Rapid quantitative detection of chloramphenicol in milk by microfluidic immunoassay.

Chloramphenicol (CAP) is a toxic substance for human health, and detection of CAP residues in milk is necessary. However, most of the traditional CAP detection methods including high performance liquid chromatography-tandem mass spectrometry (HPLC-MS) and enzyme-linked immunosorbent assay (ELISA) are time-consuming and complicated. Herein, an automated microfluidics system for CAP detection in milk was developed. The residual CAP of multiple milk samples was quantitatively detected via competitive immunoassay in a single microfluidic chip simultaneously and automatically, and the reliability of the method was confirmed by flow cytometry. Completion of the detection by the system required less than 20 min and the cost for the detection of ten samples was about US$2.5. The limit of detection was 0.05 µg L-1, and the recovery rate of CAP in milk ranged from 91.3% to 105.5%. The microfluidic system developed in this study exhibited considerable potential in the point-of-care testing (POCT) of CAP in milk.

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications.

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Rapid and inexpensive method for the simple fabrication of PDMS-based electrochemical sensors for detection in microfluidic devices.

The fabrication of PDMS microfluidic structures through soft lithography is widely reported. While this well-established method gives high precision microstructures and has been successfully used for many researchers, it often requires sophisticated instrumentation and expensive materials such as clean room facilities and photoresists. Thus, we present here a simple protocol that allows the rapid molding of simple linear microchannels in PDMS substrates aiming microfluidics-based applications. It might serve as an alternative to researchers that do not have access to sophisticated facilities such as clean rooms. The method developed here consists on the use of pencil graphite leads as template for the molding of PDMS channels. It yields structures that can be used for several applications, such as housing support for electrochemical sensors or channels for flow devices. Here, the microdevices produced through this protocol were employed for the accommodation of carbon black paste, which was utilized for the first time as amperometric sensor in microchip electrophoresis. This platform was successfully used for the separation and detection of model analytes. Ascorbic acid and iodide were separated within 45 s with peak resolution of 1.2 and sensitivities of 198 and 492 pA/µM, respectively. The background noise was ca. 84 pA. The analytical usefulness of the system developed was successfully tested through the quantification of iodide in commercial pharmaceutical formulations. It demonstrates good efficiency of the microfabrication protocol developed and enables its use for the easy and rapid prototyping of PDMS structures over a low fabrication cost.

High precision microfluidic microencapsulation of bacteriophages for enteric delivery.

A Salmonella specific bacteriophage Felix O1 (Myoviridae) was microencapsulated in a pH responsive polymer formulation. The formulation incorporated a pH responsive methacrylic acid copolymer Eudragit® S100 (10% (w/v)) with the addition of the biopolymer sodium alginate, the composition of which was varied in the range (0.5% (w/v)-2% (w/v)). The microencapsulation process employed commercially available microfluidic droplet generation devices. We have used readily available low cost microfluidic chips instead of bespoke in-house fabricated glass capillary devices which are accessible only in specialist research facilities. We show that these co-flow microfluidic devices can easily be used to prepare phage encapsulated microparticles making them suitable for use by both the phage research community and industry in order to evaluate and optimise phage compatible formulations for microencapsulation. A novelty of the work reported here is that the size of the generated monodispersed droplets could be precisely controlled in the range 50 µm-200 µm by varying the flow rates of the dispersed and continuous phases. Consequently, alginate concentration and microparticle size were shown to influence the phage release profile and the degree of acid protection afforded to phages upon exposure to simulated gastric fluid (SGF). Bigger microparticles (∼100 µm) showed better acid protection compared with smaller beads (∼50 µm) made from the same formulation. Increasing the alginate composition resulted in improved acid protection of phages for similar particle sizes. The high viscosity formulations containing higher amounts of alginate (e.g. 2% (w/v)) negatively affected ease of droplet generation in the microfluidic device thereby posing a limitation in terms of process scale-up. Felix O1 encapsulated in the formulation containing 10% (w/v) ES100 and 1% (w/v) alginate showed excellent protection upon exposure of the gelled microparticles to SGF (pH 1 for 2 h) without the use of any antacids in the encapsulation matrix. Encapsulated phages previously exposed to SGF (pH 1 for 2 h) were released at elevated pH in simulated intestinal fluid (SIF) and were shown to arrest bacterial growth in the log growth phase. We have therefore demonstrated the microencapsulation of phages using readily available microfluidic chips to produce solid dosage microcapsule forms with a rapid pH triggered release profile suitable for targeted delivery and controlled release in the gastrointestinal tract.

