Monitoring the Switching of Single BSA-ATTO 488 Molecules Covalently End-Attached to a pH-Responsive PAA Brush.

We describe a novel combination of a responsive polymer brush and a fluorescently labeled biomolecule, where the position of the biomolecule can be switched from inside to outside the brush and vice versa by a change in pH. For this, we grafted ultrathin, amino-terminated poly(acrylic acid) brushes to glass and silicon substrates. Individual bovine serum albumin (BSA) molecules labeled with fluorophore ATTO 488 were covalently end-attached to the polymers in this brush using a bis-N-succinimidyl-(pentaethylene glycol) linker. We investigated the dry layer properties of the brush-protein ensemble, and it is swelling behavior using spectroscopic ellipsometry. Total internal reflection fluorescence (TIRF) microscopy enabled us to study the distance-dependent switching of the fluorescently labeled protein molecules. The fluorescence emission from the labeled proteins ceased (out-state) when the polymer chains stretched away from the interface under basic pH conditions, and fluorescence recurred (in-state) when the chains collapsed under acidic conditions. Moreover, TIRF allowed us to study the fluorescence switching behavior of fluorescently labeled BSA molecules down to the single-molecule level, and we demonstrate that this switching is fast but that the exact intensity during the in-state is the result of a more random process. Control experiments verify that the switching behavior is directly correlated to the responsive behavior of the polymer brush. We propose this system as a platform for switchable sensor applications but also as a method to study the swelling and collapse of individual polymer chains in a responsive polymer brush.

Conductance switching and organization of two structurally related molecular wires on gold.

The self-assembly and electron transfer properties of adsorbed organic molecules are of interest for the construction of miniaturized molecular circuitries. We have investigated with scanning probe microscopy the self-organization of two structurally related molecular wires embedded within a supportive alkanethiol matrix. Our results evidence heterogeneous adsorption patterns of the molecular wires on gold with either incommensurate unit cells driven into assembly by lateral interactions or a dynamic, commensurate distribution on gold, along with formation of distinct 2D phases. We also observed diffusion-based conductance switching for one of the molecular wires, due to its propensity toward weaker lateral interactions and Au-S adatom formation. We have further demonstrated through the use of scanning tunneling spectroscopy differential current-voltage response for each molecular wire, despite their close structural similarity. Such molecular wires embedded in alkanethiol matrix and exhibiting conductance-switching phenomena have the potential to be used for the functionalization of electrodes in bioelectronic devices.

Discovery and Characterization of BAY-805, a Potent and Selective Inhibitor of Ubiquitin-Specific Protease USP21.

USP21 belongs to the ubiquitin-specific protease (USP) subfamily of deubiquitinating enzymes (DUBs). Due to its relevance in tumor development and growth, USP21 has been reported as a promising novel therapeutic target for cancer treatment. Herein, we present the discovery of the first highly potent and selective USP21 inhibitor. Following high-throughput screening and subsequent structure-based optimization, we identified BAY-805 to be a non-covalent inhibitor with low nanomolar affinity for USP21 and high selectivity over other DUB targets as well as kinases, proteases, and other common off-targets. Furthermore, surface plasmon resonance (SPR) and cellular thermal shift assays (CETSA) demonstrated high-affinity target engagement of BAY-805, resulting in strong NF-κB activation in a cell-based reporter assay. To the best of our knowledge, BAY-805 is the first potent and selective USP21 inhibitor and represents a valuable high-quality in vitro chemical probe to further explore the complex biology of USP21.

Single-molecule biosensors: Recent advances and applications.

Single-molecule biosensors serve the unmet need for real time detection of individual biological molecules in the molecular crowd with high specificity and accuracy, uncovering unique properties of individual molecules which are hidden when measured using ensemble averaging methods. Measuring a signal generated by an individual molecule or its interaction with biological partners is not only crucial for early diagnosis of various diseases such as cancer and to follow medical treatments but also offers a great potential for future point-of-care devices and personalized medicine. This review summarizes and discusses recent advances in nanosensors for both in vitro and in vivo detection of biological molecules offering single-molecule sensitivity. In the first part, we focus on label-free platforms, including electrochemical, plasmonic, SERS-based and spectroelectrochemical biosensors. We review fluorescent single-molecule biosensors in the second part, highlighting nanoparticle-amplified assays, digital platforms and the utilization of CRISPR technology. We finally discuss recent advances in the emerging nanosensor technology of important biological species as well as future perspectives of these sensors.

Avidity-Based Affinity Enhancement Using Nanoliposome-Amplified SPR Sensing Enables Low Picomolar Detection of Biologically Active Neuregulin 1.

