An inexpensive, high-throughput µPAD assay of microbial growth rate and motility on solid surfaces using Saccharomyces cerevisiae and Escherichia coli as model organisms.

Many microbial phenotypes are differentially or exclusively expressed on agar surfaces, including biofilms, motility, and sociality. However, agar-based assays are limited by their low throughput, which increases costs, lab waste, space requirements, and the time required to conduct experiments. Here, we demonstrate the use of wax-printed microfluidic paper-based analytical devices (µPADs) to measure linear growth rate of microbes on an agar growth media as a means of circumventing the aforementioned limitations. The main production materials of the proposed µPAD design are a wax printer, filter paper, and empty pipet boxes. A single wax-printed µPAD allowing 8 independent, agar-grown colonies costs $0.07, compared to $0.20 and $9.37 for the same number of replicates on traditional microtiter/spectrophotometry and Petri dish assays, respectively. We optimized the µPAD design for channel width (3 mm), agar volume (780 µL/channel), and microbe inoculation method (razor-blade). Comparative analyses of the traditional and proposed µPAD methods for measuring growth rate of nonmotile (Saccharomyces cerevisiae) and motile (flagellated Escherichia coli) microorganisms suggested the µPAD assays conferred a comparable degree of accuracy and reliability to growth rate measurements as their traditional counterparts. We substantiated this claim with strong, positive correlations between the traditional and µPAD assay, a significant nonzero slope in the model relating the two assays, a nonsignificant difference between the relative standard errors of the two techniques, and an analysis of inter-device reliability. Therefore, µPAD designs merit consideration for the development of enhanced-throughput, low-cost microbial growth and motility assays.

High-Yielding Radiosynthesis of [68Ga]Ga-PSMA-11 Using a Low-Cost Microfluidic Device.

PURPOSE: Current PET radiotracer production models result in facility and operational costs that scale prohibitively with the number of tracers synthesized, particularly those made as a single dose-on-demand. Short of a paradigm shift in the technology and economics of radiotracer production, the impact of PET on precision medicine will be limited. Inexpensive, microfluidic radiochemistry platforms have the potential to significantly reduce costs associated with dose-on-demand production and expand the breadth of PET tracers accessible for molecular imaging. PROCEDURES: To produce a miniaturized dose-on-demand device for [68Ga]Ga-PSMA-11 production, a microfluidic chip was assembled in polydimethylsiloxane (PDMS), combining all components of tracer production in an integrated, compact, and easily utilized platform. On-chip radionuclide concentration, as well as radionuclide and precursor starting material mixing and reaction were incorporated. The radionuclide was sourced from a standard, commercially available 68Ge/68Ga generator. Optimal reaction conditions were determined, which included precursor concentration (5 µg/mL), temperature (95 °C), and reaction time (1 min). RESULTS: The total trapping efficiency of combined on-chip SCX and SAX columns was greater than 70 % and could be accomplished in ~ 12 min. Under optimized conditions, [68Ga]Ga-PSMA-11 could be reliably synthesized starting from a complete generator elution (1100 MBq [29.7 mCi]) in ~ 12 min, with an average radiochemical yield of 70 %, radiochemical purity > 99 %, and specific activity > 740 MBq/µg (20 mCi/µg). Quality control testing demonstrated that tracer produced using this platform met or exceeded all typical FDA requirements for human use. CONCLUSIONS: A simple, low-cost, dose-on-demand radiosynthesis strategy, such as the chip presented here, represents an opportunity to reduce the financial barriers associated with PET imaging. While this study focused on a device for [68Ga]Ga-PSMA-11, the technology is also applicable to a wide range of other tracers where low-cost, automated, dose-on-demand production is highly desirable.

A low-cost smartphone controlled portable system with accurately confined on-chip 3D electrodes for flow-through cell electroporation.

