used in repairing the human body have to reproduce the correct signals that guide the cells towards a desirable behaviour. Nanotechnology is not only an excellent tool to produce material structures that mimic the biological ones but also holds the promise of providing efficient delivery systems. The application of nanotechnology to regenerative medicine is a wide issue and this short review will only focus on aspects of nanotechnology relevant to biomaterials science. Specifically, the fabrication of materials, such as nanoparticles and scaffolds for tissue engineering, and the nanopatterning of surfaces aimed at eliciting specific biological responses from the host tissue will be addressed.     

Arraying prostate specific antigen PSA and Fab anti-PSA using light-assisted molecular immobilization technology

We here report for the first time the creation of prostate specific antigen (PSA) and Fab anti‐PSA biosensor arrays using UV light‐assisted molecular immobilization (LAMI), aiming at the detection and quantification of PSA, a cancer marker. The technology involves formation of free, reactive thiol groups upon UV excitation of protein aromatic residues located in spatial proximity of disulphide bridges, a conserved structural feature in both PSA and Fab molecules. The created thiol groups bind onto thiol reactive surfaces leading to oriented covalent protein immobilization. Protein activity was confirmed carrying out immunoassays: immobilized PSA was recognized by Fab anti‐PSA in solution and immobilized Fab anti‐PSA cross‐reacted with PSA in solution. LAMI technology proved successful in immobilizing biomedically relevant molecules while preserving their activity, highlighting that insight into how light interacts with biomolecules may lead to new biophotonic technologies. Our work focused on the application of our new engineering principles to the design, analysis, construction, and manipulation of biological systems, and on the discovery and application of new engineering principles inspired by the properties of biological systems.     

Bionanoscience at the plant virus-inorganic chemistry interface

Bionanoscience is an inter-disciplinary area of research that sits at the interface of chemistry, biology, materials science, engineering and medicine. During the past 10 years the suitability and applicability of using plant viruses as building blocks, synthons, scaffolds or templates in bionanoscience/technology have begun to be explored. This short review describes how the plant Cowpea mosaic virus can be functionalised on its outer surface to form electroactive nanoparticles, can be used to construct monolayers on solid surfaces and multilayer arrays by a bottom-up, layer-by-layer approach, and how it can template mineralization processes to give new routes to monodisperse nanoparticles. Potential applications of virus-derived nanoparticles include nanoelectronics, sensory devices, catalysis, photonics and medical applications from imaging to the targeting and delivery of therapeutic agents.     

A modular approach to the design of protein-based smart gels

The modular nature of repeat proteins makes them a versatile platform for the design of smart materials with predetermined properties. Here, we present a general strategy for combining protein modules with specified stability and function into arrays for the assembly of stimuli-responsive gels. We have designed tetratricopeptide repeat (TPR) arrays which contain peptide-binding modules that specify the strength and reversibility of network crosslinking in combination with spacer modules that specify crosslinking geometry and overall stability of the array. By combining such arrays with multivalent peptide ligands, self-supporting stimuli-responsive gels are formed. Using microrheology, we characterized the kinetics of gelation as a function of concentration and stoichiometry of the components. We also show that such gels are effective in encapsulating and releasing small molecules. Moreover, TPR gels alone are fully compatible with cell growth, whereas gels loaded with an anticancer compound release the compound, resulting in cell death. Thus, we have demonstrated that this new class of tunable biomaterials is ripe for further development as tissue engineering and drug delivery platform.