Fully integrated digital microfluidics platform for automated immunoassay; a versatile tool for rapid, specific detection of a wide range of pathogens

© 2018 Elsevier Ltd. All rights reserved. This manuscript is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International Licence http://creativecommons.org/licenses/by-nc-nd/4.0/.With the tangible threat posed by the release of chemical and biological warfare (CBW) agents, detection of airborne pathogens is a critical military and security concern. Recent air sampling techniques developed for biocollection take advantage of Electrowetting on Dielectric (EWOD) to recover material, producing highly concentrated droplet samples. Bespoke EWOD-based digital microfluidics platforms are very well suited to take full advantage of the microlitre concentrated droplet resulting from this recovery process. In this paper we present a free-standing, fully automated DMF platform for immunoassay. Using this system, we demonstrate the automated detection of four classes of CBW agent simulant biomolecules and organisms each representing credible threat agents. Taking advantage of the full magnetic separation process with antibody-bound microbeads, rapid and complete separation of specific target antigen can be achieved with minimal washing steps allowing for very rapid detection. Here, we report clear detection of four categories of antigens achieved with assay completion times of between six and ten minutes. Detection of HSA, Bacillus atrophaeus (BG spores), MS2 bacteriophage and Escherichia coli are demonstrated with estimated limit of detection of respectively 30 ng ml -1, 4 × 10 4 cfu ml -1, 10 6 pfu ml -1 and 2 × 10 7 cfu ml -1. The fully-integrated portable platform described in this paper is highly compatible with the next generation of electrowetting-coupled air samplers and thus shows strong potential toward future in-field deployable biodetection systems and could have key implication in life-changing sectors such as healthcare, environment or food security.Peer reviewe

Optical fluid and biomolecule transport with thermal fields

A long standing goal is the direct optical control of biomolecules and water for applications ranging from microfluidics over biomolecule detection to non-equilibrium biophysics. Thermal forces originating from optically applied, dynamic microscale temperature gradients have shown to possess great potential to reach this goal. It was demonstrated that laser heating by a few Kelvin can generate and guide water flow on the micrometre scale in bulk fluid, gel matrices or ice without requiring any lithographic structuring. Biomolecules on the other hand can be transported by thermal gradients, a mechanism termed thermophoresis, thermal diffusion or Soret effect. This molecule transport is the subject of current research, however it can be used to both characterize biomolecules and to record binding curves of important biological binding reactions, even in their native matrix of blood serum. Interestingly, thermophoresis can be easily combined with the optothermal fluid control. As a result, molecule traps can be created in a variety of geometries, enabling the trapping of small biomolecules, like for example very short DNA molecules. The combination with DNA replication from thermal convection allows us to approach molecular evolution with concurrent replication and selection processes inside a single chamber: replication is driven by thermal convection and selection by the concurrent accumulation of the DNA molecules. From the short but intense history of applying thermal fields to control fluid flow and biological molecules, we infer that many unexpected and highly synergistic effects and applications are likely to be explored in the future

Hydrogel microparticles for biosensing

Due to their hydrophilic, biocompatible, and highly tunable nature, hydrogel materials have attracted strong interest in the recent years for numerous biotechnological applications. In particular, their solution-like environment and non-fouling nature in complex biological samples render hydrogels as ideal substrates for biosensing applications. Hydrogel coatings, and later, gel dot surface microarrays, were successfully used in sensitive nucleic acid assays and immunoassays. More recently, new microfabrication techniques for synthesizing encoded particles from hydrogel materials have enabled the development of hydrogel-based suspension arrays. Lithography processes and droplet-based microfluidic techniques enable generation of libraries of particles with unique spectral or graphical codes, for multiplexed sensing in biological samples. In this review, we discuss the key questions arising when designing hydrogel particles dedicated to biosensing. How can the hydrogel material be engineered in order to tune its properties and immobilize bioprobes inside? What are the strategies to fabricate and encode gel particles, and how can particles be processed and decoded after the assay? Finally, we review the bioassays reported so far in the literature that have used hydrogel particle arrays and give an outlook of further developments of the field. Keywords: Hydrogel; Biosensor; Microparticle; Multiplex assayNovartis Institutes of Biomedical Research (Presidential Fellowship)Novartis Institutes of Biomedical Research (Education Office)National Cancer Institute (U.S.) (Grant 5R21CA177393-02)National Science Foundation (U.S.) (Grant CMMI-1120724)Institute for Collaborative Biotechnologies (Grant W911NF-09-0001)United States. Army Research Offic