A microfluidics platform for combinatorial drug screening on cancer biopsies.

Screening drugs on patient biopsies from solid tumours has immense potential, but is challenging due to the small amount of available material. To address this, we present here a plug-based microfluidics platform for functional screening of drug combinations. Integrated Braille valves allow changing the plug composition on demand and enable collecting >1200 data points (56 different conditions with at least 20 replicates each) per biopsy. After deriving and validating efficient and specific drug combinations for two genetically different pancreatic cancer cell lines and xenograft mouse models, we additionally screen live cells from human solid tumours with no need for ex vivo culturing steps, and obtain highly specific sensitivity profiles. The entire workflow can be completed within 48 h at assay costs of less than US$ 150 per patient. We believe this can pave the way for rapid determination of optimal personalized cancer therapies.

An Automated Induction Microfluidics System for Synthetic Biology.

The expression of a recombinant gene in a host organism through induction can be an extensively manual and labor-intensive procedure. Several methods have been developed to simplify the protocol, but none has fully replaced the traditional IPTG-based induction. To simplify this process, we describe the development of an autoinduction platform based on digital microfluidics. This system consists of a 600 nm LED and a light sensor to enable the real-time monitoring of  the optical density (OD) samples coordinated with the semicontinuous mixing of a bacterial culture. A hand-held device was designed as a microbioreactor to culture cells and to measure the OD of the bacterial culture. In addition, it serves as a platform for the analysis of regulated protein expression in E. coli without the requirement of standardized well-plates or pipetting-based platforms. Here, we report for the first time, a system that offers great convenience without the user to physically monitor the culture or to manually add inducer at specific times. We characterized our system by looking at several parameters (electrode designs, gap height, and growth rates) required for an autoinducible system. As a first step, we carried out an automated induction optimization assay using a RFP reporter gene to identify conditions suitable for our system. Next, we used our system to identify active thermophilic ß-glucosidase enzymes that may be suitable candidates for biomass hydrolysis. Overall, we believe that this platform may be useful for synthetic biology applications that require regulating and analyzing expression of heterologous genes for strain optimization.

Single-cell RNA-seq of rheumatoid arthritis synovial tissue using low-cost microfluidic instrumentation.

Droplet-based single-cell RNA-seq has emerged as a powerful technique for massively parallel cellular profiling. While this approach offers the exciting promise to deconvolute cellular heterogeneity in diseased tissues, the lack of cost-effective and user-friendly instrumentation has hindered widespread adoption of droplet microfluidic techniques. To address this, we developed a 3D-printed, low-cost droplet microfluidic control instrument and deploy it in a clinical environment to perform single-cell transcriptome profiling of disaggregated synovial tissue from five rheumatoid arthritis patients. We sequence 20,387 single cells revealing 13 transcriptomically distinct clusters. These encompass an unsupervised draft atlas of the autoimmune infiltrate that contribute to disease biology. Additionally, we identify previously uncharacterized fibroblast subpopulations and discern their spatial location within the synovium. We envision that this instrument will have broad utility in both research and clinical settings, enabling low-cost and routine application of microfluidic techniques.

Use of Ice-Nucleating Proteins To Improve the Performance of Freeze-Thaw Valves in Microfluidic Devices.

