| 1. |
- Decrop, Deborah, et al.
(författare)
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Single-step imprinting of femtoliter microwell arrays allows digital bioassays with attomolar limit of detection
- 2017
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Ingår i: ACS Applied Materials and Interfaces. - : American Chemical Society (ACS). - 1944-8244 .- 1944-8252. ; 9:12, s. 10418-10426
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Tidskriftsartikel (refereegranskat)abstract
- Bead-based microwell array technology is growing as an ultrasensitive target detection tool. However, dissemination of the technology and its commercial use are hampered by current fabrication methods for hydrophilic-in-hydrophobic microwell arrays, which are either expensive or labour-intensive to manufacture, or which results in low bead seeding efficiencies. In this paper, we present a novel single-step manufacturing method for imprinting cheap and disposable hydrophilic-in-hydrophobic microwell arrays suitable for single-molecule detection. Single-step imprinting of hydrophilic-in-hydrophobic microwell arrays is made possible using an innovative surface energy replication approach by means of a hydrophobic thiol-ene polymer formulation. In this polymer, hydrophobic-moiety-containing monomers self-assemble against the hydrophobic surface of the imprinting stamp, which results in a hydrophobic replica surface after polymerization. After removing the stamp, hydrophilic wells are obtained with the well bottoms consisting of glass substrate. We demonstrate that the hydrophilic-in-hydrophobic imprinted microwell arrays enable successful and efficient self-assembly of individual water droplets and seeding of magnetic beads with loading efficiencies up to 96%. We also demonstrate the suitability of the microwell arrays for the isolation and detection of single-molecules achieving a limit of detection of 17.4 aM when performing a streptavidin-biotin binding assay. The ease of manufacturing demonstrated here is expected to allow translation of digital microwell array technology towards diagnostic applications.
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| 2. |
- Decrop, Deborah, et al.
(författare)
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Single-step manufacturing of femtoliter microwell arrays in a novel surface energy mimicking polymer
- 2015
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Ingår i: 18th International Conference on Solid-State Sensors, Actuators and Microsystems (IEEE TRANSDUCER 2015). - : IEEE.
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Konferensbidrag (refereegranskat)abstract
- We report a novel polymer material formulation and stamp-molding technique that enable rapid single-step manufacturing of hydrophilic-in-hydrophobic microwell arrays. We developed a modified thiol-ene-epoxy polymer (mOSTE+) formulation that mimics the surface energy of its mold during polymerization. The polymer inherits the surface energy from the mold through molecular self-assembly, in which functional monomers self-assemble at the interface between the liquid prepolymer and the mold surface. Combining this novel mOSTE+ material with a stamp-molding process leads to simultaneous surface energy mimicking and micro-structuring. This method was used to manufacture microwells with hydrophilic bottom and hydrophobic sidewall, depressed in a surrounding hydrophobic surface. The microwell arrays were successfully tested for the self-assembly of 62’000 femtoliter-droplets. Such femtoliter droplet arrays are useful for, e.g., digital ELISA and single cell/molecule analysis applications.
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| 3. |
- Horta, Sara, et al.
(författare)
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Evaluation of Immuno-Rolling Circle Amplification for Multiplex Detection and Profiling of Antigen-Specific Antibody Isotypes
- 2021
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 93:15, s. 6169-6177
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Tidskriftsartikel (refereegranskat)abstract
- Antibody characterization is essential for understanding the immune system and development of diagnostics and therapeutics. Current technologies are mainly focusing on the detection of antigen-specific immunoglobulin G (IgG) using bulk singleplex measurements, which lack information on other isotypes and specificity of individual antibodies. Digital immunoassays based on nucleic acid amplification have demonstrated superior performance by allowing the detection of single molecules in a multiplex and sensitive manner. In this study, we demonstrate for the first time an immuno-rolling circle amplification (immunoRCA) assay for the multiplex detection of three antigen-specific antibody isotypes (IgG, IgA, and IgM) and its integration with microengraving. To validate this approach, we used the autoimmune disease immune-mediated thrombotic thrombocytopenic purpura (iTTP) as the model disease with anti-ADAMTS13 autoantibodies as the diagnostic target molecules. To identify the anti-ADAMTS13 autoantibody isotypes, we designed a pool of three unique antibody-oligonucleotide conjugates for identification and subsequent amplification and visualization via RCA. To validate this approach, we first confirmed an assay specificity of >88% and a low limit of detection of 0.3 ng/mL in the spiked buffer. Subsequently, we performed a dilution series of an iTTP plasma sample for the multiplex detection of the three isotypes with higher sensitivity compared to an enzyme-linked immunosorbent assay. Finally, we demonstrated single-cell analysis of human B cells and hybridoma cells for the detection of secreted antibodies using microengraving and achieved a detection of 23.3 pg/mL secreted antibodies per hour. This approach could help to improve the understanding of antibody isotype distributions and their roles in various diseases.
