| 1. |
- Hernández-Neuta, Iván, et al.
(author)
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Microfluidic magnetic fluidized bed for DNA analysis in continuous flow mode
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Other publication (other academic/artistic)abstract
- Magnetic solid phase substrates for biomolecule manipulation have become a valuable tool for simplification and automation of molecular biology protocols. However, the handling of magnetic particles inside microfluidic chips for miniaturized assays is often challenging due to inefficient mixing, aggregation, and the advanced instrumentation required for effective actuation. Here, we describe the use of a microfluidic magnetic fluidized bed approach that enables dynamic, highly efficient and simplified magnetic bead actuation for DNA processing in a continuous flow platform with minimal technical requirements. We evaluate the performance of this approach by testing the efficiency of individual steps of a DNA assay based on padlock probes and rolling circle amplification (RCA). This assay comprises common nucleic acid analysis principles, such as hybridization, ligation, amplification and restriction digestion. We obtained efficiencies of up to 90% for these reactions and high throughput capabilities, with flow rates up to 5 L/min without compromising performance. The obtained efficiency values using the fluidized bed were superior to a commercially available solution for microfluidic manipulation of magnetic beads. Moreover, to demonstrate the potential of this approach for integration into micro-total analysis systems, we optimized the production of a low-cost polymer based micro arrayand tested its analytical performance for integrated single-molecule digital read-out. Finally, we provide the proof-of-concept for a single-chamber microfluidic chip that combines the fluidized bed with the polymer microarray for a highly simplified and integrated magnetic bead-based DNA analyzer, with potential applications in diagnostic systems.
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| 2. |
- Hernández-Neuta, Iván, et al.
(author)
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Microfluidic magnetic fluidized bed for DNA analysis in continuous flow mode
- 2018
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In: Biosensors & bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 102, s. 531-539
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Journal article (peer-reviewed)abstract
- Magnetic solid phase substrates for biomolecule manipulation have become a valuable tool for simplification and automation of molecular biology protocols. However, the handling of magnetic particles inside microfluidic chips for miniaturized assays is often challenging due to inefficient mixing, aggregation, and the advanced instrumentation required for effective actuation. Here, we describe the use of a microfluidic magnetic fluidized bed approach that enables dynamic, highly efficient and simplified magnetic bead actuation for DNA analysis in a continuous flow platform with minimal technical requirements. We evaluate the performance of this approach by testing the efficiency of individual steps of a DNA assay based on padlock probes and rolling circle amplification. This assay comprises common nucleic acid analysis principles, such as hybridization, ligation, amplification and restriction digestion. We obtained efficiencies of up to 90% for these reactions with high throughput processing up to 120 mu L of DNA dilution at flow rates ranging from 1 to 5 mu L/min without compromising performance. The fluidized bed was 20-50% more efficient than a commercially available solution for microfluidic manipulation of magnetic beads. Moreover, to demonstrate the potential of this approach for integration into micro-total analysis systems, we optimized the production of a low-cost polymer based microarray and tested its analytical performance for integrated single-molecule digital read-out. Finally, we provide the proof-of-concept for a single-chamber microfluidic chip that combines the fluidized bed with the polymer microarray for a highly simplified and integrated magnetic bead-based DNA analyzer, with potential applications in diagnostics.
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| 3. |
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| 4. |
- Kühnemund, Malte, et al.
(author)
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Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy
- 2017
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8
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Journal article (peer-reviewed)abstract
- Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies.
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| 5. |
- Neumann, Felix, et al.
(author)
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QCM mass underestimation in molecular biotechnology : Proximity ligation assay for norovirus detection as a case study
- 2018
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In: Sensors and actuators. B, Chemical. - : Elsevier BV. - 0925-4005 .- 1873-3077. ; 273, s. 742-750
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Journal article (peer-reviewed)abstract
- The development of piezoelectric mass-sensitive devices is based on the shift in resonance frequency that is proportional to the deposited mass. However, this holds true only for small, rigid masses, while it can result in mass underestimation for heavy, non-rigid masses. In this work, we demonstrate this 'missing mass' phenomenon by measurement of high molecular weight biomolecules on a Quartz Crystal Microbalance (QCM) platform. For this, we present a model bioassay consisting of a sandwich-type proximity ligation assay for the detection of norovirus-like particles, and its real-time build-up on QCM as an experimental evidence. Upon combination with a localized QCM platform, we explain the pronounced slipping effect in multilayer biological systems resulting in energy dissipation and subsequent mass underestimation. This helps in pointing out the limitations of mega-gravity field sensors for molecular diagnostics where absolute quantification of pathogen load becomes indispensable towards biosensing applications.
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