Currently, reliable valving on integrated microfluidic devices fabricated from rigid materials is confined to expensive and complex methods. Freeze-thaw valves (FTVs) can provide a low cost, low complexity valving mechanism, but reliable implementation of them has been greatly hindered by the lack of ice nucleation sites within the valve body's small volume. Work to date has required very low temperatures (on the order of -40 °C or colder) to induce freezing without nucleation sites, making FTVs impractical due to instrument engineering challenges. Here, we report the use of ice-nucleating proteins (INPs) to induce ice formation at relatively warm temperatures in microfluidic devices. Microfluidic channels were filled with buffers containing femtomolar INP concentrations from Pseudomonas syringae. The channels were cooled externally with simple, small-footprint Peltier thermoelectric coolers (TECs), and the times required for channel freezing (valve closure) and thawing (valve opening) were measured. Under optimized conditions in plastic chips, INPs made sub-10 s actuations possible at TEC temperatures as warm as -13 °C. Additionally, INPs were found to have no discernible inhibitory effects in model enzyme-linked immunosorbent assays or polymerase chain reactions, indicating their compatibility with microfluidic systems that incorporate these widely used bioassays. FTVs with INPs provide a much needed reliable valving scheme for rigid plastic devices with low complexity, low cost, and no moving parts on the device or instrument. The reduction in freeze time, accessible actuation temperatures, chemical compatibility, and low complexity make the implementation of compact INP-based FTV arrays practical and attractive for the control of integrated biochemical assays.

Scaling by shrinking: empowering single-cell 'omics' with microfluidic devices.

Recent advances in cellular profiling have demonstrated substantial heterogeneity in the behaviour of cells once deemed 'identical', challenging fundamental notions of cell 'type' and 'state'. Not surprisingly, these findings have elicited substantial interest in deeply characterizing the diversity, interrelationships and plasticity among cellular phenotypes. To explore these questions, experimental platforms are needed that can extensively and controllably profile many individual cells. Here, microfluidic structures - whether valve-, droplet- or nanowell-based - have an important role because they can facilitate easy capture and processing of single cells and their components, reducing labour and costs relative to conventional plate-based methods while also improving consistency. In this article, we review the current state-of-the-art methodologies with respect to microfluidics for mammalian single-cell 'omics' and discuss challenges and future opportunities.

A Low-Cost, Accurate, and High-Precision Fluid Dispensing System for Microscale Application.

We present here the development of a low-cost, accurate, and precise fluid dispensing system. It can be used with peristaltic or any other pump to improve the flow characteristics. The dispensing system has a range of 1 to 100 µL with accuracy of ~99.5% and standard deviation at ~150 nL over the entire range. The system developed does not depend on the accuracy or precision of the driving pump; therefore, any positive displacement pump can be used to get similar accuracy and precision, which gives an opportunity to reduce the cost of the system. The dispensing system does not require periodic calibration and can also be miniaturized for microfluidic application. Although primarily designed for aqueous liquid, it can be extended for different nonconductive liquids as well with modifications. The unit is further used for near real-time measurement of lactate from microdialysate. The individual components can easily be made disposable or sterilized for use in biomedical applications.

Low cost and compact analytical microsystem for carbon dioxide determination in production processes of wine and beer.

The design, construction and evaluation of a low cost, cyclic olefin copolymer (COC)-based continuous flow microanalyzer, with optical detection, to monitor carbon dioxide in bottled wines and beers as well as in fermentation processes, is presented. The microsystem, constructed by computer numerically controlled (CNC) micromilling and using a multilayer approach, integrates microfluidics, gas-diffusion module and an optical flow-cell in a single polymeric substrate. Its size is slightly bigger than a credit card, exactly 45 × 60 × 4 mm in the microfluidic and diffusion module zone and 22.5 × 40 × 3 mm in the flow-cell zone. The gas-diffusion module is based on a hydrophobic polyvinylidene fluoride (PVDF) membrane, which allows the transfer of the carbon dioxide present in the sample to a bromothymol blue (BTB) pH-sensitive acceptor solution, where the color change is measured optically. The detection system consisted of a LED with an emission peak at 607 nm and a photodiode integrated in a printed circuit board (PCB). The obtained analytical features after the optimization of the microfluidic platform and hydrodynamic variables are a linear range from 255 to 10000 mg L(-1) of CO2 and a detection limit of 83 mg L(-1) with a sampling rate of 30 samples h(-1).

