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.

ASFV epitope mapping by high density peptides microarrays.

African swine fever (ASF) is an acute, highly contagious and deadly infectious disease. It is a threat to animal health with major potential economic and societal impact. Despite decades of ASF vaccine research, still some gaps in knowledge are hindering the development of a functional vaccine. Worth mentioning are gaps in understanding the mechanism of ASF infection and immunity, as well as the fact that - in case of this disease - virus proteins, so-called protective antigens, responsible for inducing protective immune responses in pigs are not identified yet. In this paper we elaborate on a methodology to identify protective antigens based on epitope mapping by microarray technology. High density peptide microarrays, combined with fluorescence scanning, have been used to analyze the interaction of peptide sequences of African swine fever virus (ASFV) proteins with antibodies present in inactivated serum from infected and healthy animals. The study evidenced ASFV proteins already under the radar for vaccine development, such as p54, and identified specific sequences in those proteins that may become the focus for future vaccine candidates. Such methodology is amenable to automation and high-throughput and may help developing better targeting for next generation vaccines.

Rapid prototyping of a polymer MEMS droplet dispenser by laser-assisted 3D printing.

In this work, we introduce a polymer version of a previously developed silicon MEMS drop deposition tool for surface functionalization that consists of a microcantilever integrating an open fluidic channel and a reservoir. The device is fabricated by laser stereolithography, which offers the advantages of low-cost and fast prototyping. Additionally, thanks to the ability to process multiple materials, a magnetic base is incorporated into the cantilever for convenient handling and attachment to the holder of a robotized stage used for spotting. Droplets with diameters ranging from ∼50 µm to ∼300 µm are printed upon direct contact of the cantilever tip with the surface to pattern. Liquid loading is achieved by fully immersing the cantilever into a reservoir drop, where a single load results in the deposition of more than 200 droplets. The influences of the size and shape of the cantilever tip and the reservoir on the printing outcome are studied. As a proof-of-concept of the biofunctionalization capability of this 3D printed droplet dispenser, microarrays of oligonucleotides and antibodies displaying high specificity and no cross-contamination are fabricated, and droplets are deposited at the tip of an optical fiber bundle.

Inter-Individual Variations: A Challenge for the Standardisation of Complement Activation Assays.

Introduction: The role of the human immune system in pathologic responses to chemicals including nanomaterials was identified as a gap in current hazard assessments. However, the complexity of the human immune system as well as interspecies variations make the development of predictive toxicity tests challenging. In the present study, we have analysed to what extent fluctuations of the complement system of different individuals will have an impact on the standardisation of immunological tests. Methods: We treated commercially available pooled sera (PS) from healthy males, individual sera from healthy donors and from patients suffering from cancer, immunodeficiency and allergies with small molecules and liposomes. Changes of iC3b protein levels measured in enzyme-linked immunosorbent assays served as biomarker for complement activation. Results: The level of complement activation in PS differed significantly from responses of individual donors (p < 0.01). Only seven out of 32 investigated sera from healthy donors responded similarly to the pooled serum. This variability was even more remarkable when investigating the effect of liposomes on the complement activation in sera from donors with pre-existing pathologies. Neither the 26 sera of donors with allergies nor sera of 16 donors with immunodeficiency responded similar to the PS of healthy donors. Allergy sufferers showed an increase in iC3b levels of 4.16-fold changes when compared to PS treated with liposomes. Discussion: Our studies demonstrate that the use of pooled serum can lead to an over- or under-estimation of immunological response in particular for individuals with pre-existing pathologies. This is of high relevance when developing medical products based on nanomaterials and asks for a review of the current practice to use PS from healthy donors for the prediction of immunological effects of drugs in patients. A better understanding of individual toxicological responses to xenobiotics should be an essential part in safety assessments.

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.

Ligand-Receptor Interactions of Galectin-9 and VISTA Suppress Human T Lymphocyte Cytotoxic Activity.

Acute myeloid leukemia (AML), a blood/bone marrow cancer, is a severe and often fatal malignancy. AML cells are capable of impairing the anti-cancer activities of cytotoxic lymphoid cells. This includes the inactivation of natural killer (NK) cells and killing of T lymphocytes. Here we report for the first time that V-domain Ig-containing suppressor of T cell activation (VISTA), a protein expressed by T cells, recognizes galectin-9 secreted by AML cells as a ligand. Importantly, we found that soluble VISTA released by AML cells enhances the effect of galectin-9, most likely by forming multiprotein complexes on the surface of T cells and possibly creating a molecular barrier. These events cause changes in the plasma membrane potential of T cells leading to activation of granzyme B inside cytotoxic T cells, resulting in apoptosis.

Deposition of environmentally relevant nanoplastic models in sand during transport experiments.

