| 1. |
- Akkilic, Namik, et al.
(författare)
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Avidity-Based Affinity Enhancement Using Nanoliposome-Amplified SPR Sensing Enables Low Picomolar Detection of Biologically Active Neuregulin 1
- 2019
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Ingår i: ACS Sensors. - : American Chemical Society (ACS). - 2379-3694. ; 4:12, s. 3166-3174
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Akkilic, Namik, et al.
(författare)
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Single-molecule biosensors: Recent advances and applications
- 2020
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Ingår i: Biosensors and Bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 151
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Forskningsöversikt (refereegranskat)abstract
- 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.
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| 3. |
- Gunnarsson, Anders, 1981, et al.
(författare)
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Affinity Capturing and Surface Enrichment of a Membrane Protein Embedded in a Continuous Supported Lipid Bilayer
- 2016
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Ingår i: ChemistryOpen. - : Wiley. - 2191-1363. ; 5:5, s. 445-449
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Tidskriftsartikel (refereegranskat)abstract
- Investigations of ligand-binding kinetics to membrane proteins are hampered by their poor stability and low expression levels, which often translates into sensitivity-related limitations impaired by low signal-to-noise ratios. Inspired by affinity capturing of water-soluble proteins, which utilizes water as the mobile phase, we demonstrate affinity capturing and local enrichment of membrane proteins by using a fluid lipid bilayer as the mobile phase. Specific membrane-protein capturing and enrichment in a microfluidic channel was accomplished by immobilizing a synthesized trivalent nitrilotriacetic acid (tris-NTA)-biotin conjugate. A polymer-supported lipid bilayer containing His(6)-tagged b-secretase (BACE) was subsequently laterally moved over the capture region by using a hydrodynamic flow. Specific enrichment of His(6)-BACE in the Ni2+-NTA-modified region of the substrate resulted in a stationary three-fold increase in surface coverage, and an accompanied increase in ligand-binding response.
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| 4. |
- Gunnarsson, Anders, 1981, et al.
(författare)
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Drug Discovery at the Single Molecule Level: Inhibition-in-Solution Assay of Membrane-Reconstituted beta-Secretase Using Single-Molecule Imaging
- 2015
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 87:8, s. 4100-4103
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Tidskriftsartikel (refereegranskat)abstract
- Inhibition-in-solution assays (ISA) employing surface-based biosensors such as surface plasmon resonance (SPR) are an effective screening approach in drug discovery. However, analysis of potent binders remains a significant hurdle due to limited sensitivity and accompanied depletion of the inhibiting compounds due to high protein concentrations needed for detectable binding signals. To overcome this limitation, we explored a microscopy-based single-molecule ISA compatible with liposome-reconstituted membrane proteins. Using a set of validated small molecule inhibitors against beta-secretase 1 (BACE1), the assay was benchmarked with respect to sensitivity and dynamic range against SPR. We demonstrate that the dynamic range of measurable affinities is greatly extended by more than 2 orders of magnitude as compared to SPR, thus facilitating measurements of highly potent (K-d
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| 5. |
- Pace, Hudson, 1982, et al.
(författare)
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Preserved Transmembrane Protein Mobility in Polymer-Supported Lipid Bilayers Derived from Cell Membranes
- 2015
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 87:18, s. 9194-9203
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Tidskriftsartikel (refereegranskat)abstract
- Supported lipid bilayers (SLBs) have contributed invaluable information about the physiochemical properties of cell membranes, but their compositional simplicity often limits the level of knowledge that can be gained about the structure and function of transmembrane proteins in their native environment. Herein, we demonstrate a generic protocol for producing polymer-supported lipid bilayers on glass surfaces that contain essentially all naturally occurring cell-membrane components of a cell line while still retaining transmembrane protein mobility and activity. This was achieved by merging vesicles made from synthetic lipids (PEGylated lipids and POPC lipids) with native cell-membrane vesicles to generate hybrid vesicles which readily rupture into a continuous polymer-supported lipid bilayer. To investigate the properties of these complex hybrid SLBs and particularly the behavior of their integral membrane-proteins, we used total internal reflection fluorescence imaging to study a transmembrane protease, β-secretase 1 (BACE1), whose ectoplasmic and cytoplasmic domains could both be specifically targeted with fluorescent reporters. By selectively probing the two different orientations of BACE1 in the resulting hybrid SLBs, the role of the PEG-cushion on transmembrane protein lateral mobility was investigated. The results reveal the necessity of having the PEGylated lipids present during vesicle adsorption to prevent immobilization of transmembrane proteins with protruding domains. The proteolytic activity of BACE1 was unadulterated by the sonication process used to merge the synthetic and native membrane vesicles; importantly it was also conserved in the SLB. The presented strategy could thus serve both fundamental studies of membrane biophysics and the production of surface-based bioanalytical sensor platforms.
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| 6. |
- Wahlsten, Olov, 1989, et al.
(författare)
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Equilibrium-Fluctuation Analysis for Interaction Studies between Natural Ligands and Single G Protein-Coupled Receptors in Native Lipid Vesicles
- 2015
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 31:39, s. 10774-10780
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Tidskriftsartikel (refereegranskat)abstract
- G protein-coupled receptors (GPCRs) constitute the most versatile family of cell-membrane receptors and have been increasingly identified as important mediators of many physiological functions. They also belong to one of the most central drug target classes, but current screening technologies are limited by the requirements of overexpression and stabilization of GPCRs. This calls for sensitivity-increased detection strategies preferably meeting single-molecule detection limits. This challenge is here addressed by employing total internal reflection fluorescence microscopy to characterize the interaction kinetics between CXCR3, a GPCR involved in inflammatory responses, and two of its chemokine ligands, CXCL10 and CXCL11. Fluorescence labeling of the lipid membrane, rather than the membrane protein itself, of GPCR-containing native vesicles, and immobilization of the corresponding ligand on the surface, enabled determination of the interaction kinetics using single-molecule equilibrium-fluctuation analysis. With a limit of detection of GPCR-containing vesicles in the low picomolar concentration regime, the results demonstrate the possibility to use inhibition in solution screening of high affinity ligands/drug candidates, which due to target-binding depletion of the inhibiting compounds is demanding using assays with more moderate detection limits.
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