SARS-CoV-2 Virus Detection Via a Polymeric Nanochannel-Based Electrochemical Biosensor.

The development of simple, cost-effective, rapid, and quantitative diagnostic tools remains critical to monitor infectious COVID-19 disease. Although numerous diagnostic platforms, including rapid antigen tests, are developed and used, they suffer from limited accuracy, especially when tested with asymptomatic patients. Here, a unique approach to fabricate a nanochannel-based electrochemical biosensor that can detect the entire virion instead of virus fragments, is demonstrated. The sensing platform has uniform nanoscale channels created by the convective assembly of polystyrene (PS) beads on gold electrodes. The PS beads are then functionalized with bioreceptors while the gold surface is endowed with anti-fouling properties. When added to the biosensor, SARS-CoV-2 virus particles block the nanochannels by specific binding to the bioreceptors. The nanochannel blockage hinders the diffusion of a redox probe; and thus, allows quantification of the viral load by measuring the changes in the oxidation current before and after virus incubation. The biosensor shows a low limit of detection of ≈1.0 viral particle mL-1 with a wide detection range up to 108 particles mL-1 in cell culture media. Moreover, the biosensor is able to differentiate saliva samples with SARS-CoV-2 from those without, demonstrating the potential of this technology for translation into a point-of-care biosensor product.

Efficient metal ion sieving in rectifying subnanochannels enabled by metal-organic frameworks.

Biological ion channels have remarkable ion selectivity, permeability and rectification properties, but it is challenging to develop artificial analogues. Here, we report a metal-organic framework-based subnanochannel (MOFSNC) with heterogeneous structure and surface chemistry to achieve these properties. The asymmetrically structured MOFSNC can rapidly conduct K+, Na+ and Li+ in the subnanometre-to-nanometre channel direction, with conductivities up to three orders of magnitude higher than those of Ca2+ and Mg2+, equivalent to a mono/divalent ion selectivity of 103. Moreover, by varying the pH from 3 to 8 the ion selectivity can be tuned further by a factor of 102 to 104. Theoretical simulations indicate that ion-carboxyl interactions substantially reduce the energy barrier for monovalent cations to pass through the MOFSNC, and thus lead to ultrahigh ion selectivity. These findings suggest ways to develop ion selective devices for efficient ion separation, energy reservation and power generation.

Sulfonated Sub-1-nm Metal-Organic Framework Channels with Ultrahigh Proton Selectivity.

Biological proton channels are sub-1-nm protein pores with ultrahigh proton (H+) selectivity over other ions. Inspired by biological proton channels, developing artificial proton channels with biological-level selectivity is of fundamental significance for separation science. Herein we report synthetic proton channels fabrication based on sulfonated metal-organic frameworks (MOFs), UiO-66-X, X = SAG, NH-SAG, (NH-SAG)2 (SAG: sulfonic acid groups), which have sub-1-nm windows and a high density of sulfonic acid groups mimicking natural proton channels. The ion conductance of UiO-66-X channels follows the sequence: H+ ≫ K+ > Na+> Li+, and the sulfonated UiO-66 derivative channels show proton selectivity much higher than that of the pristine UiO-66 channels. Particularly, the UiO-66-(NH-SAG)2 channels exhibit ultrahigh proton selectivities, H+/Li+ up to ∼100, H+/Na+ of ∼80, and H+/K+ of ∼70, which are ∼3 times of that of UiO-66-NH-SAG channels, and ∼15 times of that of UiO-66@SAG channels. The ultrahigh proton selectivity in the sulfonated sub-1-nm MOF channels is mainly attributed to the narrow window-cavity pore structure functionalized with nanoconfined high-density sulfonic acid groups that facilitate fast proton transport and simultaneously exclude other cations. Our work opens an avenue to develop functional MOF channels for selective ion conduction and efficient ion separation.

Thermally Reduced Nanoporous Graphene Oxide Membrane for Desalination.