Microscale flow-through electroporation at DC voltage has advantages in delivering small molecules. Yet, electroporation based on constant voltage are liable to generate electrolysis products which limits the voltage-operating window. Parallel on-chip 3D electrodes with close and uniform spacing are required to cut down voltage as well as provide enough electric field for electroporation. Here we present a simple electrode fabrication method based on capillary restriction valves in Z-axis to achieve parallel 3D electrodes with controllable electrode spacing in PDMS chips. With electrodes accurately placed in close range, a low voltage of only 1.5 V can generate enough electric field (>400 V/cm) to make cell membrane permeable. Squeeze flow is introduced to produce higher electric field (>800 V/cm) at a fixed voltage for more efficient electroporation. Benefit from the electrode fabrication method and application of squeeze flow, we develop a smartphone controlled microfluidic electroporation system which integrate functions of sample injection, pressure regulating, real-time observation and constant DC power supply. The system is used to electroporate two cell lines, showing a permeabilization percentage of 63% for HEK-293 cells and 58% for CHO-K1 cells with optimal parameters. Thus, the portable microfluidic system provides a cost-effective and user-friendly flow-through cell electroporation platform.

Paper-Based Microfluidic Device (DON-Chip) for Rapid and Low-Cost Deoxynivalenol Quantification in Food, Feed, and Feed Ingredients.

Mycotoxin contamination causes over $5 billion of economic loss per year in the North American food and feed industry. A rapid, low-cost, portable, and reliable method for on-site detection of deoxynivalenol (DON), a representative mycotoxin predominantly occurring in grains, would be helpful to control mycotoxin contamination. In this study, a paper-based microfluidic chip capable of measuring DON (DON-Chip) in food, feed, and feed ingredients was developed. The DON-Chip incorporated a colorimetric competitive immunoassay into a paper microfluidic device and used gold nanoparticles as a signal indicator. Furthermore, a novel ratiometric analysis method was proposed to improve detection resolvability. Detection of DON in the aqueous extracts from solid food, feed, or feed ingredients was successfully validated with a detection range of 0.01-20 ppm (using dilution factors from 10 to 104). Compared with conventional methods, the DON-Chip method could greatly reduce the cost and time of mycotoxin detection in the food and feed industry.

Ultra-low-cost fabrication of polymer-based microfluidic devices with diode laser ablation.

In this work, a diode laser ablation approach was used for the fabrication of PMMA-based microfluidic devices. Compared with the conventional CO2 or femtosecond laser fabrication method, the proposed laser ablation method based on diode laser significantly lowered the cost in the fabrication of polymer-based microfluidic devices with comparable resolution and surface quality. PMMA substrate was used for the laser ablation process, due to the transparency of PMMA in the diode laser's working wavelength, a layer of Kraft tape was applied on the surface of PMMA for the absorption of laser energy, and microchannels were then achieved on the surface of PMMA with the proposed low-cost diode laser system. The comparison between the proposed method and the CO2 laser ablation method was also conducted in this study. The profile of the fabricated microchannels was carefully characterized, several microfluidic devices were also fabricated for the demonstration of the proposed fabrication method using a diode laser.

Interferometric Reflectance Imaging Sensor (IRIS) for Molecular Kinetics with a Low-Cost, Disposable Fluidic Cartridge.

The determination of kinetic information and appropriate binding pairs is fundamental to the proper optimization and selection of ligands used in immunoassays, diagnostics, and therapeutics. However, the ability to estimate such parameters in a multiplexed and inexpensive format remains difficult and modification of the ligand is often necessary. Here, we detail the methods and materials necessary to evaluate hundreds of unlabeled ligands simultaneously using the interferometric reflectance imaging sensor (IRIS). The incorporation of a low-cost fluidic cartridge that integrates on the top of the sensor simplifies reagent handling considerably.

Development of a multiplex fully automated assay for rapid quantification of CD4+ T cells from whole blood.

The development of cost-effective and rapid assays for the accurate counting of CD4 cells has remained prime focus for disease management. The lack of such assays has severely affected people living in resource-limited disease prevalent areas. CD4 count information plays a vital role in the effective management of HIV disease. There is an unmet need to develop rapid, cost-effective, portable and user-friendly point-of-care (POC) disease diagnostic platform technology for CD4+ T cell counting. Here, we have developed a flow-free magnetic actuation platform that uses antibody-coated magnetic beads to efficiently capture CD4+ T cells from a 30 µL drop of whole blood. On-chip cell lysate electrical impedance spectroscopy has been utilized to quantify the isolated CD4 cells. The developed assay has a limit of detection of 25 cells per µL and provides accurate CD4 counts in the range of 25-800 cells per µL. The whole immunoassay along with the enumeration process is very rapid and provides CD4 quantification results within 5 min time frame. The assay does not require off-chip sample preparation steps and minimizes human involvement to a greater extent. The developed impedance-based immunoassay has potential to significantly improve the CD4 enumeration process especially for POC settings.

