Valveless On-Chip Aliquoting for Molecular Diagnosis.

The detection of nucleic acids as specific markers of infectious diseases is commonly implemented in molecular biology laboratories. The translation of these benchtop assays to a lab-on-a-chip format demands huge efforts of integration and automation. The present work is motivated by a strong requirement often posed by molecular assays that combine isothermal amplification and CRISPR/Cas-based detection: after amplification, a 2-8 microliter aliquot of the reaction products must be taken for the subsequent reaction. In order to fulfill this technical problem, we have designed and prototyped a microfluidic device that is able to meter and aliquot in the required range during the stepped assay. The operation is achieved by integrating a porous material that retains the desired amount of liquid after removing the excess reaction products, an innovative solution that avoids valving and external actuation. The prototypes were calibrated and experimentally tested to demonstrate the overall performance (general fluidics, metering, aliquoting, mixing and reaction). The proposed aliquoting method is fully compatible with additional functions, such as sample concentration or reagent storage, and could be further employed in alternative applications beyond molecular diagnosis.

Coupling ELISA to smartphones for POCT of chronic and congenital Chagas disease.

Chagas disease (CD) affects about 7 million people worldwide, presents a large prevalence in Latin America, and is growing in the rest of the world, where congenital CD is the main mode of transmission. Point-of-care testing (POCT) methods are increasingly required to ease early diagnostics and increase treatment success. This work presents the development and validation of a smartphone-integrated ELISA-based POCT system for the detection of both chronic and congenital CD. Expensive and bulky equipment used for ELISA in conventional laboratories was replaced as follows. A miniaturized device was fabricated for incubation of commercial ELISA plates, achieving ∼±1 °C uniformity and stability. The ELISA plate reader was replaced by smartphone camera and image processing, comprising algorithms to account for variability sources and spatial light non-uniformity; thus, additional hardware like a dark-box is not required. The agreement between samples classified with this novel reading method vs. ELISA plate reader was found to be 99.7% and 95.4% for chronic and congenital CD, respectively. Furthermore, a smartphone application was designed and implemented to guide the user during the assay, provide connectivity, and access databases, facilitating patient monitoring and health-policy making. The whole system is aimed to be used as a practical diagnostic tool in primary health care settings, as well as to facilitate patients' follow-up to provide better treatment. Concerning the technology itself, the proposed POCT platform is versatile enough to be readily adapted for the detection of other infectious diseases.

A free customizable tool for easy integration of microfluidics and smartphones.

The integration of smartphones and microfluidics is nowadays the best possible route to achieve effective point-of-need testing (PONT), a concept increasingly demanded in the fields of human health, agriculture, food safety, and environmental monitoring. Nevertheless, efforts are still required to integrally seize all the advantages of smartphones, as well as to share the developments in easily adoptable formats. For this purpose, here we present the free platform appuente that was designed for the easy integration of microfluidic chips, smartphones, and the cloud. It includes a mobile app for end users, which provides chip identification and tracking, guidance and control, processing, smart-imaging, result reporting and cloud and Internet of Things (IoT) integration. The platform also includes a web app for PONT developers, to easily customize their mobile apps and manage the data of administered tests. Three application examples were used to validate appuente: a dummy grayscale detector that mimics quantitative colorimetric tests, a root elongation assay for pesticide toxicity assessment, and a lateral flow immunoassay for leptospirosis detection. The platform openly offers fast prototyping of smartphone apps to the wide community of lab-on-a-chip developers, and also serves as a friendly framework for new techniques, IoT integration and further capabilities. Exploiting these advantages will certainly help to enlarge the use of PONT with real-time connectivity in the near future.

Numerical simulations of paper-based electrophoretic separations with open-source tools.

A new tool for the solution of electromigrative separations in paper-based microfluidics devices is presented. The implementation is based on a recently published complete mathematical model for describing these types of separations, and was developed on top of the open-source toolbox electroMicroTransport, based on OpenFOAM® , inheriting all its features as native 3D problem handling, support for parallel computation, and a GNU GPL license. The presented tool includes full support for paper-based electromigrative separations (including EOF and the novel mechanical and electrical dispersion effects), compatibility with a well-recognized electrolyte database, and a novel algorithm for computing and controlling the electric current in arbitrary geometries. Additionally, the installation on any operating system is available due to its novel installation option in the form of a Docker image. A validation example with data from literature is included, and two extra application examples are provided, including a 2D free-flow IEF problem, which demonstrates the capabilities of the toolbox for dealing with computational and physicochemical modeling challenges simultaneously. This tool will enable efficient and reliable numerical prototypes of paper-based electrophoretic devices to accompany the contemporary fast growth in paper-based microfluidics.

Precise electroosmotic flow measurements on paper substrates.

A novel method for electroosmotic flow (EOF) measurement on paper substrates is presented; it is based on dynamic mass measurements by simply using an analytical balance. This technique provides a more reliable alternative to other EOF measurement methods on porous media. The proposed method is used to increase the amount and quality of the available information about physical parameters that characterize fluid flow on microfluidic paper-based analytical devices (µPADs). Measurements were performed on some of the most frequently used materials for µPADs, i.e., Whatman #1 , S&S, and Muntktell 00A filter paper. Obtained experimental results are consistent with the few previously reported data, either experimental or numerical, characterizing EOF in paper substrates. Moreover, a thorough analysis is presented for the quantification of the different effects that affect the measurements such as Joule effect and evaporation. Experimental results enabled, for the first time, to establish well-defined electroosmotic characteristics for the three substrates in terms of the magnitude of EOF as funtion of pH, enabling researchers to make a rational choice of the substrate depending on the electrophoretic technique to be implemented. The measurement method can be described as robust, reliable, and affordable enough for being adopted by researchers and companies devoted to electrophoretic µPADs and related technologies.

