Fabrication of molecular nanopatterns at aluminium oxide surfaces by nanoshaving of self-assembled monolayers of alkylphosphonates

Nanoshaving, by tracing an atomic force microscope probe across a surface at elevated load, has been used to fabricate nanostructures in self-assembled monolayers of alkylphosphonates adsorbed at aluminium oxide surfaces. The simple process is implemented under ambient conditions. Because of the strong bond between the alkylphosphonates and the oxide surface, loads in excess of 400 nN are required to pattern the monolayer. Following patterning of octadecylphosphonate SAMs, adsorption of aminobutyl phosphonate yielded features as small as 39 nm. Shaving of monolayers of aryl azide-terminated alkylphosphonates, followed by attachment of polyethylene glycol to unmodified regions in a photochemical coupling reaction, yielded 102 nm trenches into which NeutrAvidin coated, dye-labelled, polymer nanospheres could be deposited, yielding bright fluorescence with little evidence of nonspecific adsorption to other regions of the surface. Structures formed in alkylphosphonate films by nanoshaving were used to etch structures into the underlying metal. Because of the isotropic nature of the etch process, and the large grain size, some broadening was observed, but features 25–35 nm deep and 180 nm wide were fabricated

Interference lithographic nanopatterning of plant and bacterial light-harvesting complexes on gold substrates

We describe a facile approach for nanopatterning of photosynthetic light-harvesting complexes over macroscopic areas, and use optical spectroscopy to demonstrate retention of native properties by both site-specifically and non-specifically attached photosynthetic membrane proteins. A Lloyd's mirror dual-beam interferometer was used to expose self-assembled monolayers of amine-terminated alkylthiolates on gold to laser irradiation. Following exposure, photo-oxidized adsorbates were replaced by oligo(ethylene glycol) terminated thiols, and the remaining intact amine-functionalized regions were used for attachment of the major light-harvesting chlorophyll–protein complex from plants, LHCII. These amine patterns could be derivatized with nitrilotriacetic acid (NTA), so that polyhistidine-tagged bacteriochlorophyll–protein complexes from phototrophic bacteria could be attached with a defined surface orientation. By varying parameters such as the angle between the interfering beams and the laser irradiation dose, it was possible to vary the period and widths of NTA and amine-functionalized lines on the surfaces; periods varied from 1200 to 240 nm and linewidths as small as 60 nm (λ/4) were achieved. This level of control over the surface chemistry was reflected in the surface topology of the protein nanostructures imaged by atomic force microscopy; fluorescence imaging and spectral measurements demonstrated that the surface-attached proteins had retained their native functionality

From Monochrome to Technicolor: Simple Generic Approaches to Multicomponent Protein Nanopatterning Using Siloxanes with Photoremovable Protein-Resistant Protecting Groups.

We show that sequential protein deposition is possible by photodeprotection of films formed from a tetraethylene-glycol functionalized nitrophenylethoxycarbonyl-protected aminopropyltriethoxysilane (NPEOC-APTES). Exposure to near-UV irradiation removes the protein-resistant protecting group, and allows protein adsorption onto the resulting aminated surface. The protein resistance was tested using proteins with fluorescent labels and microspectroscopy of two-component structures formed by micro- and nanopatterning and deposition of yellow and green fluorescent proteins (YFP/GFP). Nonspecific adsorption onto regions where the protecting group remained intact was negligible. Multiple component patterns were also formed by near-field methods. Because reading and writing can be decoupled in a near-field microscope, it is possible to carry out sequential patterning steps at a single location involving different proteins. Up to four different proteins were formed into geometric patterns using near-field lithography. Interferometric lithography facilitates the organization of proteins over square cm areas. Two-component patterns consisting of 150 nm streptavidin dots formed within an orthogonal grid of bars of GFP at a period of ca. 500 nm could just be resolved by fluorescence microscopy

Versatile thiol-based reactions for micrometer- and nanometer-scale photopatterning of polymers and biomolecules

Thiol-based chemistry provides a mild and versatile tool for surface functionalization. In the present work, mercaptosilane films were patterned by utilizing UV-induced photo-oxidation of the thiol to yield sulfonate groups via contact and interferometric lithography (IL). These photo-generated sulfonic acid groups were used for selective immobilization of amino-functionalized molecules after activation with triphenylphosphine ditriflate (TPPDF). Moreover, protein-resistant poly(oligoethyleneglycolmethacrylate) (POEGMA) brushes were grown from the intact thiol groups by a surface-induced polymerization reaction. Exploiting both reactions it is possible to couple amino-labelled nitrilotriacetic acid (NH2-NTA) to sulfonate-functionalized regions, enabling the site-specific binding of green fluorescent protein (GFP) to regions defined lithographically, while exploiting the protein-resistant character of POEGMA brushes to prevent non-specific protein adsorption to previously masked areas. The outstanding reactivity of thiol groups paves the way towards novel strategies for the fabrication of complex protein nanopatterns beyond thiol–ene chemistry

