Antifouling Polymer Brushes Displaying Antithrombogenic Surface Properties.

The contact of blood with artificial materials generally leads to immediate protein adsorption (fouling), which mediates subsequent biological processes such as platelet adhesion and activation leading to thrombosis. Recent progress in the preparation of surfaces able to prevent protein fouling offers a potential avenue to mitigate this undesirable effect. In the present contribution, we have prepared several types of state-of-the-art antifouling polymer brushes on polycarbonate plastic substrate, and investigated their ability to prevent platelet adhesion and thrombus formation under dynamic flow conditions using human blood. Moreover, we compared the ability of such brushes--grafted on quartz via an adlayer analogous to that used on polycarbonate--to prevent protein adsorption from human blood plasma, assessed for the first time by means of an ultrahigh frequency acoustic wave sensor. Results show that the prevention of such a phenomenon constitutes one promising route toward enhanced resistance to thrombus formation, and suggest that antifouling polymer brushes could be of service in biomedical applications requiring extensive blood-material surface contact.

Utilizing a Key Aptamer Structure-Switching Mechanism for the Ultrahigh Frequency Detection of Cocaine.

Aptasensing of small molecules remains a challenge as detection often requires the use of labels or signal amplification methodologies, resulting in both difficult-to-prepare sensor platforms and multistep, complex assays. Furthermore, many aptasensors rely on the binding mechanism or structural changes associated with target capture by the aptameric probe, resulting in a detection scheme customized to each aptamer. It is in this context that we report herein a sensitive cocaine aptasensor that offers both real-time and label-free measurement capabilities. Detection relies on the electromagnetic piezoelectric acoustic sensor (EMPAS) platform. The sensing interface consists of a S-(11-trichlorosilyl-undecanyl)benzenethiosulfonate (BTS) adlayer-coated quartz disc onto which a structure-switching cocaine aptamer (MN6) is immobilized, completing the preparation of the MN6 cocaine aptasensor (M6CA). The EMPAS system has recently been employed as the foundation of a cocaine aptasensor based on a structurally rigid cocaine aptamer variant (MN4), an aptasensor referred to by analogy as M4CA. M6CA represents a significant increase in terms of analytical performance, compared to not only M4CA but also other cocaine aptamer-based sensors that do not rely on signal amplification, producing an apparent K(d) of 27 ± 6 µM and a 0.3 µM detection limit. Remarkably, the latter is in the range of that achieved by cocaine aptasensors relying on signal amplification. Furthermore, M6CA proved to be capable not only of regaining its cocaine-binding ability via simple buffer flow over the sensing interface (i.e., without the necessity to implement an additional regeneration step, such as in the case of M4CA), but also of detecting cocaine in a multicomponent matrix possessing potentially assay-interfering species. Finally, through observation of the distinct shape of its response profiles to cocaine injection, demonstration was made that the EMPAS system in practice offers the possibility to distinguish between the binding mechanisms of structure-switching (MN6) vs rigid (MN4) aptameric probes, an ability that could allow the EMPAS to provide a more universal aptasensing platform than what is ordinarily observed in the literature.

A survey of state-of-the-art surface chemistries to minimize fouling from human and animal biofluids.

Upon contact with bodily fluids, synthetic materials spontaneously acquire a layer of various species (most notably proteins) on their surface. The concern with respect to biomedical equipment, implants or devices resides in the possibility for biological processes with potentially harmful effects to ensue. In biosensor technology, the issue with this natural fouling phenomenon is that of non-specific adsorption to sensing platforms, which generates an often overwhelming interference signal that prevents the detection, not to mention the quantification, of target analytes present at considerably lower concentration. To alleviate this ubiquitous, recurrent problem - this genuine biotechnological plague - considerable research efforts have been devoted over the last few decades to engineer antifouling coatings. Extensive literature now exists that describes stealth organic adlayers capable of reducing fouling surface coverage Γ down to a few ng cm(-2)- however from biotechnologically irrelevant buffered solutions free or nearly depleted of any potentially interfering species. Regrettably indeed, few coatings are known to display/retain such level of performance when exposed to otherwise more complex, real-life biosamples (even diluted). Herein, we comprehensively review the state-of-the-art surface chemistries developed to date (January 2015) to minimize fouling from 8 such uncomparatively more challenging biological media (blood plasma, blood serum, cell lysate, cerebrospinal fluid, egg, milk, saliva, and urine) - whether of human or animal origin. Literature search for another 25 biological milieux generated no (exploitable) hit. Also discussed in this Review are the identification of the species responsible for fouling, and the dependence of antifouling properties on biosample source variability.