Rapid identification of antibiotic resistance using droplet microfluidics.

Culturing bacteria and monitoring bacterial cell growth is a critical issue when dealing with patients who present with bacterial infections. One of the main challenges that arises is the time taken to identify the particular strain of bacteria and consequently, decide the correct treatment. In the majority of cases, broad spectrum antibiotics are used to target infections when a narrow spectrum drug would be more appropriate. The efficient monitoring of bacterial growth and potential antibiotic resistance is necessary to identify the best treatment options for patients. Minturising the reactions into microfluidic droplets offers a novel method to rapidy analyze bacteria. Microfluidics facilitates low volume reactions that provide a unique system where each droplet reaction acts as an individual bioreactor. Here, we designed and built a novel platform that allowed us to create and monitor E.coli microfluidic droplet cultures. Optical capacity was built in and measurements of bacterial cultures were captured facilitating the continuous monitoring of individual reactions. The capacity of the instrument was demonstrated by the application of treatments to both bacteria and drug resistant strains of bacteria. We were able to detect responses within one hour in the droplet cultures, demonstrating the capacity of this workflow to the culture and rapid characterization of bacterial strains.

Biomedical microfluidic devices by using low-cost fabrication techniques: A review.

One of the most popular methods to fabricate biomedical microfluidic devices is by using a soft-lithography technique. However, the fabrication of the moulds to produce microfluidic devices, such as SU-8 moulds, usually requires a cleanroom environment that can be quite costly. Therefore, many efforts have been made to develop low-cost alternatives for the fabrication of microstructures, avoiding the use of cleanroom facilities. Recently, low-cost techniques without cleanroom facilities that feature aspect ratios more than 20, for fabricating those SU-8 moulds have been gaining popularity among biomedical research community. In those techniques, Ultraviolet (UV) exposure equipment, commonly used in the Printed Circuit Board (PCB) industry, replaces the more expensive and less available Mask Aligner that has been used in the last 15 years for SU-8 patterning. Alternatively, non-lithographic low-cost techniques, due to their ability for large-scale production, have increased the interest of the industrial and research community to develop simple, rapid and low-cost microfluidic structures. These alternative techniques include Print and Peel methods (PAP), laserjet, solid ink, cutting plotters or micromilling, that use equipment available in almost all laboratories and offices. An example is the xurography technique that uses a cutting plotter machine and adhesive vinyl films to generate the master moulds to fabricate microfluidic channels. In this review, we present a selection of the most recent lithographic and non-lithographic low-cost techniques to fabricate microfluidic structures, focused on the features and limitations of each technique. Only microfabrication methods that do not require the use of cleanrooms are considered. Additionally, potential applications of these microfluidic devices in biomedical engineering are presented with some illustrative examples.

Automated Microfluidic Platform for Serial Polymerase Chain Reaction and High-Resolution Melting Analysis.

We report the development of an automated genetic analyzer for human sample testing based on microfluidic rapid polymerase chain reaction (PCR) with high-resolution melting analysis (HRMA). The integrated DNA microfluidic cartridge was used on a platform designed with a robotic pipettor system that works by sequentially picking up different test solutions from a 384-well plate, mixing them in the tips, and delivering mixed fluids to the DNA cartridge. A novel image feedback flow control system based on a Canon 5D Mark II digital camera was developed for controlling fluid movement through a complex microfluidic branching network without the use of valves. The same camera was used for measuring the high-resolution melt curve of DNA amplicons that were generated in the microfluidic chip. Owing to fast heating and cooling as well as sensitive temperature measurement in the microfluidic channels, the time frame for PCR and HRMA was dramatically reduced from hours to minutes. Preliminary testing results demonstrated that rapid serial PCR and HRMA are possible while still achieving high data quality that is suitable for human sample testing.