Zwitterionic Poly(amino acid methacrylate) Brushes

A new cysteine-based methacrylic monomer (CysMA) was conveniently synthesized via selective thia-Michael addition of a commercially available methacrylate-acrylate precursor in aqueous solution without recourse to protecting group chemistry. Poly(cysteine methacrylate) (PCysMA) brushes were grown from the surface of silicon wafers by atom-transfer radical polymerization. Brush thicknesses of ca. 27 nm were achieved within 270 min at 20 °C. Each CysMA residue comprises a primary amine and a carboxylic acid. Surface zeta potential and atomic force microscopy (AFM) studies of the pH-responsive PCysMA brushes confirm that they are highly extended either below pH 2 or above pH 9.5, since they possess either cationic or anionic character, respectively. At intermediate pH, PCysMA brushes are zwitterionic. At physiological pH, they exhibit excellent resistance to biofouling and negligible cytotoxicity. PCysMA brushes undergo photodegradation: AFM topographical imaging indicates significant mass loss from the brush layer, while XPS studies confirm that exposure to UV radiation produces surface aldehyde sites that can be subsequently derivatized with amines. UV exposure using a photomask yielded sharp, well-defined micropatterned PCysMA brushes functionalized with aldehyde groups that enable conjugation to green fluorescent protein (GFP). Nanopatterned PCysMA brushes were obtained using interference lithography, and confocal microscopy again confirmed the selective conjugation of GFP. Finally, PCysMA undergoes complex base-catalyzed degradation in alkaline solution, leading to the elimination of several small molecules. However, good long-term chemical stability was observed when PCysMA brushes were immersed in aqueous solution at physiological pH

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

Background: Many patients with COVID-19 have been treated with plasma containing anti-SARS-CoV-2 antibodies. We aimed to evaluate the safety and efficacy of convalescent plasma therapy in patients admitted to hospital with COVID-19. Methods: This randomised, controlled, open-label, platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]) is assessing several possible treatments in patients hospitalised with COVID-19 in the UK. The trial is underway at 177 NHS hospitals from across the UK. Eligible and consenting patients were randomly assigned (1:1) to receive either usual care alone (usual care group) or usual care plus high-titre convalescent plasma (convalescent plasma group). The primary outcome was 28-day mortality, analysed on an intention-to-treat basis. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936. Findings: Between May 28, 2020, and Jan 15, 2021, 11558 (71%) of 16287 patients enrolled in RECOVERY were eligible to receive convalescent plasma and were assigned to either the convalescent plasma group or the usual care group. There was no significant difference in 28-day mortality between the two groups: 1399 (24%) of 5795 patients in the convalescent plasma group and 1408 (24%) of 5763 patients in the usual care group died within 28 days (rate ratio 1·00, 95% CI 0·93–1·07; p=0·95). The 28-day mortality rate ratio was similar in all prespecified subgroups of patients, including in those patients without detectable SARS-CoV-2 antibodies at randomisation. Allocation to convalescent plasma had no significant effect on the proportion of patients discharged from hospital within 28 days (3832 [66%] patients in the convalescent plasma group vs 3822 [66%] patients in the usual care group; rate ratio 0·99, 95% CI 0·94–1·03; p=0·57). Among those not on invasive mechanical ventilation at randomisation, there was no significant difference in the proportion of patients meeting the composite endpoint of progression to invasive mechanical ventilation or death (1568 [29%] of 5493 patients in the convalescent plasma group vs 1568 [29%] of 5448 patients in the usual care group; rate ratio 0·99, 95% CI 0·93–1·05; p=0·79). Interpretation: In patients hospitalised with COVID-19, high-titre convalescent plasma did not improve survival or other prespecified clinical outcomes. Funding: UK Research and Innovation (Medical Research Council) and National Institute of Health Research

Tocilizumab in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Background: In this study, we aimed to evaluate the effects of tocilizumab in adult patients admitted to hospital with COVID-19 with both hypoxia and systemic inflammation. Methods: This randomised, controlled, open-label, platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing several possible treatments in patients hospitalised with COVID-19 in the UK. Those trial participants with hypoxia (oxygen saturation <92% on air or requiring oxygen therapy) and evidence of systemic inflammation (C-reactive protein ≥75 mg/L) were eligible for random assignment in a 1:1 ratio to usual standard of care alone versus usual standard of care plus tocilizumab at a dose of 400 mg–800 mg (depending on weight) given intravenously. A second dose could be given 12–24 h later if the patient's condition had not improved. The primary outcome was 28-day mortality, assessed in the intention-to-treat population. The trial is registered with ISRCTN (50189673) and ClinicalTrials.gov (NCT04381936). Findings: Between April 23, 2020, and Jan 24, 2021, 4116 adults of 21 550 patients enrolled into the RECOVERY trial were included in the assessment of tocilizumab, including 3385 (82%) patients receiving systemic corticosteroids. Overall, 621 (31%) of the 2022 patients allocated tocilizumab and 729 (35%) of the 2094 patients allocated to usual care died within 28 days (rate ratio 0·85; 95% CI 0·76–0·94; p=0·0028). Consistent results were seen in all prespecified subgroups of patients, including those receiving systemic corticosteroids. Patients allocated to tocilizumab were more likely to be discharged from hospital within 28 days (57% vs 50%; rate ratio 1·22; 1·12–1·33; p<0·0001). Among those not receiving invasive mechanical ventilation at baseline, patients allocated tocilizumab were less likely to reach the composite endpoint of invasive mechanical ventilation or death (35% vs 42%; risk ratio 0·84; 95% CI 0·77–0·92; p<0·0001). Interpretation: In hospitalised COVID-19 patients with hypoxia and systemic inflammation, tocilizumab improved survival and other clinical outcomes. These benefits were seen regardless of the amount of respiratory support and were additional to the benefits of systemic corticosteroids. Funding: UK Research and Innovation (Medical Research Council) and National Institute of Health Research

Spectroscopic and atomic force microscopy characterization of the electrografting of 3,5-bis(4-diazophenoxy)benzoicacid on gold surfaces

10.1016/j.bios.2012.07.036The synthesis of a bipodal diazonium salt, 3,5-bis(4-diazophenoxy)benzoic acid, and the study of its electrochemical deposition on gold surfaces is presented. The presence of the organic layer on the gold surface was characterized using atomic force microscopy and X-ray photoelectron spectroscopy, demonstrating the presence of phenyl groups, indicative of the grafted layer as well as the formation of multilayers, dependent on the electrografting conditions