Material-specific binding peptides empower sustainable innovations in plant health, biocatalysis, medicine and microplastic quantification.

Material-binding peptides (MBPs) have emerged as a diverse and innovation-enabling class of peptides in applications such as plant-/human health, immobilization of catalysts, bioactive coatings, accelerated polymer degradation and analytics for micro-/nanoplastics quantification. Progress has been fuelled by recent advancements in protein engineering methodologies and advances in computational and analytical methodologies, which allow the design of, for instance, material-specific MBPs with fine-tuned binding strength for numerous demands in material science applications. A genetic or chemical conjugation of second (biological, chemical or physical property-changing) functionality to MBPs empowers the design of advanced (hybrid) materials, bioactive coatings and analytical tools. In this review, we provide a comprehensive overview comprising naturally occurring MBPs and their function in nature, binding properties of short man-made MBPs (<20 amino acids) mainly obtained from phage-display libraries, and medium-sized binding peptides (20-100 amino acids) that have been reported to bind to metals, polymers or other industrially produced materials. The goal of this review is to provide an in-depth understanding of molecular interactions between materials and material-specific binding peptides, and thereby empower the use of MBPs in material science applications. Protein engineering methodologies and selected examples to tailor MBPs toward applications in agriculture with a focus on plant health, biocatalysis, medicine and environmental monitoring serve as examples of the transformative power of MBPs for various industrial applications. An emphasis will be given to MBPs' role in detecting and quantifying microplastics in high throughput, distinguishing microplastics from other environmental particles, and thereby assisting to close an analytical gap in food safety and monitoring of environmental plastic pollution. In essence, this review aims to provide an overview among researchers from diverse disciplines in respect to material-(specific) binding of MBPs, protein engineering methodologies to tailor their properties to application demands, re-engineering for material science applications using MBPs, and thereby inspire researchers to employ MBPs in their research.

Directed Evolution of Material Binding Peptide for Polylactic Acid-specific Degradation in Mixed Plastic Wastes.

In order to preserve our livelihood for future generations, responsible use of plastics in a climate-neutral and circular economy has to be developed so that plastics can be used in an environmentally friendly way by future generations. The prerequisite is that bioplastic polymers such as polylactic acid (PLA) can be efficiently recycled from petrochemical based plastic. Here, a concept in which accelerated PLA degradation in the mixed suspension of PLA and polystyrene (PS) nanoparticles has been achieved through an engineered material binding peptide. After comparison of twenty material binding peptides, Cg-Def is selected due to its PLA binding specificity. Finally, a suitable high-throughput screening system is developed for enhancing material-specific binding toward PLA in presence of PS. Through KnowVolution campaign, a variant Cg-Def YH (L9Y/S19H) with 2.0-fold improved PLA binding specificity compared to PS is generated. Contact angle and surface plasmon resonance measurements validated higher surface coverage of Cg-Def YH on PLA surface and the fusion of Cg-Def YH with PLA degrading enzyme confirmed the accelerated PLA depolymerization (two times higher than only enzyme) in mixed PLA/PS plastics.

A Competitive High-Throughput Screening Platform for Designing Polylactic Acid-Specific Binding Peptides.

Among biobased polymers, polylactic acid (PLA) is recognized as one of the most promising bioplastics to replace petrochemical-based polymers. PLA is typically blended with other polymers such as polypropylene (PP) for improved melt processability, thermal stability, and stiffness. A technical challenge in recycling of PLA/PP blends is the sorting/separation of PLA from PP. Material binding peptides (MBPs) can bind to various materials. Engineered MBPs that can bind in a material-specific manner have a high potential for material-specific detection or enhanced degradation of PLA in mixed PLA/PP plastics. To obtain a material-specific MBP for PLA binding (termed PLAbodies ), protein engineering of MBP Cg-Def for improved PLA binding specificity is reported in this work. In detail, a 96-well microtiter plate based high-throughput screening system for PLA specific binding (PLABS) was developed and validated in a protein engineering (KnowVolution) campaign. Finally, the Cg-Def variant V2 (Cg-Def S19K/K10L/N13H) with a 2.3-fold improved PLA binding specificity compared to PP was obtained. Contact angle and surface plasmon resonance measurements confirmed improved material-specific binding of V2 to PLA (1.30-fold improved PLA surface coverage). The established PLABS screening platform represents a general methodology for designing PLAbodies for applications in detection, sorting, and material-specific degradation of PLA in mixed plastics.