Graphene-based laminar membranes open new avenues for water treatment; in particular, reduced graphene oxide (rGO) membranes with high stability in aqueous solutions are gaining increased attention for desalination. However, the low water permeability of these membranes significantly limits their applications. In this study, the water permeability of thermally reduced GO membrane was increased by a factor of 26 times by creating in-plane nanopores with an average diameter of ∼3 nm and a high density of 2.89 × 1015 m-2 via H2O2 oxidation. These in-plane nanopores provide additional transport channels and shorten the transport distance for water molecules. Meanwhile, salt rejection of this membrane is dominated by both the Donnan effect and the size exclusion of the interspaces. Besides, the water permeability and salt rejection of the thermally reduced nanoporous GO membrane can also be simply tuned by adjusting the thermal treatment time and membrane thickness. Additionally, the fabricated membrane exhibited a relatively stable rejection of Na2SO4 during the long-term testing. This work demonstrates a novel and effective strategy for fabricating high-performance laminar rGO membranes for desalination applications.

High-Throughput Production of Chromium(III) Complexes for Antibody Immobilization.

A robot-assisted high-throughput methodology was employed to produce chromium(III) complexes suitable for the surface modification of the commercially available PerkinElmer Optiplate96 well plate for use in enzyme-linked immunosorbent assays (ELISAs). The complexes were immobilized to the native functionality of the well plate and first screened using a horseradish-peroxidase-tagged (HRP) mouse antibody to quantify binding. The top "hits" were further assessed for their ability to present the antibody in a functional state using an ELISA. "Hits" from the second screen yielded four complexes capable of improving the signal intensity of the ELISA by greater than 500%. The metal/base ratio of these complexes was also investigated, and we isolated the most stable and reproducible candidate, [Cr(OH)6]3-, which was formed from chromium(III) perchlorate and pH adjusted with ethylenediamine. This chromium solution was employed in a clinically relevant setting for the detection of bovine TNFα producing up to a 200% increase in signal intensity.

Shear Assisted Electrochemical Exfoliation of Graphite to Graphene.

The exfoliation characteristics of graphite as a function of applied anodic potential (1-10 V) in combination with shear field (400-74 400 s(-1)) have been studied in a custom-designed microfluidic reactor. Systematic investigation by atomic force microscopy (AFM) indicates that at higher potentials thicker and more fragmented graphene sheets are obtained, while at potentials as low as 1 V, pronounced exfoliation is triggered by the influence of shear. The shear-assisted electrochemical exfoliation process yields large (∼10 µm) graphene flakes with a high proportion of single, bilayer, and trilayer graphene and small ID/IG ratio (0.21-0.32) with only a small contribution from carbon-oxygen species as demonstrated by X-ray photoelectron spectroscopy measurements. This method comprises intercalation of sulfate ions followed by exfoliation using shear induced by a flowing electrolyte. Our findings on the crucial role of hydrodynamics in accentuating the exfoliation efficiency suggest a safer, greener, and more automated method for production of high quality graphene from graphite.

Boundary flow on end-grafted PEG brushes.

We investigated the boundary conditions for flow of a Newtonian liquid over soft interfaces by measuring hydrodynamic drainage forces with colloid probe atomic force microscopy in a viscous liquid. The investigated soft surfaces are end-grafted brushes of thiolated poly(ethylene glycol) (PEG), of molecular weight 1k and 30k, grafted-to gold. The conditions for brush preparation were optimized as to meet the stringent conditions required for surface force measurements, namely reproducible and uniform surface composition and roughness. The fit of a slip model to the experimental data returned a slip length of 16 nm on the PEG 1k brush and 25 nm on the PEG30k brush. The slip length can be interpreted as a penetration length, which accounts for flow within the top half of the brush for the PEG30k case, and within the brush and surface roughness for the PEG1k case. These findings confirm earlier simulation studies by our group on the flow of liquids within polymer brushes.

One-Reactant Photografting of ATRP Initiators for Surface-Initiated Polymerization.