Adlayer-mediated antibody immobilization to stainless steel for potential application to endothelial progenitor cell capture.

This work describes the straightforward surface modification of 316L stainless steel with BTS, S-(11-trichlorosilylundecanyl)-benzenethiosulfonate, a thiol-reactive trichlorosilane cross-linker molecule designed to form intermediary coatings with subsequent biofunctionalization capability. The strategy is more specifically exemplified with the immobilization of intact antibodies and their Fab' fragments. Both surface derivatization steps are thoroughly characterized by means of X-ray photoelectron spectroscopy. The antigen binding capability of both types of biofunctionalized surfaces is subsequently assessed by fluorescence microscopy. It was determined that BTS adlayers achieve robust immobilization of both intact and fragmented antibodies, while preserving antigen binding activity. Another key finding was the observation that the Fab' fragment immobilization strategy would constitute a preferential option over that involving intact antibodies in the context of in vivo capture of endothelial progenitor cells in stent applications.

Ultra-high frequency piezoelectric aptasensor for the label-free detection of cocaine.

This paper describes a label-free and real-time piezoelectric aptasensor for the detection of cocaine. The acoustic wave sensing platform is a quartz substrate functionalized with an adlayer of S-(11-trichlorosilyl-undecanyl)-benzenethiosulfonate (BTS) cross-linker onto which the anti-cocaine MN4 DNA aptamer is next immobilized. Preparation of the sensor surface was monitored using X-ray photoelectron spectroscopy (XPS), while the binding of cocaine to surface-attached MN4 was evaluated using the electromagnetic piezoelectric acoustic sensor (EMPAS). The MN4 aptamer, unlike other cocaine aptamer variants, has its secondary structure preformed in the unbound state with only tertiary structure changes occurring during target binding. It is postulated that the highly sensitive EMPAS detected the binding of cocaine through target mass loading coupled to aptamer tertiary structure folding. The sensor achieved an apparent Kd of 45 ± 12 µM, and a limit of detection of 0.9 µM. Repeated regenerability of the sensor platform was also demonstrated. This work constitutes the first application of EMPAS technology in the field of aptasensors. Furthermore, it is so far one of the very few examples of a bulk acoustic wave aptasensor that is able to directly detect the binding interaction between an aptamer and a small molecule in a facile one-step protocol without the use of a complex assay or signal amplification step.

Ultrathin Surface Chemistry to Delay Anion Fouling.

The unwanted fouling of surfaces by ionic adsorption has received little research attention. In this context, ultrathin organic adlayer surface chemistry-featuring monoethylene glycol based molecular residues-is described that is capable of noticeably decreasing the rate of anion depletion from solution. The strategy is exemplified with glass as the substrate material and fluoride as the anion foulant.

Prevention of surface-induced thrombogenesis on poly(vinyl chloride).

Much biomedical equipment consisting of or containing plastic polymer(s) must come into contact with blood - an interaction that, at the molecular level, may unfortunately prompt biological processes with potentially deleterious, short- or long-term effects such as thrombosis. In the present investigation, this problem is alleviated for poly(vinyl chloride) (PVC) through chemical surface modification with an ultrathin, monoethylene glycol-based coating - a transformation that is characterized using X-ray photoelectron spectroscopy (XPS) supplemented by contact angle goniometry (CAG). Antithrombogenic properties are assessed through calculation (for the first 10 min, and after 60 min) of the surface coverage percentage due to platelet adhesion, aggregation and thrombus formation upon continuous exposure to fluorescently-labelled whole human blood. At all shear rates investigated (300, 900, and 1500 s-1), surface coverage decreases by >99% with respect to bare PVC (10 min, short-term contact with blood). Most importantly, antithrombogenic performance is retained for longer-term exposure experiments (60 min), regardless of applied shear rate as well.