Nanoplastics (NPTs) are defined as colloids that originated from the unintentional degradation of plastic debris. To understand the possible risks caused by NPTs, it is crucial to determine how they are transported and where they may finally accumulate. Unfortunately, although most sources of plastic are land-based, risk assessments concerning NPTs in the terrestrial environmental system (soils, aquifers, freshwater sediments, etc.) have been largely lacking compared to studies concerning NPTs in the marine system. Furthermore, an important limitation of environmental fate studies is that the NPT models used are questionable in terms of their environmental representativeness. This study describes the fate of different NPT models in a porous media under unfavorable (repulsive) conditions, according to their physical and chemical properties: average hydrodynamic diameters (200-460 nm), composition (polystyrene with additives or primary polystyrene) and shape (spherical or polymorphic). NPTs that more closely mimic environmental NPTs present an inhomogeneous shape (i.e., deviating from a sphere) and are more deposited in a sand column by an order of magnitude. This deposition was attributed in part to physical retention, as confirmed by the straining that occurred for the larger size fractions. Additionally, different Derjaguin-Landau-Verwey-Overbeek (DLVO) models -the extended DLVO (XDLVO) and a DLVO modified by surface element integration (SEI) method-suggest that the environmentally relevant NPT models may alter its orientation to diminish repulsion from the sand surface and may find enough kinetic energy to deposit in the primary energetic minimum. These results point to the importance of choosing environmentally relevant NPT models.

Multiplexed Remote SPR Detection of Biological Interactions through Optical Fiber Bundles.

The development of sensitive methods for in situ detection of biomarkers is a real challenge to bring medical diagnosis a step forward. The proof-of-concept of a remote multiplexed biomolecular interaction detection through a plasmonic optical fiber bundle is demonstrated here. The strategy relies on a fiber optic biosensor designed from a 300 µm diameter bundle composed of 6000 individual optical fibers. When appropriately etched and metallized, each optical fiber exhibits specific plasmonic properties. The surface plasmon resonance phenomenon occurring at the surface of each fiber enables to measure biomolecular interactions, through the changes of the retro-reflected light intensity due to light/plasmon coupling variations. The functionalization of the microstructured bundle by multiple protein probes was performed using new polymeric 3D-printed microcantilevers. Such soft cantilevers allow for immobilizing the probes in micro spots, without damaging the optical microstructures nor the gold layer. We show here the potential of this device to perform the multiplexed detection of two different antibodies with limits of detection down to a few tenths of nanomoles per liter. This tool, adapted for multiparametric, real-time, and label free monitoring is minimally invasive and could then provide a useful platform for in vivo targeted molecular analysis.

Dark Field Microscopy-Based Biosensors for the Detection of E. coli in Environmental Water Samples.

Development of sensitive methods for the determination of E. coli bacteria contamination in water distribution systems is of paramount importance to ensure the microbial safety of drinking water. This work presents a new sensing platform enabling the fast detection of bacteria in field samples by using specific antibodies as the biorecognition element and dark field microscopy as the detection technique. The development of the sensing platform was performed using non-pathogenic bacteria, with the E. coli DH5α strain as the target, and Bacillus sp. 9727 as the negative control. The identification of the captured bacteria was made by analyzing the dark field microscopy images and screening the detected objects by using object circularity and size parameters. Specificity tests revealed the low unspecific attachment of either E. coli over human serum albumin antibodies (negative control for antibody specificity) and of Bacillus sp. over E. coli antibodies. The system performance was tested using field samples, collected from a wastewater treatment plant, and compared with two quantification techniques (i.e., Colilert-18 test and quantitative polymerase chain reaction (qPCR)). The results showed comparable quantification capability. Nevertheless, the present method has the advantage of being faster, is easily adaptable to in-field analysis, and can potentially be extended to the detection of other bacterial strains.

Characterisation of nanomaterial hydrophobicity using engineered surfaces.

Characterisation of engineered nanomaterials (NMs) is of outmost importance for the assessment of the potential risks arising from their extensive use. NMs display indeed a large variety of physico-chemical properties that drastically affect their interaction with biological systems. Among them, hydrophobicity is an important property that is nevertheless only slightly covered by the current physico-chemical characterisation techniques. In this work, we developed a method for the direct characterisation of NM hydrophobicity. The determination of the nanomaterial hydrophobic character is carried out by the direct measurement of the affinity of the NMs for different collectors. Each collector is an engineered surface designed in order to present specific surface charge and hydrophobicity degrees. Being thus characterised by a combination of surface energy components, the collectors enable the NM immobilisation with surface coverage in relation to their hydrophobicity. The experimental results are explained by using the extended DLVO theory, which takes into account the hydrophobic forces acting between NMs and collectors. Graphical abstractDetermination of hydrophobicity character of nanomaterials by measuring their affinity to engineered surfaces.