Biomarkers serve as indicators of disease progression or therapeutic response of an medical intervention, and means for enabling a reliable and sensitive biomarker detection are therefore vital in clinical settings. Most biosensor assays require high-affinity interactions in combination with an enzyme or fluorescent tag to enable detection and frequently employ extensive washing procedures prior to signal readout. Attempts to overcome this limitation by using natural biological partners tend to be demanding, because their very low affinity is frequently not compatible with the need of reaching low limits of detection (LODs), especially for circulating biomarkers that possess short half-lives. To address these challenges, we developed a label-free surface plasmon resonance (SPR) platform for the detection of neuregulin 1 (NRG1) using ErbB4-modified liposomes offering both signal amplification and affinity enhancement via functional multivalent interactions. Through the functional avidity interaction between NRG1 and ErbB4, an LOD of 3.5 picomolar was reached, which is about 60-fold higher than traditional SPR and miniaturized immunoassays. The biosensor displays also an 8-fold higher sensitivity when compared with a single-molecule immunoassay employing the natural binding partner rather than a high-affinity antibody as one of the interaction partners. In fact, the liposome-induced avidity between NRG1 and ErbB4 offered an LOD that was comparable to that obtained using a high-affinity antibody and enabled detection of NRG1 in plasma with a LOD of 36 pM. Employing the liposome-enhanced platform in conjunction with a low-affinity biomarker receptor thus enables the assessment of the functional state of the biomarker at competitive LODs and eliminates the need for high-affinity antibodies.

DNA-Based Photoacoustic Nanosensor for Interferon Gamma Detection.

Tracking protein levels in the body is vital in both research and medicine, where understanding their physiological roles provides insight into their regulation in homeostasis and diseases. In medicine, protein levels are actively sampled since they continuously fluctuate, reflecting the status of biological systems and provide insight into patient health. One such protein is interferon gamma, a clinically relevant protein with immunoregulatory functions that play critical roles against infection. New tools for continuously monitoring protein levels in vivo are invaluable in monitoring real-time conditions of patients to allow better care. Here, we developed a DNA-based nanosensor for the photoacoustic detection of interferon gamma. This work demonstrates how we transformed a simple DNA motif, receptors, and a novel phthalocyanine dye into a proof-of-concept photoacoustic nanosensor for protein detection. Surface plasmon resonance kinetic analysis demonstrated that the nanosensor is responsive and reversible to interferon gamma with an affinity in the nanomolar range, KD1 = 167 nM and KD2 = 316 nM. As a reporter, our design includes a novel phthalocyanine-based photoacoustic dye that stacks in a J-aggregate, causing a 22.5% increase in signal. Upon receptor binding, the DNA structure bends to induce phthalocyanine dye stacking, resulting in a 55% increase in photoacoustic signal in the presence of 10 µM interferon gamma. This proof-of-concept nanosensor is a novel approach to the development of a photoacoustic sensor and may be adapted for other proteins of interest in the future for in vivo tracking.

Responsive polymer brushes for controlled nanoparticle exposure.

We propose the design of a novel mixed polymer brush system that could act as a selective sensor with a distinct on-off switch. In the proposed system, a (single) nanoparticle (such as an antibody) is end-attached to a responsive chain, which is surrounded by a brush of nonresponsive chains. The collapse of the responsive chain leads to a protected state, where the nanoparticle is hidden in the polymer brush, while swelling of the responsive chain brings the nanoparticle outside of the brush into an exposed and active state. We investigate this system by numerical self-consistent field theory and predict a first-order like transition between the active state and the protective state at a critical decrease in solvent quality for the responsive chain. We show that by careful design of the brush parameters such as grafting density and chain length, for a given particle size, it is possible to fine-tune the desired switching mechanism.

Voltage-controlled fluorescence switching of a single redox protein.

Heterogeneous electron transfer (ET) of the redox protein, wild-type azurin (wt-Az) from Pseudomonas aeruginosa, was monitored at the single-molecule (SM) level by fluorescence resonance energy transfer (FRET), one electron at a time. Azurin molecules were labeled with an organic fluorophore (Cy5), and the FRET-coupling between Cy5 and the redox center (copper) was used to study ET to a semi-transparent, 10nm thin gold electrode in an optical configuration. By using a confocal microscope and a bipotentiostat for control of the electrode potential, the oxidation and reduction processes of individual Az-Cy5 molecules were monitored. In the oxidized state of the redox center of the azurin molecule, the fluorescence emission of the covalently attached Cy5 was largely quenched by FRET ('off'-state), whereas the emission was recovered upon reduction ('on'-state). The work presented here, shows directly controlled single redox switching events of an individual redox protein and its thermodynamic dispersion. We show that the distribution of midpoint potentials (E0) of individual azurin molecules peaks at 45.7±0.5 mV with a full width at half maximum of 15 mV vs saturated calomel electrode (SCE).

RETRACTED: Monolayers of pigment-protein complexes on a bare gold electrode: Orientation controlled deposition and comparison of electron transfer rate for two configurations.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief and the Corresponding Author, Muhammad Kamran. This paper has been withdrawn as the authors did not fully consult with their project collaborators prior to publication and failed to include them as co-authors of the article. This is acknowledged by the corresponding author. The authors and the Publisher would like to apologise for any inconvenience caused.

Chemically-induced redox switching of a metalloprotein reveals thermodynamic and kinetic heterogeneity, one molecule at a time.

Oxidation (off state) and reduction (on state) of a single azurin molecule is monitored, one electron at a time, which depend on the chemical redox potential. By analysing the fluorescence time traces from individual azurin molecules, reaction kinetics and redox thermodynamics were determined.