Magnetic Nanoparticle Enhanced Actuation Strategy for mixing, separation, and detection of biomolecules in a Microfluidic Lab-on-a-Chip System

Magnetic nanoparticle (MNP) combined with biomolecules in a microfluidic system can be efficiently used in various applications such as mixing, pre-concentration, separation and detection. They can be either integrated for point-of care applications or used individually in the area of bio-defense, drug delivery, medical diagnostics, and pharmaceutical development. The interaction of magnetic fields with magnetic nanoparticles in microfluidic flows will allow simplifying the complexity of the present generation separation and detection systems. The ability to understand the dynamics of these interactions is a prerequisite for designing and developing more efficient systems. Therefore, in this work proof-of-concept experiments are combined with advanced numerical simulation to design, develop and optimize the magnetic microfluidic systems for mixing, separation and detection. Different strategies to combine magnetism with microfluidic technology are explored; a time-dependent magnetic actuation is used for efficiently mixing low volume of samples whereas tangential microfluidic channels were fabricated to demonstrate a simple low cost magnetic switching for continuous separation of biomolecules. A simple low cost generic microfluidic platform is developed using assembly of readily available permanent magnets and electromagnets. Microfluidic channels were fabricated at much lower cost and with a faster construction time using our in-house developed micromolding technique that does not require a clean room. Residence-time distribution (RTD) analysis obtained using dynamic light scattering data from samples was successfully used for the first time in microfluidic system to characterize the performance. Both advanced multiphysics finite element models and proof of concept experimentation demonstrates that MNPs when tagged with biomolecules can be easily manipulated within the microchannel. They can be precisely captured, separated or detected with high efficiency and ease of operation. Presence of MNPs together with time-dependent magnetic actuation also helps in mixing as well as tagging biomolecules on chip, which is useful for point-of-care applications. The advanced mathematical model that takes into account mass and momentum transport, convection & diffusion, magnetic body forces acting on magnetic nanoparticles further demonstrates that the performance of microfluidic surface-based bio-assay can be increased by incorporating the idea of magnetic actuation. The numerical simulations were helpful in testing and optimizing key design parameters and demonstrated that fluid flow rate, magnetic field strength, and magnetic nanoparticle size had dramatic impact on the performance of microfluidic systems studied. This work will also emphasize the importance of considering magnetic nanoparticles interactions for a complete design of magnetic nanoparticle-based Lab-on-a-chip system where all the laboratory unit operations can be easily integrated. The strategy demonstrated in this work will not only be easy to implement but also allows for versatile biochip design rules and provides a simple approach to integrate external elements for enhancing mixing, separation and detection of biomolecules. The vast applications of this novel concept studied in this work demonstrate its potential of to be applied to other kinds of on-chip immunoassays in future. We think that the possibility of integrating magnetism with microfluidic-based bioassay on a disposable chip is a very promising and versatile approach for point-of care diagnostics especially in resource-limited settings

Microfluidic for human health: a versatile tool for new progress in cancer diagnosis and contaminants detection