Self-initiated photografting polymerization is used to couple the polymerizable initiator monomer 2-(2-chloropropanoyloxy)ethyl acrylate to a range of polymeric substrates. The technique requires only UV light to couple the initiator to surfaces. The initiator surface density can be varied by inclusion of a diluent monomer or via selection of initiator and irradiation parameters. The functionality of the initiator surface is demonstrated by subsequent surface-initiated atom transfer radical polymerization. Surfaces are characterized by x-ray photoelectron spectroscopy (XPS), ellipsometry, and atomic force microscopy (AFM), and UV-induced changes to the initiator are assessed by (1) H NMR and gel permeation chromatography (GPC). This is the first time this one-reactant one-step technique has been demonstrated for creating an initiator surface of variable density.

Tailored donor-acceptor polymers with an A-D1-A-D2 structure: controlling intermolecular interactions to enable enhanced polymer photovoltaic devices.

Extensive efforts have been made to develop novel conjugated polymers that give improved performance in organic photovoltaic devices. The use of polymers based on alternating electron-donating and electron-accepting units not only allows the frontier molecular orbitals to be tuned to maximize the open-circuit voltage of the devices but also controls the optical band gap to increase the number of photons absorbed and thus modifies the other critical device parameter-the short circuit current. In fact, varying the nonchromophoric components of a polymer is often secondary to the efforts to adjust the intermolecular aggregates and improve the charge-carrier mobility. Here, we introduce an approach to polymer synthesis that facilitates simultaneous control over both the structural and electronic properties of the polymers. Through the use of a tailored multicomponent acceptor-donor-acceptor (A-D-A) intermediate, polymers with the unique structure A-D1-A-D2 can be prepared. This approach enables variations in the donor fragment substituents such that control over both the polymer regiochemistry and solubility is possible. This control results in improved intermolecular π-stacking interactions and therefore enhanced charge-carrier mobility. Solar cells using the A-D1-A-D2 structural polymer show short-circuit current densities that are twice that of the simple, random analogue while still maintaining an identical open-circuit voltage. The key finding of this work is that polymers with an A-D1-A-D2 structure offer significant performance benefits over both regioregular and random A-D polymers. The chemical synthesis approach that enables the preparation of A-D1-A-D2 polymers therefore represents a promising new route to materials for high-efficiency organic photovoltaic devices.

Protein micropatterns by PEG grafting on Dewetted PLGA films.

The ability to control protein and cell positioning on a microscopic scale is crucial in many biomedical applications, such as single cell studies. We have developed and investigated the grafting of poly(ethylene glycol) (PEG) brushes onto poly(d,l-lactide-co-glycolide) (PLGA) thin films, which can be micropatterned by exploiting their spontaneous dewetting on top of polystyrene (PS) films. Dense PEG brushes with excellent protein repellence were achieved on PLGA by using cloud point grafting conditions, and selective adsorption of proteins on the micropatterned substrates was achieved by exploiting the different affinity protein adsorption onto the PEG brushes and the PS holes. PEG-grafted PLGA films showed better resistance against spontaneous degradation in buffer than bare PLGA films, due to passivation by the thin PEG coating. The simplicity of dewetting and subsequent grafting approaches, coupled with the ability to coat and pattern nonplanar substrates give rise to possible applications of PEG-grafted PLGA films in single cell studies and cell cultures for tissue engineering.

Accurate prediction of solvent flux in sub-1-nm slit-pore nanosheet membranes.

Nanosheet-based membranes have shown enormous potential for energy-efficient molecular transport and separation applications, but designing these membranes for specific separations remains a great challenge due to the lack of good understanding of fluid transport mechanisms in complex nanochannels. We synthesized reduced MXene/graphene hetero-channel membranes with sub-1-nm pores for experimental measurements and theoretical modeling of their structures and fluid transport rates. Our experiments showed that upon complete rejection of salt and organic dyes, these membranes with subnanometer channels exhibit remarkably high solvent fluxes, and their solvent transport behavior is very different from their homo-structured counterparts. We proposed a subcontinuum flow model that enables accurate prediction of solvent flux in sub-1-nm slit-pore membranes by building a direct relationship between the solvent molecule-channel wall interaction and flux from the confined physical properties of a liquid and the structural parameters of the membranes. This work provides a basis for the rational design of nanosheet-based membranes for advanced separation and emerging nanofluidics.

