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Barrier-Film Based Reagent Storage and Release on Microfluidic Platforms for Sample-To-Answer Automation of Bioassays
Abstract
Sample-to-answer automation of multiplexed bioassay panels represents a key selling point of microfluidic Lab-on-a-chip devices. For real-world point-of-use scenarios, on-chip reagent storage is a key requirement for enabling the downstream microfluidic operations (e.g., resuspension of dried or lyophilized reagents using stored buffers) [1]. Several methods have been developed to this end with issues around cost, manufacturability and reliability being the key factors [2, 3]. This paper reports manufacture, assembly and characterization of a novel, low-cost and scalable technology for reagent storage and release based on sacrificial barrier films. We demonstrate an average evaporative volume loss of stored DI water at room temperature (21 o C) of 0.38% (± 0.3) for 42 days at a significantly low current materials cost of 30 cents per unit. The outlet of a chip-based reagent-storage chamber is transiently blocked by a barrier film composed of a distinct fluoropolymer on pressure sensitive adhesive. Due to its excellent hydrophobic, biocompatible and water / vapor barrier properties, the film allows for minimization of evaporation related losses of aqueous reagents. An immiscible and biocompatible oil-based liquid is layered on top of the aqueous phase. During storage and shipping, laminar conditions prevent the oil phase from reaching the barrier film. Under rotationally induced artificial gravity, the denser ancillary liquid displaces the reagent to contact the sacrificial film (Figure 1). It then retained in a volume-matched capture chamber so that the aqueous reagent is isolated further downstream. We demonstrate the ability of this actitation mechanism within a band of ±1.40 Hz for varying radial positions and release frequencies. Key characteristics of the reagent storage technology (Figure 2): a) Average evaporative losses of 0.38% (± 0.3) (test units with 500 µl DI water) when stored at room temperature (21 o C) for 42 days. b) Ability of the reagent storage units to handle real time transport was tested by air + ground shipping of 31 individual units on 6 discs over 10 days in transit sent in 3 different packages. No significant losses (when compared to unshipped units) in either the final amount of reagent released post-transport or the release frequencies were observed. c) Cumulative systemic losses of reagents post release within the microfluidic chips were 2.5µl (± 1.3 µl). All the release liquid is isolated after release in the capture chamber thus delivering only the aqueous reagent to the final chamber. d) Current cost of a barrier film insert is ~ €0.30. e) Centrifugal release frequency bands are characterized as a function of radial position and the depth of the upper and lower parts of the dual depth channel (Fig 2 B, C, D). Word Count: 490 References: 1. Smith et al. CD-Based Microfluidics for Primary Care in Extreme Point-of-Care Settings. Micromachines 2016, 7(2), 22. 2. Oordt et al. Miniature stick-packaging-an industrial technology for pre-storage and release of reagents in lab on-a-chip systems. Lab Chip, 2013,13, 2888-2892. 3. Smith et al. Blister pouches for effective reagent storage on microfluidic chips for blood cell counting.
... This paper focuses on a novel type of rotationally actuated valve; during storage of an aqueous reagent, a (water) dissolvable film (DF) presents a diffusion barrier which is initially protected by an oleophilic, ancillary liquid having a certain, specific density (Gaughran et al. 2016;Mishra et al. 2015Mishra et al. , 2017Mishra et al. , 2020Ducrée 2021d;Lu et al. 2020). Upon spinning, a centrifugo-hydrostatic equilibrium is reached, in which the interface between the two immiscible liquids contacts and thus dissolves the DF. ...
... Stiction of the progressing meniscus, e.g., caused by capillary pinning owing to manufacturing-related artefacts or dust, may be overcome by choosing a sufficiently elevated spin rate ≫ 0 for reaching hydrostatic equilibrium (5) at . In our own, naturally limited set of similar assay implementations Mishra et al. 2020), we did not observe any adverse effect of the ancillary liquid on the bioanalytical performance. Yet, a general, blanket guarantee cannot be issued a priori. ...
... Specific configurations of the reagent storage technique have been tested for premature opening upon possibly adverse conditions during storage, manual handling and transport. The valve proved to be stable, the main impact was evaporation, which was therefore included in the digital twin simulation (Fig. 6) Mishra et al. 2020). It is assumed that a malicious, brute force approach would be required for inducing valve opening by deformation of the disc. ...
