Simplified immobilisation method for histidine-tagged enzymes in poly(methyl methacrylate) microfluidic devices

Article in press. Kulsharova, G., New BIOTECHNOLOGY (2017), https://doi.org/10.1016/j.nbt.2017.12.004Poly(methyl methacrylate) (PMMA) microfluidic devices have become promising platforms for a wide range of applications. Here we report a simple method for immobilising histidine-tagged enzymes suitable for PMMA microfluidic devices. The 1-step-immobilisation described is based on the affinity of the His-tag/Ni-NTA interaction and does not require prior amination of the PMMA surface, unlike many existing protocols. We compared it with a 3-step immobilisation protocol involving amination of PMMA and linking NTA via a glutaraldehyde cross-linker. These methods were applied to immobilise transketolase (TK) in PMMA microfluidic devices. Binding efficiency studies showed that about 15% of the supplied TK was bound using the 1-step method and about 26% of the enzyme was bound by the 3-step method. However, the TK-catalysed reaction producing l-erythrulose performed in microfluidic devices showed that specific activity of TK in the device utilising the 1-step immobilisation method was approximately 30% higher than that of its counterpart. Reusability of the microfluidic device produced via the 1-step method was tested for three cycles of enzymatic reaction and at least 85% of the initial productivity was maintained. The device could be operated for up to 40 h in a continuous flow and on average 70% of the initial productivity was maintained. The simplified immobilisation method required fewer chemicals and less time for preparation of the immobilised microfluidic device compared to the 3-step method while achieving higher specific enzyme activity. The method represents a promising approach for the development of immobilised enzymatic microfluidic devices and could potentially be applied to combine protein purification with immobilisation.Peer reviewe

Electrowetting-Based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H 2O 2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.Peer reviewe

Rotational-pulse actuated dissolvable-film valves for automated purification of total RNA from E. Coli

In this work we report for the first time on a repertoire of valving technologies which are combined to enable automated purification of total RNA from cell homogenate. Process control is implemented us-ing rotational-pulse actuated dissolvable-film (DF) valves; where the order of valve actuation is deter-mined by the disc architecture while the timing of valve actuation is governed by pulses in the spin rate. Selective liquid routing is enabled by combining a heavy, inert and immiscible liquid plug with a DF. The combination of these technologies enables bead-based extraction of amplifiable RNA, with a yield comparable to gold-standard bench-top protocols

Automated on-disc total RNA extraction from whole blood towards point-of-care for early-stage diagnostics

We present a novel integrated, centrifugo-pneumatic micro-homogenizer (“μHomogenizer”) for automated sample preparation and total RNA extraction from whole blood. Using a Trizol based protocol, this novel μHomogenizer efficiently lyses whole blood spiked with E. coli, retains the organic-mixed fraction and yields the aqueous phase with the total RNA content. By the interplay of microfluidic design and a protocol of rotational frequencies, we concatenate (and parallelize) a sequence of five subsequent liquid handling operations that complete in less than 10 minutes. A comparison of the total nucleotide content yields similar performance as conventional, essentially manual off-disc sample preparation methods

Mimicking insect communication: Release and detection of pheromone, biosynthesized by an alcohol acetyl transferase immobilized in a microreactor

Infochemical production, release and detection of (Z,E)-9,11-tetradecadienyl acetate, the major component of the pheromone of the moth Spodoptera littoralis is achieved in a novel microfluidic system, designed to mimic the final step of the pheromone biosynthesis by immobilized recombinant alcohol acetyl transferase. The microfluidic system is part of an "artificial gland", i.e. a chemoemitter that comprises a microreactor connected to a microevaporator and is able to produce and release a pre-defined amount of the major component of the pheromone from the corresponding (Z,E)-9,11-tetradecadienol.. Performance of the entire chemoemitter has been assessed in electrophysiological and behavioral experiments. Electroantennographic depolarizations of the pheromone produced by the chemoemitter were ca. 40% relative to that evoked by the synthetic pheromone. In a wind tunnel, the pheromone released from the evaporator elicited on males a similar attraction behaviour as 3 virgin females in most of the parameters considered.Peer reviewe

Development of an on-disc isothermal in vitro amplification and detection of bacterial RNA

This document is the Accepted Manuscript version, made available under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License CC BY NC-ND 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/). The final, published version is available online at doi: https://doi.org/10.1016/j.snb.2016.08.018. Published by Elsevier B. V.We present a centrifugal microfluidic “Lab-on-a-Disc” (LoaD) system capable of implementing nucleic acid in vitro amplification using non-contact heating and fluorescence detection. The system functionality is verified by implementing a Nucleic Acid Sequence Based Amplification (NASBA) reaction, targeting the tmRNA transcript of Haemophilus influenzae. The NASBA assay incorporates fluorescent molecular beacon probes reporting target tmRNA amplification for endpoint detection. The system implements non-contact IR heating to heat the NASBA reaction to the required target temperatures during denaturation and amplification steps. The LoaD control system facilitates spin speed and chamber positioning for heating and fluorescence detection. The LoaD alignment system uses magnetic fields to locate and lock the chamber in the required position (heating or detection). The NASBA assay was implemented on the system using Haemophilus influenzae tmRNA over the range 102–104 cell equivalent (CE) units. For comparison, identical qNASBA assays were implemented on a Roche LightCycler 2.0 over this concentration range.Peer reviewe