Label-Free Exosome Detection Based on a Low-Cost Plasmonic Biosensor Array Integrated with Microfluidics.

Localized surface plasmon resonance-based plasmonic biosensors are interesting candidates for the design of portable optical biosensor platforms owing to their integration, miniaturization, multiparameter, real-time, and label-free detection characteristics. Plasmonic biosensor arrays that have been combined with microfluidics have been developed herein to detect exosomes label-free. Gold nano-ellipsoid arrays were fabricated with low-cost anodic aluminum oxide thin films that act as shadow masks for evaporation of Au. The nano-ellipsoid arrays were integrated with a microfluidic chip to achieve multiparameter detection. The anti-CD63 antibody that is specific to the exosome transmembrane protein CD63 is modified on the surface of the nano-ellipsoids. Exosome samples were injected into the biosensor platform at different concentrations and detected successfully. The detection limit was 1 ng/mL. The proposed plasmonic biosensor array can be universally applicable for the detection of other biomarkers by simply changing the antibody on the surface of the Au nano-ellipsoids. Moreover, this biosensor platform is envisaged to be potentially useful in the development of low-cost plasmonic-based biosensors for biomarker detection and for the investigation of exosomes for noninvasive disease diagnoses.

A portable microfluidic platform for rapid determination of microbial load and somatic cell count in milk.

Microfluidics systems that have been emerged in the last 20 years and used for processing the fluid in a microchannel structure at microliter levels are alternative to the conventional methods. The objective of the study is to develop a microfluidic platform for determination of the microbial load and the number of somatic cells in milk. For this purpose, a polydimethylsiloxane (PDMS) chip with a channel size of 300 µm × 60 µm was produced. Cells/bacteria labeled with fluorescent stain in milk were counted with the proposed microfluidic platform and the results were compared with the reference cell concentration/the bacterial counts by conventional method. It was found that our platform could count somatic and bacterial cells with an accuracy above 80% in 20 min run for each analysis. The portable overall platform has an overall dimension of 25x25x25 cm and weighs approximately 9 kg.

A low-cost, non-invasive phase velocity and length meter and controller for multiphase lab-in-a-tube devices.

Opportunities for accessible microfluidic device integration have sharply grown with the rise of readily available lab-in-a-tube strategies. Herein, we present a facile, non-invasive, plug-and-play phase velocity and length measuring strategy for rapid deployment onto tube-based microfluidic systems, enabling quick and accurate residence (reaction) time measurement and tuning. Our approach utilizes inexpensive off-the-shelf optical phase sensors and requires no prior knowledge of the fluid composition or physical properties. Compared to camera-based measurements in fluoropolymer tubing, the optical phase sensor-based technique shows mean absolute percentage errors of 1.3% for velocity and 3.3% for length. Utilizing the developed multiphase flow monitoring technique, we screen the accessible parameter space of gas-liquid segmented flows. To further demonstrate the functionality of this process monitoring strategy, we implement two feedback controllers to establish simultaneous setpoint control for phase velocity and length. Next, to showcase the effectiveness and versatility of the developed multiphase flow process controller, we apply it to systematic studies of the effect of liquid slug velocity (controlling precursor mixing timescale) on the colloidal synthesis of cesium lead tribromide nanocrystals. By varying the liquid slug velocity and maintaining constant precursor composition, liquid slug length, and residence time, we observe a bandgap tunability from 2.43 eV (510 nm) to 2.52 eV (494 nm).

Single-Cell RNA Sequencing with Drop-Seq.

Drop-Seq is a low-cost, high-throughput platform to profile thousands of cells by encapsualting them into individual droplets. Uniquely barcoded mRNA capture microparticles and cells are coconfined through a microfluidic device within the droplets where they undergo cell lysis and RNA hybridiztion. After breaking the droplets and pooling the hybridized particles, reverse transcription, PCR, and sequencing in single reactions allow to generate data from thousands of single-cell transcriptomes while maintaining information on the cellular origin of each transcript.