A Flow Cytometry-Based Ultrahigh-Throughput Screening Method for Directed Evolution of Oxidases.

Oxidases are of interest to chemical and pharmaceutical industries because they catalyze highly selective oxidations. However, oxidases found in nature often need to be re-engineered for synthetic applications. Herein, we developed a versatile and robust flow cytometry-based screening platform "FlOxi" for directed oxidase evolution. FlOxi utilizes hydrogen peroxide produced by oxidases expressed in E. coli to oxidize Fe2+ to Fe3+ (Fenton reaction). Fe3+ mediates the immobilization of a His6 -tagged eGFP (eGFPHis ) on the E. coli cell surface, ensuring the identification of beneficial oxidase variants by flow cytometry. FlOxi was validated with two oxidases-a galactose oxidase (GalOx) and a D-amino acid oxidase (D-AAO)-yielding a GalOx variant (T521A) with a 4.4-fold lower Km value and a D-AAO variant (L86M/G14/A48/T205) with a 4.2-fold higher kcat than their wildtypes. Thus, FlOxi can be used for the evolution of hydrogen peroxide-producing oxidases and applied for non-fluorescent substrates.

Plastibodies for multiplexed detection and sorting of microplastic particles in high-throughput.

Sensitive high-throughput analytic methodologies are needed to quantify microplastic particles (MPs) and thereby enable routine monitoring of MPs to ultimately secure animal, human, and environmental health. Here we report a multiplexed analytical and flow cytometry-based high-throughput methodology to quantify MPs in aqueous suspensions. The developed analytic MPs-quantification platform provides a sensitive as well as high-throughput detection of MPs that relies on the material binding peptide Liquid Chromatography Peak I (LCI) conjugated to Alexa-fluorophores (LCIF16C-AF488, LCIF16C-AF594, and LCIF16C-AF647). These fluorescent material-binding peptides (also termed plastibodies) were used to fluorescently label polystyrene MPs, whereas Alexa-fluorophores alone exhibited a negligible background fluorescence. Mixtures of polystyrene MPs that varied in size (500 nm to 5 µm) and varied in labeled populations were analyzed and sorted into distinct populations reaching sorting efficiencies >90 % for 1 × 106 sorted events. Finally, a multiplexed quantification and sorting with up to three plastibodies was successfully achieved to validate that the combination of plastibodies and flow cytometry is a powerful and generally applicable methodology for multiplexed analysis, quantification, and sorting of microplastic particles.

Rational Design Yields Molecular Insights on Leaf-Binding of Anchor Peptides.

In times of a constantly growing world population and increasing demand for food, sustainable agriculture is crucial. The rainfastness of plant protection agents is of pivotal importance to reduce the amount of applied nutrients, herbicides, and fungicides. As a result of protective agent wash-off, plant protection is lost, and soils and groundwater are severely polluted. To date, rainfastness of plant protection products has been achieved by adding polymeric adjuvants to the agrochemicals. However, polymeric adjuvants will be regarded as microplastics in the future, and environmentally friendly alternatives are needed. Anchor peptides (APs) are promising biobased and biodegradable adhesion promoters. Although the adhesion of anchor peptides to artificial surfaces, such as polymers, has already been investigated in theory and experimentally, exploiting the adhesion to biological surfaces remains challenging. The complex nature and composition of biological surfaces such as plant leaves and fruit surfaces complicate the generation of accurate models. Here, we present the first detailed three-layered atomistic model of the surface of apple leaves and use it to compute free energy profiles of the adhesion and desorption of APs to and from that surface. Our model is validated by a novel fluorescence-based microtiter plate (MTP) assay that mimics these complex processes and allows for quantifying them. For the AP Macaque Histatin, we demonstrate that aromatic and positively charged amino acids are essential for binding to the waxy apple leaf surface. The established protocols should generally be applicable for tailoring the binding properties of APs to biological interfaces.

Real-Time Label-Free Direct Electronic Monitoring of Topoisomerase Enzyme Binding Kinetics on Graphene.

