An Innovative AI-based primer design tool for precise and accurate detection of SARS-CoV-2 variants of concern.

As the COVID-19 pandemic winds down, it leaves behind the serious concern that future, even more disruptive pandemics may eventually surface. One of the crucial steps in handling the SARS-CoV-2 pandemic was being able to detect the presence of the virus in an accurate and timely manner, to then develop policies counteracting the spread. Nevertheless, as the pandemic evolved, new variants with potentially dangerous mutations appeared. Faced by these developments, it becomes clear that there is a need for fast and reliable techniques to create highly specific molecular tests, able to uniquely identify VOCs. Using an automated pipeline built around evolutionary algorithms, we designed primer sets for SARS-CoV-2 (main lineage) and for VOC, B.1.1.7 (Alpha) and B.1.1.529 (Omicron). Starting from sequences openly available in the GISAID repository, our pipeline was able to deliver the primer sets for the main lineage and each variant in a matter of hours. Preliminary in-silico validation showed that the sequences in the primer sets featured high accuracy. A pilot test in a laboratory setting confirmed the results: the developed primers were favorably compared against existing commercial versions for the main lineage, and the specific versions for the VOCs B.1.1.7 and B.1.1.529 were clinically tested successfully.

Optimal self-assembly of lipid nanoparticles (LNP) in a ring micromixer.

Lipid nanoparticles (LNPs) for RNA and DNA delivery have attracted considerable attention for their ability to treat a broad range of diseases and to vectorize mRNA for COVID vaccines. LNPs are produced by mixing biomolecules and lipids, which self-assemble to form the desired structure. In this domain, microfluidics shows clear advantages: high mixing quality, low-stress conditions, and fast preparation. Studies of LNPs produced in micromixers have revealed, in certain ranges of flow rates, a degradation in performance in terms of size, monodispersity and encapsulation efficiency. In this study, we focus on the ring micromixer, which is well adapted to high throughput. We reveal three regimes, side-by-side, transitional and highly mixed, that control the mixing performance of the device. Furthermore, using cryo-TEM and biochemical analysis, we show that the mixing performances are strongly correlated to the characteristics of the LNPs we produce. We emphasize the importance of the flow-rate ratio and propose a physical criterion based on the onset of temporal instabilities for producing LNPs with optimal characteristics in terms of geometry, monodispersity and encapsulation yield. These criteria are generally applicable.

Freeze-Dried Microfluidic Monodisperse Microbubbles as a New Generation of Ultrasound Contrast Agents.

We succeeded in freeze-drying monodisperse microbubbles without degrading their performance, that is, their monodispersity in size and echogenicity. We used microfluidic technology to generate cryoprotected highly monodisperse microbubbles (coefficient of variation [CV] <5%). By using a novel retrieval technique, we were able to freeze-dry the microbubbles and resuspend them without degradation, that is, keeping their size distribution narrow (CV <6%). Acoustic characterization performed in two geometries (a centimetric cell and a millichannel) revealed that the resuspended bubbles conserved the sharpness of the backscattered resonance peak, leading to CVs ranging between 5% and 10%, depending on the geometry. As currently observed with monodisperse bubbles, the peak amplitudes are one order of magnitude higher than those of commercial ultrasound contrast agents. Our work thus solves the question of storage and transportation of highly monodisperse bubbles. This work might open pathways toward novel clinical non-invasive measurements, such as local pressure, impossible to carry out with the existing commercial ultrasound contrast agents.

Dendritic growth of protein gel in the course of sweat pore plugging by aluminium salts under physiological conditions.

Are aluminium ions unavoidable in antiperspirants? To answer this question, we present confocal microscopy images of dendritic plugs appearing in sweat flowing across a microfluidic channel in the presence of aluminium salts. By comparing with numerical simulations, we identify the mechanisms forming this structured protein gel inside the pore.

Real time observation of the interaction between aluminium salts and sweat under microfluidic conditions.

Aluminium salts such as aluminium chlorohydrate (ACH) are the active ingredients of antiperspirant products. Their mechanism of action involves a temporary and superficial plugging of eccrine sweat pores at the skin surface. We developed a microfluidic system that allows the real time observation of the interactions between sweat and ACH in conditions mimicking physiological sweat flow and pore dimensions. Using artificial sweat containing bovine serum albumin as a model protein, we performed experiments under flowing conditions to demonstrate that pore clogging results from the aggregation of proteins by aluminium polycations at specific location in the sweat pore. Combining microfluidic experiments, confocal microscopy and numerical models helps to better understand the physical chemistry and mechanisms involved in pore plugging. The results show that plugging starts from the walls of sweat pores before expanding into the centre of the channel. The simulations aid in explaining the influence of ACH concentration as well as the impact of flow conditions on the localization of the plug. Altogether, these results outline the potential of both microfluidic confocal observations and numerical simulations at the single sweat pore level to understand why aluminium polycations are so efficient for sweat channel plugging.

Performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification.

To respond to the urgent need for COVID-19 testing, countries perform nucleic acid amplification tests (NAAT) for the detection of SARS-CoV-2 in centralized laboratories. Real-time RT-PCR (Reverse transcription-Polymerase Chain Reaction), used to amplify and detect the viral RNA., is considered, as the current gold standard for diagnostics. It is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings [1]. In the present work, by harnessing progress made in the past two decades in isothermal amplification and paper microfluidics, we created a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT-LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or fluorescent probes. Depending on the viral load in the tested samples, the detection takes between twenty minutes and one hour. Using a set of 16 pools of naso-pharyngal swab eluates, we estimated a limit of detection comparable to real-time RT-PCR (i.e. 1 genome copies per microliter of clinical sample) and no cross-reaction with eight major respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called "COVIDISC" to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment will expedite the widespread dissemination of this device. What is proposed here is a new efficient tool to help managing the pandemics.

Unraveling driving regimes for destabilizing concentrated emulsions within microchannels.

Coalescence is the most widely demonstrated mechanism for destabilizing emulsion droplets in microfluidic chambers. However, we find that depending on the channel wall surface functionalization, surface zeta potential, type of surfactant, characteristics of the oil as a dispersed phase, or even the presence of externally-induced stress, other different destabilization mechanisms can occur in subtle ways. In general, we observe four regimes leading to destabilization of concentrated emulsions: (i) coalescence, (ii) emulsion bursts, (iii) a combination of the two first mechanisms, attributed to the simultaneous occurrence of coalescence and emulsion bursts; and (iv) compaction of the droplet network that eventually destabilizes to fracture-like behavior. We correlate various physico-chemical properties (zeta potential, contact angle, interfacial tension) to understand their respective influence on the destabilization mechanisms. This work provides insights into possible ways to control or inflict emulsion droplet destabilization for different applications.

Colloidal particle deposition on microchannel walls, for attractive and repulsive surface potentials.

Surface interactions are an interplay of van der Waals adhesion forces with electrostatic charges. In colloidal deposition, at low ionic strengths, the Debye layer is sufficiently large to prevent particles from approaching the surface. It is only with the addition of higher salt concentrations, typically above 0.1 M, that surface charges are screened for interactions to take place via van der Waals-adhesion forces. This is true for repulsive charges, when both surfaces have similar charges and signs of the zeta potential are the same. However, with attractive charges, where zeta potential signs are opposite, the result is also opposite. By combining microfluidic experiments, theory, and numerical simulations, results show that when charges are attractive, particle deposition instead increases at low ionic strengths (at greater Debye lengths), at rates controlled by van der Waals forces but assisted by electrostatic forces. We propose a mechanism where particles approach the wall, mobilized by electrostatic attraction, up to a distance where van der Waals forces come into play, collecting the particles at the wall, which electrostatic forces alone are unable to achieve, owing to hindered diffusion. The present work thus allows us to understand the different mechanisms that govern deposition in the case where surface charges are opposite.

Micrometric Monodisperse Solid Foams as Complete Photonic Bandgap Materials.

Solid foams with micrometric pores are used in different fields (filtering, 3D cell culture, etc.), but today, controlling their foam geometry at the pore level, their internal structure, and the monodispersity, along with their mechanical properties, is still a challenge. Existing attempts to create such foams suffer either from slow speed or size limitations (above 80 µm). In this work, by using a temperature-regulated microfluidic process, 3D solid foams with highly monodisperse open pores (PDI lower than 5%), with sizes ranging from 5 to 400 µm and stiffnesses spanning 2 orders of magnitude, are created for the first time. These features open the way for exciting applications, in cell culture, filtering, optics, etc. Here, the focus is set on photonics. Numerically, these foams are shown to open a 3D complete photonic bandgap, with a critical index of 2.80, thus compatible with the use of rutile TiO2. In the field of photonics, such structures represent the first physically realizable self-assembled FCC (face-centered cubic) structure that possesses this functionality.

Stable liquid foams from a new polyfluorinated surfactant.

Liquid foams exhibiting long-term stability are a key-challenge in material design. Based on this perspective, new pyridinium polyfluorinated surfactants were synthesized from simple building blocks enabling unusually stable liquid foams. While the batch-generated foams were used for qualitative foaming evaluation, microfluidics allowed a quantitative insight into the aging effects of monodisperse foams.