Sampling and multiplexing in lab-on-paper bioelectroanalytical devices for glucose determination.

In this work, we present a multiplexed (eight simultaneous measurements) paper-based electrochemical device developed in a very simple way and using low-cost materials, such as paper, carbon ink and multifunctional connector headers. Meanwhile, we have also combined the paper-based electrochemical platform with a glass-fiber strip in order to integrate easily a sampling step. Both approaches, simultaneous measuring and sampling, have been applied to the determination of glucose using bienzymatic biosensors. They are fabricated by adsorbing the mixture of enzymes (glucose oxidase and horseradish peroxidase), as well as the ferrocyanide, mediator of the electron transfer, on the paper-based electrode. After drying, the measuring solution (containing either glucose standards or samples) is added and the eight corresponding chronoamperograms are recorded. In the case of the microfluidic approach for sampling purposes, the glass-fiber pad (sampler) is immersed in a container with the solution, which flows by capillarity until it reaches the working electrode. The integration of one more step of the analytical process advances towards real and useful lab-on-a-chip devices. With these designs, a linear range comprised between 0.5 and 15 mM was achieved for glucose determination, with an excellent precision. If the sampler is employed, it is not necessary to use micropipettes and, nevertheless, precise measurements are obtained. The RSD of the slopes obtained for different calibrations performed in different days, with different arrays of electrochemical cells and different solutions is ca. 1%. Accurate results are obtained in the determination of glucose in real samples (orange fruit and cola beverages).

Thermopneumatic suction integrated microfluidic blood analysis system.

Blood tests provide crucial diagnostic information regarding several diseases. A key factor that affects the precision and accuracy of blood tests is the interference of red blood cells; however, the conventional methods of blood separation are often complicated and time consuming. In this study, we devised a simple but high-efficiency blood separation system on a self-strained microfluidic device that separates 99.7 ± 0.3% of the plasma in only 6 min. Parameters, such as flow rate, design of the filter trench, and the relative positions of the filter trench and channel, were optimized through microscopic monitoring. Moreover, this air-difference-driven device uses a cost-effective and easy-to-use heater device that creates a low-pressure environment in the microchannel within minutes. With the aforementioned advantages, this blood separation device could be another platform choice for point-of-care testing.

Cost analysis of oil cake-to-biodiesel production in packed-bed micro-flow reactors with immobilized lipases.

Biodiesel production depends to a great extent on the use of cheap raw materials, since biodiesel itself is a mass product, not a high-value product. New processing methods, such as micro-flow continuous processing combined with enzymatic catalysis, open doors to the latter. As reported here, the window of opportunity in enzyme-catalyzed biodiesel production is the conversion of waste cooking oil. The main technological challenge for this is to obtain efficient immobilization of the lipase catalyst on beads. The beads can be filled into tubular reactors where designed packed-bed provide porous channels, forming micro-flow. It turns out, that in this way, the immobilization costs become the decisive economic factor. This paper reports a solution to that issue. The use of oil cake enables economic viability, which is not given by any of the commercial polymeric substrates used so far for enzyme immobilization. The costs of immobilization are mirrored in the earnings and cash flow of the new biotechnological process.

Label-free detection of pepsinogen 1 and 2 by polyethylene coating Lamb microfluidic device.

Early screening of gastric cancer is a critical importance for the improvement of patients' survival rate. Here, a polyethylene coating Lamb (PE-Lamb) microfluidic device with immune layer for gastric cancer label-free detection was constructed. Two serum pepsinogen 1 (PG1) and pepsinogen 2 (PG2) biomarkers were applied to screen and predict the appearance of gastric cancer. Compared with enzyme-linked immunosorbent assay (ELISA), this method achieved a higher sensitivity and less time (40 min vs 120 min). The limit of detections (LOD) were reached 60 pg/mL for PG1 and 30 pg/mL for PG2, which have two orders of magnitude lower than traditional ELISA. The linearity coefficient indexes (R2) for PG1 and PG2 were 0.992 and 0.953 respectively, which is similar to that of ELISA. In addition, PG1 and PG2 mixed antigens sample with human serum was detected by PE-Lamb approach, and the frequency response showed high reproducibility and specificity. The results indicate that PE-lamb diagnostic technique is a novel and promising method for high-throughput screening and early diagnosis of gastric cancer.