Nanotopographic surfaces with defined surface chemistries from amyloid fibril networks can control cell attachment.

We show for the first time the possibility of using networks of amyloid fibrils, adsorbed to solid supports and with plasma polymer coatings, for the fabrication of chemically homogeneous surfaces with well-defined nanoscale surface features reminiscent of the topography of the extracellular matrix. The robust nature of the fibrils allows them to withstand the plasma polymer deposition conditions used with no obvious deleterious effect, thus enabling the underlying fibril topography to be replicated at the polymer surface. This effect was seen despite the polymer coating thickness being an order of magnitude greater than the fibril network. The in vitro culture of fibroblast cells on these surfaces resulted in increased attachment and spreading compared to flat plasma polymer films with the same chemical composition. The demonstrated technique allows for the rapid and reproducible fabrication of substrates with nanoscale fibrous topography that we believe will have applications in the development of new biomaterials allowing, for example, the investigation of the effect of extracellular matrix mimicking nanoscale morphology on cellular phenotype.

Wearable microneedle array-based sensor for transdermal monitoring of pH levels in interstitial fluid.

Microneedle-based wearable sensors offer an alternative approach to traditional invasive blood-based health monitoring and disease diagnostics techniques. Instead of blood, microneedle-based sensors target the skin interstitial fluid (ISF), in which the biomarker type and concentration profile resemble the one found in the blood. However, unlike blood, interstitial fluid does not have the same pH-buffering capacity causing deviation of pH levels from the physiological range. Information about the skin ISF pH levels can be used as a biomarker for a wide range of pathophysiological conditions and as a marker for the calibration of a wearable sensor. The ISF pH can significantly affect the detection accuracy of other biomarkers as it influences enzyme activity, aptamer affinity, and antibody-antigen interaction. Herein, we report the fabrication of a high-density polymeric microneedle array-based (PMNA) sensing patch and its optimization for the potentiometric transdermal monitoring of pH levels in ISF. The wearable sensor utilizes a polyaniline-coated PMNA having a density of ∼10,000 microneedles per cm2, containing individual microneedles with a height of ∼250 µm, and a tip diameter of ∼2 µm. To prevent interference from other body fluids like sweat, an insulating layer is deposited at the base of the PMNA. The wearable pH sensor operates from pH 4.0 to 8.6 with a sensitivity of 62.9 mV per pH unit and an accuracy of ±0.036 pH units. Furthermore, testing on a mouse demonstrates the ability of the PMNA to provide a real-time reading of the transdermal pH values. This microneedle-based system will significantly contribute to advancing transdermal wearable sensors technology, simplifying the fabrication process, and improving the cost-effectiveness of such devices.

Fluoropolymer Functionalization of Organ-on-Chip Platform Increases Detection Sensitivity for Cannabinoids.

Microfluidic technology is applied across various research areas including organ-on-chip (OOC) systems. The main material used for microfluidics is polydimethylsiloxane (PDMS), a silicone elastomer material that is biocompatible, transparent, and easy to use for OOC systems with well-defined microstructures. However, PDMS-based OOC systems can absorb hydrophobic and small molecules, making it difficult and erroneous to make quantitative analytical assessments for such compounds. In this paper, we explore the use of a synthetic fluoropolymer, poly(4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethylene) (Teflon™ AF 2400), with excellent "non-stick" properties to functionalize OOC systems. Cannabinoids, including cannabidiol (CBD), are classes of hydrophobic compounds with a great potential for the treatment of anxiety, depression, pain, and cancer. By using CBD as a testing compound, we examined and systematically quantified CBD absorption into PDMS by means of an LC-MS/MS analysis. In comparison to the unmodified PDMS microchannels, an increase of approximately 30× in the CBD signal was detected with the fluoropolymer surface modification after 3 h of static incubation. Under perfusion conditions, we observed an increase of nearly 15× in the CBD signals from the surface-modified microchannels than from the unmodified microchannels. Furthermore, we also demonstrated that fluoropolymer-modified microchannels are compatible for culturing hCMEC/D3 endothelial cells and for CBD perfusion experiments.