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| 4. |
- Horta, Sara, et al.
(författare)
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Evaluation of immuno-rolling circle amplification for multiplexed detection and profiling of antigen-specific antibody isotypes
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Antibody characterization has become essential for diagnosis and development of therapeutic solutions in autoimmune, cardiovascular and infectious diseases. The specificity, affinity and isotype are crucial information for antibody studies and can be obtained from screening plasma samples or populations of B cells. Current technologies are mainly focusing on the discovery of abundant immunoglobulins, namely IgG, and are based on bulk measurements. In this study, we present a digital screening platform utilizing rolling circle amplification (RCA) for the detection of antigen-specific antibody isotypes in solution or secreted from single cells. To validate this approach, the autoimmune disease immune-mediated thrombotic thrombocytopenic purpura (iTTP) was used as model disease and anti-ADAMTS13 antibodies were the target molecules. Antibody-oligonucleotide conjugates (AOCs) were designed for the multiplexed detection of human antibody isotypes IgA, IgG and IgM. Then, ADAMTS13 fragments were coated on glass slides and subsequently, target antibodies identified by specific AOC binding and visualized via an RCA assay. First, we validated the method by characterizing the assay specificity and limit of detection (LoD). When seeding different isotypes, we confirmed the high specificity of the assay (> 90%) and detection of monoclonal anti-ADAMTS13 IgG down to 0.3 ng/mL. A dilution series of a plasma sample of iTTP patient confirmed the multiplexed detection of the three isotypes with higher sensitivity compared to ELISA. Finally, we performed single cell analysis of human B cells and hybridoma cells for the detection of secreted antibodies using microengraving, achieving a detection of 23.3 pg/mL secreted antibodies per hour. This approach could help to improve the understanding of antibody isotype distributions and their roles in various diseases.
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| 5. |
- Kuhnemund, Malte, et al.
(författare)
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Circle-to-circle amplification on a digital microfluidic chip for amplified single molecule detection
- 2014
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 14:16, s. 2983-2992
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a novel digital microfluidic nucleic acid amplification concept which is based on padlock probe mediated DNA detection and isothermal circle-to-circle amplification (C2CA). This assay platform combines two digital approaches. First, digital microfluidic manipulation of droplets which serve as micro-reaction chambers and shuttling magnetic particles between these droplets facilitates the integration of complex solid phase multistep assays. We demonstrate an optimized novel particle extraction and transfer protocol for superparamagnetic particles on a digital microfluidic chip that allows for nearly 100% extraction efficiencies securing high assay performance. Second, the compartmentalization required for digital single molecule detection is solved by simple molecular biological means, circumventing the need for complex microfabrication procedures necessary for most, if not all, other digital nucleic acid detection methods. For that purpose, padlock probes are circularized in a strictly target dependent ligation reaction and amplified through two rounds of rotting circle amplification, including an intermediate digestion step. The reaction results in hundreds of 500 nm sized individually countable DNA nanospheres per detected target molecule. We demonstrate that integrated miniaturized digital microfluidic C2CA results in equally high numbers of C2CA products mu L-1 as off-chip tube control experiments indicating high assay performance without signal loss. As low as 1 aM synthetic Pseudomonas aeruginosa DNA was detected with a linear dynamic range over 4 orders of magnitude up to 10 fM proving excellent suitability for infectious disease diagnostics.
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| 7. |
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| 8. |
- Zandi Shafagh, Reza, et al.
(författare)
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Reaction Injection Molding of Hydrophilic-in-Hydrophobic Femtolitre-Well Arrays
- 2019
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Ingår i: Microsystems & Nanoengineering. - : Nature Publishing Group. - 2055-7434. ; :5
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Tidskriftsartikel (refereegranskat)abstract
- Patterning of micro- and nanoscale topologies and surface properties of polymer devices is of particular importance for a broad range of life science applications, including cell-adhesion assays and highly sensitive bioassays. The manufacturing of such devices necessitates cumbersome multiple-step fabrication procedures and results in surface properties which degrade over time. This critically hinders their wide-spread dissemination. Here, we simultaneously mold and surface energy pattern microstructures in off-stoichiometric thiol-ene by area-selective monomer self-assembly in a rapid micro-reaction injection molding cycle. We replicated arrays of 1,843,650 hydrophilic-in-hydrophobic femtolitre-wells with long-term stable surface properties and magnetically trapped beads with 75% and 87.2% efficiency in single- and multiple-seeding events, respectively. These results form the basis for ultrasensitive digital biosensors, specifically, and for the fabrication of medical devices and life science research tools, generally.
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