On the hydration of subnanometric antifouling organosilane adlayers: a molecular dynamics simulation.

The connection between antifouling and surface hydration is a fascinating but daunting question to answer. Herein, we use molecular dynamics (MD) computer simulations to gain further insight into the role of surface functionalities in the molecular-level structuration of water (surface kosmotropicity)--within and atop subnanometric organosilane adlayers that were shown in previous experimental work to display varied antifouling behavior. Our simulations support the hypothesized intimate link between surface hydration and antifouling, in particular the importance of both internal and interfacial hydrophilicity and kosmotropicity. The antifouling mechanism is also discussed in terms of surface dehydration energy and water dynamicity (lability and mobility), notably the crucial requirement for clustered water molecules to remain tightly bound for extensive periods of time--i.e. exhibit slow exchange dynamics. A substrate effect on surface hydration, which would also participate in endowing antifouling adlayers with hydrogel-like characteristics, is also proposed. In contrast, the role of adlayer flexibility, if any, is assigned a secondary role in these ultrathin structures made of short building blocks. The conclusions from this work are well in line with those previously drawn in the literature.

A true theranostic approach to medicine: towards tandem sensor detection and removal of endotoxin in blood.

Sepsis is one of the leading causes of death around the world. The condition occurs when a local infection overcomes the host natural defense mechanism and suddenly spreads into the circulatory system, triggering a vigorous, self-injurious inflammatory host response. The pathogenesis of sepsis is relatively well known, one of the most potent immuno-activator being bacterial lipopolysaccharide (LPS) - also known as 'endotoxin'. Tests exist to detect endotoxin in bodily fluids, but are expensive, not necessarily user-friendly and require reporter molecules. In addition, the situation for safe and effective anti-endotoxin therapy is problematical. At the present time, endotoxin removal through cartridge hemoperfusion is one of the better alternatives to combat sepsis. The capability to both measure endotoxemia levels and offer an adapted response treatment in a timely manner is crucial for better management and improved prognosis, but is currently unavailable. In this context, we describe herein preliminary research towards the development of an alternative LPS biosensor and an innovative LPS neutralization cartridge to be eventually combined in an all-integrated configuration for the theranostic, personalized treatment of blood endotoxemia/sepsis. LPS detection is performed in a real-time and label-free manner in full human blood plasma, using ultra-high frequency acoustic wave sensing in combination with ultrathin, oligoethylene glycol-based mixed surface chemistry imposed on piezoelectric quartz discs. Biosensing platforms are functionalized with polymyxin B (PMB), a cyclic peptide antibiotic with high affinity for LPS. Analogous surface modification is used on glass beads for the therapeutic cartridge component of the combined strategy. Incubation of LPS-spiked whole blood with PMB-bead chemistry resulted in a significant decrease in the production of pro-inflammatory TNF-α cytokine. LPS neutralization is discussed in relation to the perturbation of its supramolecular chemistry in solution.

Prevention of thrombogenesis from whole human blood on plastic polymer by ultrathin monoethylene glycol silane adlayer.

In contemporary society, a large percentage of medical equipment coming in contact with blood is manufactured from plastic polymers. Unfortunately, exposure may result in undesirable protein-material interactions that can potentially trigger deleterious biological processes such as thrombosis. To address this problem, we have developed an ultrathin antithrombogenic coating based on monoethylene glycol silane surface chemistry. The strategy is exemplified with polycarbonate--a plastic polymer increasingly employed in the biomedical industry. The various straightforward steps of surface modification were characterized with X-ray photoelectron spectroscopy supplemented by contact angle goniometry. Antithrombogenicity was assessed after 5 min exposure to whole human blood dispensed at a shear rate of 1000 s(-1). Remarkably, platelet adhesion, aggregation, and thrombus formation on the coated surface was greatly inhibited (>97% decrease in surface coverage) compared to the bare substrate and, most importantly, nearly nonexistent.

Probing the hydration of ultrathin antifouling organosilane adlayers using neutron reflectometry.

Neutron reflectometry data and modeling support the existence of a relatively thick, continuous phase of water stemming from within an antifouling monoethylene glycol silane adlayer prepared on oxidized silicon wafers. In contrast, this physically distinct (from bulk) interphase is much thinner and only interfacial in nature for the less effective adlayer lacking internal ether oxygen atoms. These results provide further insight into the link between antifouling and surface hydration.