Electrochemical Biosensors Based on Convectively Assembled Colloidal Crystals.

Rapid, sensitive, selective and portable virus detection is in high demand globally. However, differentiating non-infectious viral particles from intact/infectious viruses is still a rarely satisfied sensing requirement. Using the negative space within monolayers of polystyrene (PS) spheres deposited directly on gold electrodes, we fabricated tuneable nanochannels decorated with target-selective bioreceptors that facilitate the size-selective detection of intact viruses. Detection occurred through selective nanochannel blockage of diffusion of a redox probe, [Fe(CN)6]3/4-, allowing a quantifiable change in the oxidation current before and after analyte binding to the bioreceptor immobilised on the spheres. Our model system involved partial surface passivation of the mono-assembled PS spheres, by silica glancing angle deposition, to confine bioreceptor immobilisation specifically to the channels and improve particle detection sensitivity. Virus detection was first optimised and modelled with biotinylated gold nanoparticles, recognised by streptavidin immobilised on the PS layer, reaching a low limit of detection of 37 particles/mL. Intact, label-free virus detection was demonstrated using MS2 bacteriophage (~23-28 nm), a marker of microbiological contamination, showing an excellent limit of detection of ~1.0 pfu/mL. Tuneable nanochannel geometries constructed directly on sensing electrodes offer label-free, sensitive, and cost-efficient point-of-care biosensing platforms that could be applied for a wide range of viruses.

Physical vs photolithographic patterning of plasma polymers: an investigation by ToF-SSIMS and multivariate analysis.

Physical and photolithographic techniques are commonly used to create chemical patterns for a range of technologies including cell culture studies, bioarrays and other biomedical applications. In this paper, we describe the fabrication of chemical micropatterns from commonly used plasma polymers. Atomic force microscopy (AFM) imaging, time-of-flight static secondary ion mass spectrometry (ToF-SSIMS) imaging, and multivariate analysis have been employed to visualize the chemical boundaries created by these patterning techniques and assess the spatial and chemical resolution of the patterns. ToF-SSIMS analysis demonstrated that well-defined chemical and spatial boundaries were obtained from photolithographic patterning, while the resolution of physical patterning via a transmission electron microscopy (TEM) grid varied depending on the properties of the plasma system including the substrate material. In general, physical masking allowed diffusion of the plasma species below the mask and bleeding of the surface chemistries. Multivariate analysis techniques including principal component analysis (PCA) and region of interest (ROI) assessment were used to investigate the ToF-SSIMS images of a range of different plasma polymer patterns. In the most challenging case, where two strongly reacting polymers, allylamine and acrylic acid were deposited, PCA confirmed the fabrication of micropatterns with defined spatial resolution. ROI analysis allowed for the identification of an interface between the two plasma polymers for patterns fabricated using the photolithographic technique which has been previously overlooked. This study clearly demonstrated the versatility of photolithographic patterning for the production of multichemistry plasma polymer arrays and highlighted the need for complementary characterization and analytical techniques during the fabrication plasma polymer micropatterns.

Aminomalononitrile-Assisted Multifunctional Antibacterial Coatings.