Microfluidics Mediated Production of Foams for Biomedical Applications.

Within the last decade, there has been increasing interest in liquid and solid foams for several industrial uses. In the biomedical field, liquid foams can be used as delivery systems for dermatological treatments, for example, whereas solid foams are frequently used as scaffolds for tissue engineering and drug screening. Most of the foam functionalities are largely correlated to their mechanical properties and their structure, especially bubble/pore size, shape, and interconnectivity. However, the majority of conventional foaming fabrication techniques lack pore size control which can induce important inhomogeneities in the foams and subsequently decrease their performance. In this perspective, new advanced technologies have been introduced, such as microfluidics, which offers a highly controlled production, allowing for design customization of both liquid foams and solid foams obtained through liquid-templating. This short review explores both the fabrication and the characterization of foams, with a focus on solid polymer foams, and sheds the light on how microfluidics can overcome some existing limitations, playing a crucial role in their production for biomedical applications, especially as scaffolds in tissue engineering.

Deposition kinetics of bi- and tridisperse colloidal suspensions in microchannels under the van der Waals regime.

We investigate the kinetics of irreversible adsorption under the van der Waals regime, i.e. weakly Brownian polydisperse colloidal suspensions injected into shallow microchannels at high ionic strengths, where each suspension is represented by populations of particles with different particle sizes. We find that each population size of the particle in the suspension can be treated independently using an analytical solution based on the advection-diffusion equation and that the distribution of the adsorbed particles along the channel axis behaves according to a power law. The experimental measurements agree with Langevin simulations and are well accounted for by theory valid in the van der Waals regime. Operating in the van der Waals regime permits the present study to confirm the use of microfluidics as an effective in situ method to measure the Hamaker constant of particles under aqueous conditions.

Foam as a self-assembling amorphous photonic band gap material.

We show that slightly polydisperse disordered 2D foams can be used as a self-assembled template for isotropic photonic band gap (PBG) materials for transverse electric (TE) polarization. Calculations based on in-house experimental and simulated foam structures demonstrate that, at sufficient refractive index contrast, a dry foam organization with threefold nodes and long slender Plateau borders is especially advantageous to open a large PBG. A transition from dry to wet foam structure rapidly closes the PBG mainly by formation of bigger fourfold nodes, filling the PBG with defect modes. By tuning the foam area fraction, we find an optimal quantity of dielectric material, which maximizes the PBG in experimental systems. The obtained results have a potential to be extended to 3D foams to produce a next generation of self-assembled disordered PBG materials, enabling fabrication of cheap and scalable photonic devices.

Fibrin-Targeted Polymerized Shell Microbubbles as Potential Theranostic Agents for Surgical Adhesions.

The development of new therapies for surgical adhesions has proven to be difficult as there is no consistently effective way to assess treatment efficacy in clinical trials without performing a second surgery, which can result in additional adhesions. We have developed lipid microbubble formulations that use a short peptide sequence, CREKA, to target fibrin, the molecule that forms nascent adhesions. These targeted polymerized shell microbubbles (PSMs) are designed to allow ultrasound imaging of early adhesions for diagnostic purposes and for evaluating the success of potential treatments in clinical trials while acting as a possible treatment. In this study, we show that CREKA-targeted microbubbles preferentially bind fibrin over fibrinogen and are stable for long periods of time (∼48 h), that these bound microbubbles can be visualized by ultrasound, and that neither these lipid-based bubbles nor their diagnostic-ultrasound-induced vibrations damage mesothelial cells in vitro. Moreover, these bubbles show the potential to identify adhesionlike fibrin formations and may hold promise in blocking or breaking up fibrin formations in vivo.

Optimizing Hyperuniformity in Self-Assembled Bidisperse Emulsions.

We study long range density fluctuations (hyperuniformity) in two-dimensional jammed packings of bidisperse droplets. Taking advantage of microfluidics, we systematically span a large range of size and concentration ratios of the two droplet populations. We identify various defects increasing long range density fluctuations mainly due to organization of local particle environment. By choosing an appropriate bidispersity, we fabricate materials with a high level of hyperuniformity. Interesting transparency properties of these optimized materials are established based on numerical simulations.

Paper microfluidics for nucleic acid amplification testing (NAAT) of infectious diseases.

The diagnosis of infectious diseases is entering a new and interesting phase. Technologies based on paper microfluidics, coupled to developments in isothermal amplification of Nucleic Acids (NAs) raise opportunities for bringing the methods of molecular biology in the field, in a low setting environment. A lot of work has been performed in the domain over the last few years and the landscape of contributions is rich and diverse. Most often, the level of sample preparation differs, along with the sample nature, the amplification and detection methods, and the design of the device, among other features. In this review, we attempt to offer a structured description of the state of the art. The domain is not mature and there exist bottlenecks that hamper the realization of Nucleic Acid Amplification Tests (NAATs) complying with the constraints of the field in low and middle income countries. In this domain however, the pace of progress is impressively fast. This review is written for a broad Lab on a Chip audience.