In the last ten years, interest in manipulating droplets in microchannels has emerged from two important motivations. The first arise from the desire to produce well controlled droplets for material science applications, for example in the pharmaceutical or food industries. In this context, microfluidics allows for producing such droplets in a controlled and reproducible manner, also allowing complex combinations to be designed and explored. A second motivation originates with the advent of the -omics era, which has a much need for being able to carry out experiments at the smallest possible scale (if possible single cells or molecules), on a massively parallel platform and with high throughput. In this case, droplets are viewed as micro-reactor in which samples are confined, and which offer a way to manipulate small volumes. Droplet microfluidics is the most powerful microfluidic technology used to produce and manipulate monodisperse droplets. This technique addresses the need for lower costs, shorter times, and higher sensitivities to compartmentalize reactions into picolitre volume, instead of the microlitre volumes commonly used with standard methods. Droplets can provide a well-defined environment into which individual cells can be compartmentalized in a controlled way. This coupled with the advantages of droplet microfluidics has allowed the development of several methods for single-cells analysis. In this work a microfluidics label-free approach for circulating tumor cells (CTCs) detection is presented. In the last decades, CTCs have received enormous attention as a new biomarkers for cancer study, for this reason their capture and retain represents a major challenge in cancer research. Many issues regarding the detection and characterization of CTCs are owing to their extremely rarity (one CTCs for 5 x 10^9 erythrocytes/mL and for 7 x 10^6 leucocytes /mL) and their heterogeneous nature (there is no unique biologic marker for CTCs identification). Although much promising progress has been made in CTCs detection, the robustness in distinguishing between healthy cells and CTCs, and the isolation of live CTCs need to be improved further. The method developed in this work exploits the so-called Warburg effect (WE): even in the presence of oxygen cancer cells limit largely their metabolism to glycolysis leading to increased production of lactate. Using droplet microfluidics, cancer cells are compartmentalized into a picolitre droplets and lactate secreted by cells are retained in the droplet. The secretion of lactate leads to a rapid increase in the concentration of acid in cell-containing droplets. CTCs are thus detected by monitoring the pH of the droplet using a pH- sensitive dye, without the need for surface-antigen labeling. A suspension of tumor cells (A549) mixed with white blood cells were emulsified in picoliter droplets, and it was observed a clear fraction of droplets with a reduced pH, leading to a distinct population of droplets containing a cancer cell from empty or WBC containing drops. With this method a very few number (up to 10) of tumor cells in a background of 200,000 white blood cells are detected, with average detection rates in the range of 60%. To demonstrate that this is a general method for detection of cancer cells, several cancer cell lines were tested, including ovarian TOV21G, breast MDA-MB 453, glioblastoma U231, colorectal HT-29, breast MCF-7 and MDA-MB-231 and all showed acidification of droplets. With the method established, samples based on blood cancer patients with confirmed metastatic disease were tested. The results show clearly that numerous positive droplets are detected in the sample of metastatic patients. Moreover, this method is capable of retrieving live cells, opening up routes for further large scale investigation of the nature of CTCs. Another interesting area where droplet-based microfluidics is playing an increasingly important role is the synthesis of functional polymeric microparticles or microgels. They have been suggested as diagnostic tools for the rapid multiplexed screening of biomolecules, because of their advantages in detection and quantification. In the second part of this thesis, the synthesis of polymeric microparticles, functionalized with peptides, through droplet microfluidics is presented. Peptide was efficiently encapsulated into the polymeric microparticles in order to create a functional microparticles for selective protein detection in complex fluids. Protein binding occurred with higher affinity (K D 0.1-0.4 µM) than the conventional detection methods (K D 70 µM). Current work demonstrate easy and fast realization of functionalized monodisperse microgels using droplet microfluidic and how the inclusion of small molecules within polymeric network improve both the affinity and the specificity of protein capture. This work provides advances in gel particle functionalization and opens new possibilities for direct molecules detection in complex fluids. A possible application of this method was for label-free aflatoxin M 1 (AFM 1 ) detection. AFM 1 is the most toxic, carcinogenic, teratogenic and mutagenic class of aflatoxins (AFs) and can be present due to in a wide range of food and feed commodities, such as milk and dairy products, representing an important issue especially for developing countries. Currently, the detection methods used to quantify AFM 1 require complex and laborious sample pretreatments, expensive instruments and skilled operators, thus limiting their application. Driven by the need of overcoming some of these limits, poly(ethylene glycol) dyacrilate (PEGDA) functional microparticles were produced using microfluidics. Two novel peptides were synthesized for specific aflatoxin detection and encapsulated in PEGDA microparticles for AFM 1 detection. AFM 1 -binding peptides occurred with high affinity (K D 3.66-6.57 pM, respectively for the two sequences) and detection was achieved measuring AFM 1 innate fluorescence. The detection limit of this technique for AFM 1 was estimated to be 1.64 ng/Kg, with a dynamic detection range between 3.28 ng/Kg and 70 ng/Kg, which meets present legislative limits of 50 ng/Kg for AFM 1 in milk. Therefore, the developed systems provides a promising approach for rapid screening of food contaminates because it resulted to be simple, sensitive, specific, and with not need multiple separation steps, overcoming the limits of the traditional AFM 1 detection methods, which are expensive and time consuming. The use of microfluidics has allowed development of robust, label-free, sensitive and high-throughput platforms which may be used in the near future to improve the quality of life