Medical device associated infections remain a significant problem for all classes of devices at this point in time. Here, we have developed a surface modification technique to fabricate multifunctional coatings that combine antifouling and antimicrobial properties. Zwitterionic polymers providing antifouling properties and quaternary ammonium containing polymers providing antimicrobial properties were combined in these coatings. Throughout this study, aminomalononitrile (AMN) was used to achieve one-step coatings incorporating different polymers. The characterization of coatings was carried out using static water contact angle (WCA) measurements, X-ray photoelectron spectroscopy (XPS), profilometry, and scanning electron microscopy (SEM), whereas the biological response in vitro was analyzed using Staphylococcus epidermidis and Escherichia coli as well as L929 fibroblast cells. Zwitterionic polymers synthesized from sulfobetaine methacrylate and 2-aminoethyl methacrylate were demonstrated to reduce bacterial attachment when incorporated in AMN assisted coatings. However, bacteria in suspension were not affected by this approach. On the other hand, alkylated polyethylenimine polymers, synthesized to provide quaternary ammonium groups, were demonstrated to have contact killing properties when incorporated in AMN assisted coatings. However, the high bacterial attachment observed on these surfaces may be detrimental in applications requiring longer-term bactericidal activity. Therefore, AMN-assisted coatings containing both quaternary and zwitterionic polymers were fabricated. These multifunctional coatings were demonstrated to significantly reduce the number of live bacteria not only on the modified surfaces, but also in suspension. This approach is expected to be of interest in a range of biomedical device applications.

Enhanced electrochemical sensing performance by insitu electrocopolymerization of pyrrole and thiophene-grafted chitosan.

Nowadays, there is increasing number of electrochemical biosensors which utilize chitosan (Ch); as an enzyme immobilization matrix, and conductive nanomaterials; as electron carriers improving sensitivity of the biosensor. However, the challenge these sensors face is the lack of uniform dispersion of nanomaterials throughout the Ch film, which can negatively affect analytical performance of the biosensor. In this study, we report the development of an enzyme immobilization matrix that displays enhanced electrochemical performance thanks to a novel conductive thin film prepared via in situ electrocopolymerization of pyrrole (Py) and thiophene-grafted chitosan (Th-Ch). This is a simple thin film preparation method that can help overcome aforementioned challenges by providing a uniformly distributed conductive layer on the electrode. We are also for the first time reporting the synthesis and characterization of Th-Ch, where grafted Th plays an essential role as a linking group between Ch and Py. The resulting conductive Ch-based thin film was modified with glucose oxidase (GOx) which served as a model enzyme. In situ electrocopolymerization of Py with Th-Ch resulted in a highly conductive thin film enabling approximately 40% higher sensitivity when compared to a Py-Ch composite. This new type of composite thin film is promising in biosensor technology due to its biocompatibility, the chemically and physically modifiable structure, as well as its electrical conductivity.

In Situ Surface Modification of Microfluidic Blood-Brain-Barriers for Improved Screening of Small Molecules and Nanoparticles.

Here, we have developed and evaluated a microfluidic-based human blood-brain-barrier (µBBB) platform that models and predicts brain tissue uptake of small molecule drugs and nanoparticles (NPs) targeting the central nervous system. By using a photocrosslinkable copolymer that was prepared from monomers containing benzophenone and N-hydroxysuccinimide ester functional groups, we were able to evenly coat and functionalize µBBB chip channels in situ, providing a covalently attached homogenous layer of extracellular matrix proteins. This novel approach allowed the coculture of human endothelial cells, pericytes, and astrocytes and resulted in the formation of a mimic of cerebral endothelium expressing tight junction markers and efflux proteins, resembling the native BBB. The permeability coefficients of a number of compounds, including caffeine, nitrofurantoin, dextran, sucrose, glucose, and alanine, were measured on our µBBB platform and were found to agree with reported values. In addition, we successfully visualized the receptor-mediated uptake and transcytosis of transferrin-functionalized NPs. The BBB-penetrating NPs were able to target glioma cells cultured in 3D in the brain compartment of our µBBB. In conclusion, our µBBB was able to accurately predict the BBB permeability of both small molecule pharmaceuticals and nanovectors and allowed time-resolved visualization of transcytosis. Our versatile chip design accommodates different brain disease models and is expected to be exploited in further BBB studies, aiming at replacing animal experiments.