Workshop, Cost-Effective and Streamlined Fabrications of Re-Usable World-To-Chip Connectors for Handling Sample of Limited Volume and for Assembling Chip Array.

The world-to-chip interface is an essential yet intriguing part of making and employing microfluidic devices. A user-friendly connector could be expensive or difficult to make. We fabricated two ports of microfluidic chips with easily available materials including Teflon blocks, double adhesive films, coverslips, and transparency films. By using a mini grinder, coverslips were drilled to form small holes for the fluid passages between port and chip. Except for the double adhesive films, the resultant ports are durable and re-useable. The DK1 port, contains a mini three-way switch which allows users to handle fluid by a tube-connected pump, or by a manual pipette for the sample of trace amount. The other port, the DK2 port, provides secured tube-connections. Importantly, we invented a bridge made of craft cutter-treated transparency films and double adhesive films to mediate liquid flow between DK2 port and chip. With the use of a bridge, users do not need to design new ports for new chips. Also, individual chips could be linked by a bridge to form a chip array. We successfully applied DK1 port on a microfluidic chip where green fluorescent protein was immobilized. We used DK2 port on an array of fish chips where the embryos of zebra fish developed.

An automated microfluidic chemiluminescence immunoassay platform for quantitative detection of biomarkers.

A rapid, sensitive and quantitative biomarker detection platform is of great importance to the small clinic or point-of-care (POC) diagnosis. In this work, we realize that an automated diagnostic platform mainly includes two components: (1) an instrument that can complete all steps of the chemiluminescence immunoassay automatically and (2) an integrated microfluidic chip which is disposable and harmless. In the instrument, we adopt vacuum suction cups which are driven by linear motor to realize a simple, effective and convenient control. The method of acridine esterification chemiluminescence is adopted to achieve a quantitative detection, and a photomultiplier tube is used to detect photons from acridine ester producing in alkaline conditions. We use the laser cutting machine and hot press machine to accomplish the product of microfluidic chips. The automated microfluidics-based system is demonstrated by implementation of a chemiluminescence immunoassay for quantitative detection of ferritin. We observe alinear relationship between CL intensity and the concentration of ferritin from 5.1 to 1300 ng mL -1and the limit of detection (LoD) is 2.55 ng mL -1. At the same time, we also used the automated microfluidics-based system to test clinical serum samples. The whole process of chemiluminescence experiment can complete within 45 min. We realize that this lab-on-a-chip chemiluminescence immunoassay platform with features of automation and quantitation provides a promising strategy for POC diagnosis.

Low cost 3D microfluidic chips for multiplex protein detection based on photonic crystal beads.

Point-of-care testing (POCT) devices used in multiplex bioassays are in great demand for clinical, environmental and biomedical applications. Photonic crystal beads (PCBs), as structural color self-coding carriers, can be integrated with microfluidic chips to realize convenient and highly sensitive biomarker detection. Here we developed a three dimensional (3D) microfluidic chip based on PCBs, which is low cost and easy to manufacture for mass production and application. The chip was fabricated with polyethylene terephthalate, polymethyl methacrylate sheets, a Ni square mesh grid and transparent double-sided tape. In practice, the target molecules could be captured by PCBs immobilized with probes in a flow-through manner. It was found that the as-proposed chip needed less washing and its background was effectively reduced in comparison with a flow-over chip. Besides, the limit of detection (LOD) of anti-human alpha fetoprotein (AFP) was calculated to be 18.92 ng mL-1, which could meet the need of clinical detection of AFP. Furthermore, the chip demonstrated the feasibility of simultaneous detection of human immunoglobulin G, carcinoembryonic antigen and AFP, which suggests that it has a broad application prospect in multiplex bioassays.

Integrating thin film microfluidics in developing a concise synthesis of DGJNAc: A potent inhibitor of α-N-acetylgalctosaminidases.

A simple synthesis, which utilizes a thin film microfluidic reactor for a problematic step, of a potent inhibitor of α-N-acetylhexosaminidases, DGJNAc, has been developed.