Decentralized bioanalytical testing in resource-poor settings ranks among the most common applications of microfluidic systems. The high operational autonomy in such point-of-care/point-of-use scenarios requires long-term onboard storage of liquid reagents, which also need to be safely contained during transport and handling, and then reliably released just prior to their introduction to an assay protocol. Over the recent decades, centrifugal microfluidic technologies have demonstrated the capability of integrated, automated and parallelized sample preparation and detection of bioanalytical protocols. This paper presents a novel technique for onboard storage of liquid reagents which can be issued by a rotational stimulus of the system-innate spindle motor, while still aligning with the conceptual simplicity of such “Lab-on-a-Disc” (LoaD) systems. In this work, this highly configurable reagent storage technology is captured by a digital twin, which permits complex performance analysis and algorithmic design optimization according to objectives as expressed by target metrics.
We present the analysis of blister pouches for reagent storage and release into microfluidic devices towards point-of-care blood cell counting applications. Blister pouches provide an effective reagent storage mechanism and can be mounted onto microfluidic cartridges directly. Reagents can be released from blister pouches through automated or manual compression and consequent rupturing of the pouches. A microfluidic device for metering of blister pouch contents was developed and investigated as part of this work, as precise volumes of reagents are often required when performing reactions, and particularly for blood cell counting applications which are the focus of this study. The metering device shows high accuracy and repeatability with an error of 1.93% and standard deviation of 3.1% across 30 test results. This work also investigates important blister pouch characteristics for three different types of blister pouch foil materials, including forces required to burst the blister, as well as shelf life and reagent compatibility of the blisters. Typical forces required are in the range of 25–35 N depending on the blister foil material used. Blister shelf life can be greatly affected by the reagent being stored, and thus, the blister foil material choice is crucial. This work provides a clear understanding of the implementation required to ensure that the blister pouches can be effectively used on microfluidic chips, with an example application area being point-of-care diagnostics.
We review the utility of centrifugal microfluidic technologies applied to point-of-care
diagnosis in extremely under-resourced environments. The various challenges faced in these
settings are showcased, using areas in India and Africa as examples. Measures for the ability of
integrated devices to effectively address point-of-care challenges are highlighted, and centrifugal,
often termed CD-based microfluidic technologies, technologies are presented as a promising
platform to address these challenges. We describe the advantages of centrifugal liquid handling,
as well as the ability of a standard CD player to perform a number of common laboratory tests,
fulfilling the role of an integrated lab-on-a-CD. Innovative centrifugal approaches for point-of-care
in extremely resource-poor settings are highlighted, including sensing and detection strategies,
smart power sources and biomimetic inspiration for environmental control. The evolution of
centrifugal microfluidics, along with examples of commercial and advanced prototype centrifugal
microfluidic systems, is presented, illustrating the success of deployment at the point-of-care. A
close fit of emerging centrifugal systems to address a critical panel of tests for under-resourced
clinic settings, formulated by medical experts, is demonstrated. This emphasizes the potential of
centrifugal microfluidic technologies to be applied effectively to extremely challenging point-of-care
scenarios and in playing a role in improving primary care in resource-limited settings across the
developing world.
Stick-packaging of goods in tubular-shaped composite-foil pouches has become a popular technology for food and drug packaging. We miniaturized stick-packaging for use in lab-on-a-chip (LOAC) systems to pre-store and on-demand release the liquid and dry reagents in a volume range of 80-500 μl. An integrated frangible seal enables the pressure-controlled release of reagents and simplifies the layout of LOAC systems, thereby making the package a functional microfluidic release unit. The frangible seal is adjusted to defined burst pressures ranging from 20 to 140 kPa. The applied ultrasonic welding process allows the packaging of temperature sensitive reagents. Stick-packs have been successfully tested applying recovery tests (where 99% (STDV = 1%) of 250 μl pre-stored liquid is released), long-term storage tests (where there is loss of only <0.5% for simulated 2 years) and air transport simulation tests. The developed technology enables the storage of a combination of liquid and dry reagents. It is a scalable technology suitable for rapid prototyping and low-cost mass production.