Modelling a permanent magnet synchronous motor in FEniCSx for parallel high-performance simulations

© 2022 The Authors. Published by Elsevier B.V. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/There are concerns that the extreme requirements of heavy-duty vehicles and aviation will see them left behind in the electrification of the transport sector, becoming the most significant emitters of greenhouse gases. Engineers extensively use the finite element method to analyse and improve the performance of electric machines, but new highly scalable methods with a linear (or near) time complexity are required to make extreme-scale models viable. This paper introduces a three-dimensional permanent magnet synchronous motor model using FEniCSx, a finite element platform tailored for efficient computing and data handling at scale. The model demonstrates comparable magnetic flux density distributions to a verification model built in Ansys Maxwell with a maximum deviation of 7% in the motor’s static regions. Solving the largest mesh, comprising over eight million cells, displayed a speedup of 198 at 512 processes. A preconditioned Krylov subspace method was used to solve the system, requiring 92% less memory than a direct solution. It is expected that advances built on this approach will allow system-level multiphysics simulations to become feasible within electric machine development. This capability could provide the near real-world accuracy needed to bring electric propulsion systems to large vehicles.Peer reviewe

Possible mechanisms of CO₂ reduction by H₂ via prebiotic vectorial electrochemistry

Methanogens are putatively ancestral autotrophs that reduce CO2 with H2 to form biomass using a membrane-bound, proton-motive Fe(Ni)S protein called the energy-converting hydrogenase (Ech). At the origin of life, geologically sustained H+ gradients across inorganic barriers containing Fe(Ni)S minerals could theoretically have driven CO2 reduction by H2 through vectorial chemistry in a similar way to Ech. pH modulation of the redox potentials of H2, CO2 and Fe(Ni)S minerals could in principle enable an otherwise endergonic reaction. Here, we analyse whether vectorial electrochemistry can facilitate the reduction of CO2 by H2 under alkaline hydrothermal conditions using a microfluidic reactor. We present pilot data showing that steep pH gradients of approximately 5 pH units can be sustained over greater than 5 h across Fe(Ni)S barriers, with H+-flux across the barrier about two million-fold faster than OH--flux. This high flux produces a calculated 3-pH unit-gradient (equating to 180 mV) across single approximately 25-nm Fe(Ni)S nanocrystals, which is close to that required to reduce CO2. However, the poor solubility of H2 at atmospheric pressure limits CO2 reduction by H2, explaining why organic synthesis has so far proved elusive in our reactor. Higher H2 concentration will be needed in future to facilitate CO2 reduction through prebiotic vectorial electrochemistry

Event-triggered logical flow control for comprehensive process integration of multi-step assays on centrifugal microfluidic platforms

Content in the UH Research Archive is made available for personal research, educational, and non-commercial purposes only. Unless otherwise stated, all content is protected by copyright, and in the absence of an open license, permissions for further re-use should be sought from the publisher, the author, or other copyright holder.The centrifugal "lab-on-a-disc" concept has proven to have great potential for process integration of bioanalytical assays, in particular where ease-of-use, ruggedness, portability, fast turn-around time and cost efficiency are of paramount importance. Yet, as all liquids residing on the disc are exposed to the same centrifugal field, an inherent challenge of these systems remains the automation of multi-step, multi-liquid sample processing and subsequent detection. In order to orchestrate the underlying bioanalytical protocols, an ample palette of rotationally and externally actuated valving schemes has been developed. While excelling with the level of flow control, externally actuated valves require interaction with peripheral instrumentation, thus compromising the conceptual simplicity of the centrifugal platform. In turn, for rotationally controlled schemes, such as common capillary burst valves, typical manufacturing tolerances tend to limit the number of consecutive laboratory unit operations (LUOs) that can be automated on a single disc. In this paper, a major advancement on recently established dissolvable film (DF) valving is presented; for the very first time, a liquid handling sequence can be controlled in response to completion of preceding liquid transfer event, i.e. completely independent of external stimulus or changes in speed of disc rotation. The basic, event-triggered valve configuration is further adapted to leverage conditional, large-scale process integration. First, we demonstrate a fluidic network on a disc encompassing 10 discrete valving steps including logical relationships such as an AND-conditional as well as serial and parallel flow control. Then we present a disc which is capable of implementing common laboratory unit operations such as metering and selective routing of flows. Finally, as a pilot study, these functions are integrated on a single disc to automate a common, multi-step lab protocol for the extraction of total RNA from mammalian cell homogenate.Peer reviewe