Comprehensive model of electromigrative transport in microfluidic paper based analytical devices.

A complete mathematical model for electromigration in paper-based analytical devices is derived, based on differential equations describing the motion of fluids by pressure sources and EOF, the transport of charged chemical species, and the electric potential distribution. The porous medium created by the cellulose fibers is considered like a network of tortuous capillaries and represented by macroscopic parameters following an effective medium approach. The equations are obtained starting from their open-channel counterparts, applying scaling laws and, where necessary, including additional terms. With this approach, effective parameters are derived, describing diffusion, mobility, and conductivity for porous media. While the foundations of these phenomena can be found in previous reports, here, all the contributions are analyzed systematically and provided in a comprehensive way. Moreover, a novel electrophoretically driven dispersive transport mechanism in porous materials is proposed. Results of the numerical implementation of the mathematical model are compared with experimental data, showing good agreement and supporting the validity of the proposed model. Finally, the model succeeds in simulating a challenging case of free-flow electrophoresis in paper, involving capillary flow and electrophoretic transport developed in a 2D geometry.

USB powered microfluidic paper-based analytical devices.

Microfluidic paper-based analytical devices (µPADs) allow user-friendly and portable chemical determinations, although they provide limited applicability due to insufficient sensitivity. Several approaches have been proposed to address poor sensitivity in µPADs, but they frequently require bulky equipment for power and/or read-outs. Universal serial buses (USB) are an attractive alternative to less portable power sources and are currently available in many common electronic devices. Here, USB-powered µPADs (USB µPADs) are proposed as a fusion of both technologies to improve performance without adding instrumental complexity. Two ITP USB µPADs were developed, both powered by a 5 V potential provided through standard USB ports. The first device was fabricated using the origami approach. Its operation was analyzed experimentally and numerically, yielding a two-order-of-magnitude sample focusing in 15 min. The second ITP USB µPAD is a novel design, which was numerically prototyped with the aim of handling larger sample volumes. The reservoirs were moved away from the ITP channel and capillary action was used to drive the sample and electrolytes to the separation zone, predicting 25-fold sample focusing in 10 min. USB µPADs are expected to be adopted by minimally-trained personnel in sensitive areas like resource-limited settings, the point-of-care and in emergencies.

Rapid Bacteria Detection at Low Concentrations Using Sequential Immunomagnetic Separation and Paper-Based Isotachophoresis.

Detecting bacteria is important in the fields of human health, environmental monitoring, and food safety. Foodborne pathogens alone are estimated to cause 420 000 deaths annually, with low-income regions affected most. Despite improvements in bacterial detection, fast, disposable, low-cost, sensitive, and user-friendly methods are still needed. Traditional methods for detecting bacteria rely primarily on cell culturing or polymerase chain reaction (PCR), which require highly trained personnel and a central laboratory and take several hours or even days to deliver results. Low-cost methods like lateral flow immunoassays exist but frequently suffer from poor sensitivity and/or lack quantitative results. Here, a rapid method for detecting bacteria at very low concentrations is presented using two sequential preconcentration steps. In the first preconcentration step, the sample is mixed with antibody-modified magnetic particles and free antibodies conjugated to ß-galactosidase (ß-gal). The target bacteria are isolated and concentrated using immunomagnetic separation. The isolated bacteria are then incubated with chlorophenol red-ß-d-galactopyranoside (CPRG), which reacts with ß-gal to produce chlorophenol red (CPR) in a bacteria concentration-dependent manner. In the second step, CPR and CPRG are separated and focused using an isotachophoretic microfluidic paper-based analytical device, significantly improving the final detection limit relative to paper-based devices lacking the focusing mechanism. Moreover, CPR and CPRG form two visible color bands that act as test and control bands, respectively, improving assay robustness. The method was tested with E. coli DH5-α and successfully detected concentrations as low as 9.2 CFU/mL in laboratory samples and 920 CFU/mL in apple juice samples in ∼90 min.

Patterning and Modeling Three-Dimensional Microfluidic Devices Fabricated on a Single Sheet of Paper.

This work describes a method to fabricate three-dimensional paper microfluidic devices in one step, without the need of stacking layers of paper, glue, or tape. We used a nontransparent negative photoresist that allows patterning selectively (vertically) the paper, creating systems of two or three layers, including channels. To demonstrate the capabilities of this methodology, we designed, fabricated, and tested a six-level diluter. The performance of the device was also simulated using a simple numerical model implemented in the program PETSc-FEM. The resulting µPAD is small (1.6 cm × 2.2 cm), inexpensive, requires low volumes of sample (5 µL), and is able to perform mixing and dilution in 2 min.

Design keys for paper-based concentration gradient generators.

The generation of concentration gradients is an essential operation for several analytical processes implemented on microfluidic paper-based analytical devices. The dynamic gradient formation is based on the transverse dispersion of chemical species across co-flowing streams. In paper channels, this transverse flux of molecules is dominated by mechanical dispersion, which is substantially different than molecular diffusion, which is the mechanism acting in conventional microchannels. Therefore, the design of gradient generators on paper requires strategies different from those used in traditional microfluidics. This work considers the foundations of transverse dispersion in porous substrates to investigate the optimal design of microfluidic paper-based concentration gradient generators (µPGGs) by computer simulations. A set of novel and versatile µPGGs were designed in the format of numerical prototypes, and virtual experiments were run to explore the ranges of operation and the overall performance of such devices. Then physical prototypes were fabricated and experimentally tested in our lab. Finally, some basic rules for the design of optimized µPGGs are proposed. Apart from improving the efficiency of mixers, diluters and µPGGs, the results of this investigation are relevant to attain highly controlled concentration fields on paper-based devices.