Low-cost Methods for Making 3D Fluidic Polymer and Glass Chips Using Metal Templates.

Microfluidics is a rapidly growing field in which small volumes of liquid are moved through channels in a large variety of applications. Fabricating such channels can be expensive. Here, we describe an inexpensive method for making 3D channels in fluidic chips by using a sacrificial template made of coated metal wire or metal tubes. A 3D template is embedded in polymer or glass and then dissolved, leaving channels in the chip, without the need for expensive instruments. By changing the mold, chips of various shapes can be made.

Point-of-Care Diagnostics in Low Resource Settings: Present Status and Future Role of Microfluidics.

The inability to diagnose numerous diseases rapidly is a significant cause of the disparity of deaths resulting from both communicable and non-communicable diseases in the developing world in comparison to the developed world. Existing diagnostic instrumentation usually requires sophisticated infrastructure, stable electrical power, expensive reagents, long assay times, and highly trained personnel which is not often available in limited resource settings. This review will critically survey and analyse the current lateral flow-based point-of-care (POC) technologies, which have made a major impact on diagnostic testing in developing countries over the last 50 years. The future of POC technologies including the applications of microfluidics, which allows miniaturisation and integration of complex functions that facilitate their usage in limited resource settings, is discussed The advantages offered by such systems, including low cost, ruggedness and the capacity to generate accurate and reliable results rapidly, are well suited to the clinical and social settings of the developing world.

Biomarker detection for disease diagnosis using cost-effective microfluidic platforms.

Early and timely detection of disease biomarkers can prevent the spread of infectious diseases, and drastically decrease the death rate of people suffering from different diseases such as cancer and infectious diseases. Because conventional diagnostic methods have limited application in low-resource settings due to the use of bulky and expensive instrumentation, simple and low-cost point-of-care diagnostic devices for timely and early biomarker diagnosis is the need of the hour, especially in rural areas and developing nations. The microfluidics technology possesses remarkable features for simple, low-cost, and rapid disease diagnosis. There have been significant advances in the development of microfluidic platforms for biomarker detection of diseases. This article reviews recent advances in biomarker detection using cost-effective microfluidic devices for disease diagnosis, with the emphasis on infectious disease and cancer diagnosis in low-resource settings. This review first introduces different microfluidic platforms (e.g. polymer and paper-based microfluidics) used for disease diagnosis, with a brief description of their common fabrication techniques. Then, it highlights various detection strategies for disease biomarker detection using microfluidic platforms, including colorimetric, fluorescence, chemiluminescence, electrochemiluminescence (ECL), and electrochemical detection. Finally, it discusses the current limitations of microfluidic devices for disease biomarker detection and future prospects.

Fast and sensitive detection of an anthrax biomarker using SERS-based solenoid microfluidic sensor.

We report the application of a fully automated surface-enhanced Raman scattering (SERS)-based solenoid-embedded microfluidic device to the quantitative and sensitive detection of anthrax biomarker poly-γ-D-glutamic acid (PGA) in solution. Analysis is based on the competitive reaction between PGA and PGA-conjugated gold nanoparticles with anti-PGA-immobilized magnetic beads within a microfluidic environment. Magnetic immunocomplexes are trapped by yoke-type solenoids embedded within the device, and their SERS signals were directly measured and analyzed. To improve the accuracy of measurement process, external standard values for PGA-free serum were also measured through use of a control channel. This additional measurement greatly improves the reliability of the assay by minimizing the influence of extraneous experimental variables. The limit of detection (LOD) of PGA in serum, determined by our SERS-based microfluidic sensor, is estimated to be 100 pg/mL. We believe that the defined method represents a valuable analytical tool for the detection of anthrax-related aqueous samples.