Particle Deposition Kinetics of Colloidal Suspensions in Microchannels at High Ionic Strength.

Despite its considerable practical importance, the deposition of real Brownian particles transported in a channel by a liquid, at small Reynolds numbers, has never been described at a comprehensive level. Here, by coupling microfluidic experiments, theory, and numerics, we succeed in unravelling the problem for the case of straight channels at high salinity. We discover a broad regime of deposition (the van der Waals regime) in which particle-wall van der Waals interactions govern the deposition mechanism. We determine the range of existence of the regime, for which we calculate the concentration profiles, retention profiles, and deposition kinetics analytically. The retention profiles decay as the inverse of the square root of the distance from the entry, and the deposition kinetics are given by the expression [Formula: see text], where S is a dimensionless deposition function, A is the Hamaker constant, and ξL is a dimensionless parameter characterizing fluid flow properties. These findings are well supported by numerics. Experimentally, we find that the retention profiles behave as x-0.5±0.1 (where x is the distance from the channel entry) over three decades in scale, as predicted theoretically. By varying the flow conditions (speed, geometry, surface properties, and concentration) so as to cover four decades in ξL and taking the Hamaker constant as a free parameter, we accurately confirm the theoretical expression for the deposition kinetics. Operating in the van der Waals regime enables control of the deposition rates via surface chemistry. From a surface science perspective, working in the van der Waals regime enables us to measure the Hamaker constants of thousands of particles in a few minutes, a task that would take a much longer time to perform with standard AFM.

Paper-based RNA detection and multiplexed analysis for Ebola virus diagnostics.

The most performing techniques enabling early diagnosis of infectious diseases rely on nucleic acid detection. Today, because of their high technicality and cost, nucleic acid amplification tests (NAAT) are of benefit only to a small fraction of developing countries population. By reducing costs, simplifying procedures and enabling multiplexing, paper microfluidics has the potential to considerably facilitate their accessibility. However, most of the studies performed in this area have not quit the lab. This letter brings NAAT on paper closer to the field, by using clinical samples and operating in a resource-limited setting. We first performed isothermal reverse transcription and Recombinase Polymerase Amplification (RT-RPA) of synthetic Ribonucleic Acid (RNA) of Ebola virus using paper microfluidics devices. We further applied this method in Guinea to detect the presence of Ebola virus in human sample RNA extracts, with minimal facilities (carry-on detection device and freeze-dried reagents on paper). RT-RPA results were available in few minutes and demonstrate a sensitivity of 90.0% compared to the gold-standard RT-PCR on a set of 43 patient samples. Furthermore, the realization of a nine-spot multilayered device achieving the parallel detection of three distinct RNA sequences opens a route toward the detection of multiple viral strains or pathogens.

In situ targeted activation of an anticancer agent using ultrasound-triggered release of composite droplets.

The efficiency of a drug is usually highly dependent on the way it is administered or delivered. As such, targeted-therapy, which requires conceiving drug-delivery vehicles that will change their state from a relatively stable structure with a very slow leak-rate to an unstable structure with a fast release, clearly improves the pharmacokinetics, the absorption, the distribution, the metabolism and the therapeutic index of a given drug. In this context, we have developed a particularly effective double stimuli-responsive drug-delivery method allowing an ultrasound-induced release of a monomethylauristatin E-glucuronide prodrug and its subsequent activation by a ß-glucuronidase. This led to an increase of cytotoxicity of about 80% on cancer cells.

Designing Colloidal Molecules with Microfluidics.

The creation of new colloidal materials involves the design of functional building blocks. Here, a microfluidic method for designing building blocks one by one, at high throughput, with a broad range of shapes is introduced. The method exploits a coupling between hydrodynamic interactions and depletion forces that controls the configurational dynamics of droplet clusters traveling in microfluidic channels. Droplet clusters can be solidified in situ with UV. By varying the flow parameters, clusters are prescribed a given size, geometry, chemical and/or magnetic heterogeneities enabling local bonding. Compact structures (chains, triangles, diamonds, tetrahedrons,...) and noncompact structures, such as crosses and T, difficult to obtain with current techniques are produced. Size dispersions are small (2%) and throughputs are high (30 000 h-1). The work opens a new pathway, based on microfluidics, for designing colloidal building blocks with a potential to enable the creation of new materials.