Critical role of surface hydration on the dynamics of serum adsorption studied with monoethylene glycol adlayers on gold.

The dynamics of serum adsorption on bare and monoethylene glycol adlayer-modified gold surfaces is investigated using acoustic wave physics. Hydration experiments support the pivotal role ascribed to water in the antifouling of surfaces. Behavioural discrepancy is interpreted in terms of difference in water structuring properties (surface kosmotropicity).

Surface chemistry to minimize fouling from blood-based fluids.

Upon contact with bodily fluids/tissues, exogenous materials spontaneously develop a layer of proteins on their surface. In the case of biomedical implants and equipment, biological processes with deleterious effects may ensue. For biosensing platforms, it is synonymous with an overwhelming background signal that prevents the detection/quantification of target analytes present in considerably lower concentrations. To address this ubiquitous problem, tremendous efforts have been dedicated over the years to engineer protein-resistant coatings. There is now extensive literature available on stealth organic adlayers able to minimize fouling down to a few ng cm(-2), however from technologically irrelevant single-protein buffered solutions. Unfortunately, few coatings have been reported to present such level of performance when exposed to highly complex proteinaceous, real-world media such as blood serum and plasma, even diluted. Herein, we concisely review the surface chemistry developed to date to minimize fouling from these considerably more challenging blood-based fluids. Adsorption dynamics is also discussed.

New functionalizable alkyltrichlorosilane surface modifiers for biosensor and biomedical applications.

We report herein three unprecedented alkyltrichlorosilane surface modifiers bearing pentafluorophenyl ester (PFP), benzothiosulfonate (BTS), or novel ß-propiolactone (BPL) functionalizable terminal groups. Evidence is provided that these molecules can be prepared in very high purity (as assessed by NMR) through a last synthetic step of Pt-catalyzed alkene hydrosilylation then directly employed, without further purification, for the surface modification of quartz and medical grade stainless steel. Subsequent on-surface functionalizations with amine and thiol model molecules demonstrate the potential of these molecular adlayers to be important platforms for future applications in the bioanalytical and biomedical fields.

Single ether group in a glycol-based ultra-thin layer prevents surface fouling from undiluted serum.

Through systematic structural modification, it is shown that the internal, single oxygen atom of simple monoethylene glycol-based organic films is essential for radically altering the fouling behaviour of quartz against undiluted serum, as characterized by the electromagnetic piezoelectric acoustic sensor. The synergy is strongest with distal hydroxyls.

Label-free detection of HIV-2 antibodies in serum with an ultra-high frequency acoustic wave sensor.

Herein is described a label-free immunosensor dedicated to the detection of HIV-2. The biosensor platform is constructed as a mixed self-assembled monolayer-coated quartz wafer onto which HIV-2 immunodominant epitopes are immobilized. The biosensing properties, in terms of specific vs. non-specific antigen-antibody interactions, are evaluated with the electromagnetic piezoelectric acoustic sensor (EMPAS) using equimolar serum solutions of HIV-2 or HIV-1 monoclonal antibodies, respectively. This immunosensor constitutes the first real-world application of the EMPAS technology in the bioanalytical field.

Linker immobilization of protein and oligonucleotide on indium-tin-oxide for detection of probe-target interactions by Kelvin physics.

This work describes a label-free microarray analysis technique capable of detecting biomolecule target interactions with probes being anchored with a new linker-diluent system on indium-tin-oxide. The method is based on the differential work function characteristics of the substrate measured by a scanning Kelvin nanoprobe in terms of contact potential difference signals.

A palladium-catalyzed alkylation/direct arylation synthesis of nitrogen-containing heterocycles.

A norbornene-mediated palladium-catalyzed sequence is described in which an alkyl-aryl bond and an aryl-heteroaryl bond are formed in one reaction vessel. The aryl-heteroaryl bond-forming step occurs via a direct arylation reaction. A number of six-, seven-, and eight-membered ring-annulated indoles, pyrroles, pyrazoles, and azaindoles were synthesized from the corresponding bromoalkyl azole and an aryl iodide.