Monolayer graphene field-effect sensors operating in liquid have been widely deployed for detecting a range of analyte species often under equilibrium conditions. Here we report on the real-time detection of the binding kinetics of the essential human enzyme, topoisomerase I interacting with substrate molecules (DNA probes) that are immobilized electrochemically on to monolayer graphene strips. By monitoring the field-effect characteristics of the graphene biosensor in real-time during the enzyme-substrate interactions, we are able to decipher the surface binding constant for the cleavage reaction step of topoisomerase I activity in a label-free manner. Moreover, an appropriate design of the capture probes allows us to distinctly follow the cleavage step of topoisomerase I functioning in real-time down to picomolar concentrations. The presented results are promising for future rapid screening of drugs that are being evaluated for regulating enzyme activity.

Self-assembled electrical biodetector based on reduced graphene oxide.

Large-scale fabrication of graphene-based devices is an aspect of great importance for various applications including chemical and biological sensing. Toward this goal, we present here a novel chemical route for the site-specific realization of devices based on reduced graphene oxide (RGO). Electrodes patterned by photolithography are modified with amino functional groups through electrodeposition. The amine groups function as hooks for the attachment of graphene oxide flakes selectively onto the electrodes. Graphene-like electrical behavior is attained by a subsequent thermal annealing step. We show that this anchoring strategy can be scaled-up to obtain RGO devices at a wafer scale in a facile manner. The scalability of our approach coupled with the use of photolithography is promising for the rapid realization of graphene-based devices. We demonstrate one possible application of the fabricated RGO devices as electrical biosensors through the immunodetection of amyloid beta peptide.

Towards in vitro molecular diagnostics using nanostructures.

Nanostructures appear to be promising for a number of applications in molecular diagnostics, mainly due to the increased surface-to-volume ratio they can offer, the very low limit of detection achievable, and the possibility to fabricate point-of-care diagnostic devices. In this paper, we review examples of the use of nanostructures as diagnostic tools that bring in marked improvements over prevalent classical assays. The focus is laid on the various sensing paradigms that possess the potential or have demonstrated the capability to replace or augment current analytical strategies. We start with a brief introduction of the various types of nanostructures and their physical properties that determine the transduction principle. This is followed by a concise collection of various functionalization protocols used to immobilize biomolecules on the nanostructure surface. The sensing paradigms are discussed in two contexts: the nanostructure acting as a label for detection, or the nanostructure acting as a support upon which the molecular recognition events take place. In order to be successful in the field of molecular diagnostics, it is important that the nanoanalytical tools be evaluated in the appropriate biological environment. The final section of the review compiles such examples, where the nanostructure-based diagnostic tools have been tested on realistic samples such as serum, demonstrating their analytical power even in the presence of complex matrix effects. The ability of nanodiagnostic tools to detect ultralow concentrations of one or more analytes coupled with portability and the use of low sample volumes is expected to have a broad impact in the field of molecular diagnostics.

Enzyme-free sugar sensing in microfluidic channels with an affinity-based single-wall carbon nanotube sensor.

We present a novel nonenzymatic carbon nanotube sensor integrated in a microfluidic channel for the detection of sugars. The sensor is assembled as a liquid-gated field-effect transistor, with the transistor channel composed of 1 to 10 nanotubes, which are controllably functionalized with boronic acid receptors. The devices show sensitivity to glucose in a concentration range of 5 to 30 mM. Furthermore, by controlling the type of nanotube-receptor coupling (as covalent or noncovalent) and by deploying a sensitive impedance-based detection technique, we corroborate in detail the transduction mechanism of our affinity-based sensor. In the case of covalent coupling, charge carrier scattering along the nanotubes is the dominant mechanism. While in the noncovalent case, surface charge effects dominate. The identification of the mechanism along with the tunability of the chemical coupling and the cost-effective integration in microchannels constitute a solid basis for the entry of nanotube-based sensors in lab-on-a-chip applications.

Applications of the static quenching of rhodamine B by carbon nanotubes.

We report here on the interaction of the fluorescent dye rhodamine B (RB) with single-walled carbon nanotubes (SWCNTs). We observe that SWCNTs statically quench the fluorescence of RB by forming a stable ground state complex. Careful spectroscopic analysis indicates that the complex formation is efficient mainly with certain chiral forms. We propose three different applications utilizing this quenching mechanism and the associated complexation. Firstly, the quenching efficiency can be utilized as a measure for the characterization and quantification of nanotube dispersions. Secondly, we demonstrate that the specific complexation of RB can be deployed to enrich certain chiral forms in suspension. Finally, we show that RB can be effectively used to visualize nanotubes deposited on substrates.