Paper electrodes for bioelectrochemistry: Biosensors and biofuel cells.

Paper-based analytical devices (PAD) emerge in the scientific community since 2007 as low-cost, wearable and disposable devices for point-of-care diagnostic due to the widespread availability, long-time knowledge and easy manufacturing of cellulose. Rapidly, electrodes were introduced in PAD for electrochemical measurements. Together with biological components, a new generation of electrochemical biosensors was born. This review aims to take an inventory of existing electrochemical paper-based biosensors and biofuel cells and to identify, at the light of newly acquired data, suitable methodologies and crucial parameters in this field. Paper selection, electrode material, hydrophobization of cellulose, dedicated electrochemical devices and electrode configuration in biosensors and biofuel cells will be discussed.

Multiplex microarray ELISA versus classical ELISA, a comparison study of pollutant sensing for environmental analysis.

The present study describes the development, optimization and performance comparison of three ELISAs and one multiplex immunoassay in a microarray format. The developed systems were dedicated to the detection of three different classes of pollutants (pesticide, explosive and toxin) in water. The characteristics and performances of these two types of assays were evaluated and compared, in order to verify that multiplex immunoassays can replace ELISA for multiple analyte sensing. 2,4-Dichlorophenoxyacetic acid, 2,4,6-trinitrotoluene and okadaic acid were chosen as model targets and were immobilized in classical microtiter plate wells or arrayed at the surface of a microarray integrated within a classical 96-well plate. Once optimized, the classical ELISAs and microarray-based ELISA performances were evaluated and compared in terms of limit of detection, IC50, linearity range and reproducibility. Classical ELISAs provided quite good sensitivity (limit of detection down to 10 µg L(-1)), but the multiplex immunoassay was proven to be more sensitive (limit of detection down to 0.01 µg L(-1)), more reproducible and an advantageous tool in terms of cost and time expenses. This multiplex tool was then used for the successful detection of the three target molecules in spiked water samples and achieved very promising recovery rates.

High-throughput multiplexed competitive immunoassay for pollutants sensing in water.

The present study described the development and evaluation of a new fully automated multiplex competitive immunoassay enabling the simultaneous detection of five water pollutants (okadaic acid (OA), 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine (atrazine), 2.4-dichlorophenoxyacetic acid (2,4-D), 2,4,6-trinitrotoluene (TNT), and 1,3,5-trinitroperhydro-1,3,5-triazine (RDX)). The technology is taking advantage of an optical-clear pressure-sensitive adhesive on which biomolecules can be immobilized and that can be integrated within a classical 96-well format. The optimization of the microarray composition and cross-reaction was performed using an original approach where probe molecules (haptens) were conjugated to different carriers such as protein (bovine serum albumin or ovalbumin), amino-functionalized latex beads, or dextran polymer and arrayed at the surface of the adhesive. A total of 17 different probes were then arrayed together with controls on the adhesive surface and screened toward their specific reactivity and cross-reactivity. Once optimized, the complete setup was used for the detection of the five target molecules (less than 3 h for 96 samples). Limits of detection of 0.02, 0.01, 0.01, 100, and 0.02 µg L(-1) were found for OA, atrazine, 2,4-D, TNT, and RDX, respectively. The proof of concept of the multiplex competitive detection (semiquantitative or qualitative) of the five pollutants was also demonstrated on 16 spiked samples.

Multipurpose high-throughput filtering microarrays (HiFi) for DNA and protein assays.

We are reporting here a low cost colorimetric device for high-throughput multiplexed blood group genotyping and allergy diagnosis, displayed as an automated 96-well microtiter plate format. A porous polymeric membrane sealed at the bottom of each well accounts for the sensor support. For each sensing unit, a 6×6 matrix of specific probes is spotted on the external surface of the membrane resulting in 5 mm(2) microarrays. Thanks to the membrane porosity, reagents dispensed into the well can be eliminated through vacuum soaking. This unusual design drastically reduces the assay background signal. The system was first validated on robust models composed of either two complementary oligonucleotide sequences or one allergen/specific rabbit IgG pair. The quality of both oligonucleotide and protein immobilisation on the membrane substrate was then demonstrated together with the capacity to use the arrayed biomolecules as probes for the quantitative detection of specific targets (respectively complementary oligonucleotide and specific antibody). On the basis of these good results, two multiplex assays were developed for crude biological samples testing, focussing on two human in vitro diagnosis applications: a hybridisation assay for multiplex blood group genotyping and a multiparametric immunoassay for allergy diagnosis. In both cases, the transfer to crude biological samples testing was successful i.e. high signal to noise ratio of the stained membranes, reproducibility and good correlation with results obtained using routine testing procedures.