Separation and purification of biomacromolecules based on microfluidics

Separation and purification of biomacromolecules either in biopharmaceuticals and fine chemicals manufacturing, or in diagnostics and biological characterization, can substantially benefit from application of microfluidic devices. Small volumes of equipment, very efficient mass and heat transfer together with high process control result in process intensification, high throughputs, low energy consumption and reduced waste production as compared to conventional processing. This review highlights microfluidics-based separation and purification of proteins and nucleic acids with the focus on liquid-liquid extractions, particularly with biocompatible aqueous two-phase systems, which represent a cost-effective and green alternative. A variety of microflow set-ups are shown to enable sustainable and efficient isolation of target biomolecules both for preparative, as well as for analytical purposes.publishe

Microvortices In Droplets: Generation & Applications

The emerging field of droplet microfluidics deals with the manipulation of nL-fL droplets encapsulated within an immiscible carrier phase. The droplets are used as reaction containers for biochemical assays, enabling drastic reduction in assay volumes needed for modern life sciences research. To achieve this, basic laboratory processes such as mixing, detection, and metering must be emulated in the droplet format. Three important unit operations relevant to high throughput screening include 1) the concentration of particles and species within droplets, which is necessary for heterogeneous assays; 2) sensing the biochemical contents of a droplet; and 3) the sorting of droplets based on physical or chemical properties, which is important for single cell and proteomic assays. Currently, particle concentration in droplets requires active components, such as on-chip electrodes or magnets, along with charged or magnetic particles. Similarly, sensing and sorting droplets by chemical composition is based on flow cytometry, which also requires on-chip electrodes, feedback control, and chemical labeling. It is desirable to avoid active field techniques due to complexity, size, and cost constraints, and replace them with more simple and passive techniques. In this thesis, we utilize microvortices, the rotational motion of fluid, to enhance the capabilities of droplet microfluidics in the above three areas. The microvortices are generated using two methods: (i) hydrodynamic recirculation drag and (ii) tensiophoresis. In the first method, species concentration is accomplished by exploiting the shear-induced vortices that occur naturally inside a droplet/plug as it moves through a microchannel. Prior studies utilized these flows for enhancing mixing or interphase mass transfer. This work exploits microvortices together with two other independent phenomena--sedimentation of particles and interfacial adsorption of proteins--to concentrate both types of species at the rear of the droplet, where they can be extracted from the drop. In the latter case, the protein localization at the rear of drop reduces the interfacial tension locally resulting in an asymmetry in the drop shape. Under laminar flow, the shape deformation is deterministic and can serve as a sensitive, label-free indicator of protein concentration in proteomic screening. In the second method, label-free sorting of droplets is accomplished by a novel droplet actuation technique termed Tensiophoresis. A microchemical gradient across the droplet is transduced into a microvortex flow which propels the droplets up the chemical gradient. Using laminar flow to precisely control the gradient, droplets can be sorted by size with 3.3% resolution over a wide turning range. Droplets can be also sorted based on chemical composition because tensiophoresis is inhibited by surface active agents adsorbed on the droplet surface. Studies conducted using Bovine Serum Albumin (BSA) show that the droplet migration velocity scales inversely with protein concentration in the droplet, and migration velocity can be correlated to protein concentration with a 1 femtomole limit of detection. As modern life sciences research becomes increasingly reliant on high throughput workflows, microdroplet technology can meet the growing demand to perform screening at ultra-high throughputs with reduced sample volume. This thesis contributes three important unit operations which expand the capabilities of droplet-based workflows in proteomics, cell biology, and other areas of biomedical research