A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid—based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids.

An inexpensive, hand-held, point-of-care, disposable, self-contained, immunoassay cassette comprised of air pouches for pumping, a metering chamber, reagents storage chambers, a mixer, and a lateral flow strip was designed, constructed, and tested. The assay was carried out in a consecutive flow format. The detection was facilitated with up-converting, phosphor (UCP) reporter particles. The automated, timely pumping of the various reagents was driven by a spring-loaded timer. The utility of the cassette was demonstrated by detecting antibodies to HIV in saliva samples and further evaluated with a non-contagious, haptenized DNA assay. The cassette has several advantages over dip sticks such as sample preprocessing, integrated storage of reagents, and automated operation that reduces operator errors and training. The cassette and actuator described herein can readily be extended to detect biomarkers of other diseases in body fluids and other fluids at the point of care. The system is particularly suitable for resource poor countries, where funds and trained personnel are in short supply.

The building blocks for an inexpensive, disposable, luminescence-based microfluidic immunoassay cassette are described, and their integration in a point-of-care diagnostic system is demonstrated. Fluid motion in the cassette is driven by depressing finger-actuated pouches. All reagents needed for the immunoassay can be stored in the cassette in liquid form. Prior to use, the cassette consists of two separate parts. A top storage component contains pouches, sealed storage chambers, a metering chamber, and needle seats. The bottom processing component contains connection needles, a mixing chamber, and a detection chamber with immobilized proteins. Subsequent to sample introduction, the storage and processing components are mated. The needles form hydraulic connections between the two parts and, in some cases, close valves. The pouches are then actuated sequentially to induce flow of various reagents and facilitate process operations. The cassette is compatible with different detection modalities. Both a cassette with immunochromatographic-based detection and a cassette with microbead-based detection were constructed and evaluated. The immunochromatographic cassette was used to detect antibodies to HIV in saliva samples. The bead-based cassette was used to detect the proinflammatory chemokine IL-8. The experimental data demonstrates good repeatability and reasonable sensitivity.

Recently, there has been a growing interest in point-of-care devices capable of detecting nucleic acids (NA) in clinical and environmental samples. Nucleic acid detection requires, however, various sample preparation steps that complicate device operation. An attractive remedy is to integrate many, if not all, sample preparation operations and nucleic acid amplification into a single reaction chamber. A microfluidic chip that integrates, in a single chamber, polymerase chain reaction (PCR) amplification with solid-phase extraction of nucleic acids using a nanoporous, aluminium oxide membrane (AOM) is described. Samples suspected of containing target bacteria and/or viruses are mixed with lysis agents and a chaotropic salt and loaded into a plastic chip housing a nanoporous, aluminium oxide membrane. The nucleic acids in the lysate bind to the membrane. The membrane is then washed, the chamber is filled with the PCR reaction reagents, and the chamber's temperature is cycled to amplify the captured nucleic acids and produce detectable products. Both DNA and RNA (with reverse-transcription) isolation and amplification are demonstrated. Due to the dry membrane's high resistance to liquid flow, a specialized flow control system was devised to facilitate sample introduction and membrane washing.

Given the impact of NIV on respiratory function and the importance of respiratory function to quality of life and survival, understanding differences that influence NIV prescribing is critical. This information may inform future study design and identify areas warranting additional research to develop best practices for NIV implementation.

A point-of-care, diagnostic system incorporating a portable thermal cycler and a compact fluorescent detector for real-time, polymerase chain reaction (PCR) on disposable, plastic microfluidic reactors with relatively large reaction volume (ranging from 10 µL to 100 µL) is described. To maintain temperature uniformity and a relatively fast temperature ramping rate, the system utilizes double-sided heater that features a master, thermoelectric element and a thermal waveguide connected to a second thermoelectric element. The waveguide has an aperture for optical coupling between a miniature, fluorescent reader and the PCR reaction chamber. The temperature control is accomplished with a modified, feedforward, variable structural proportional-integral-derivative controller. The temperature of the liquid in the reaction chamber tracks the set-point temperature with an accuracy of ± 0.1 °C. The transition times from one temperature to another are minimized with controllable overshoots (< 2 °C) and undershoots (< 5 °C). The disposable, single-use PCR chip can be quickly inserted into a thermal cycler/reader unit for point-of-care diagnostics applications. The large reaction chamber allows convenient pre-storing of dried, paraffin-encapsulated PCR reagents (polymerase, primers, dNTPs, dyes, and buffers) in the PCR chamber. The reagents are reconstituted "just in time" by heating during the PCR process. The system was tested with viral and bacterial nucleic acid targets.

A portable, fully automated analyzer that provides actuation and flow control to a disposable, self-contained, microfluidic cassette ("chip") for point-of-care, molecular testing is described. The analyzer provides mechanical actuation to compress pouches that pump liquids in the cassette, to open and close diaphragm valves for flow control, and to induce vibrations that enhance stirring. The analyzer also provides thermal actuation for the temperature cycling needed for polymerase chain reaction (PCR) amplification of nucleic acids and for various drying processes. To improve the temperature uniformity of the PCR chamber, the system utilizes a double-sided heating/cooling scheme with a custom feedforward, variable, structural proportional-integral-derivative (FVSPID) controller. The analyzer includes a programmable central processing unit that directs the sequence and timing of the various operations and that is interfaced with a computer. The disposable cassette receives a sample, and it carries out cell lysis, nucleic acid isolation, concentration, and purification, thermal cycling, and either real time or lateral flow (LF) based detection. The system's operation was demonstrated by processing saliva samples spiked with B. cereus cells. The amplicons were detected with a lateral flow assay using upconverting phosphor reporter particles. This system is particularly suited for use in regions lacking centralized laboratory facilities and skilled personnel.

A portable, small footprint, light, general purpose analyzer (processor) to control the flow in immunoassay cassettes and to facilitate the detection of test results is described. The durable analyzer accepts disposable cassettes that contain pouches and reaction chambers for various unit operations such as hydration of dry reagents, stirring, and incubation. The analyzer includes individually controlled, linear actuators to compress the pouches in the cassette, which facilitates the pumping and mixing of sample and reagents, and to close diaphragm-based valves for flow control. The same types of actuators are used to compress pouches and actuate valves. The analyzer also houses a compact OEM scanner/reader to excite fluorescence and detect emission from labels. The analyzer is hydraulically isolated from the cassette, reducing the possibility of cross-contamination. The analyzer facilitates programmable, automated execution of a sequence of operations such as pumping and valving in a timely fashion, reducing the level of expertise required from the operator and the possibility for errors. The analyzer’s design is modular and expandable to accommodate cassettes of various complexities and additional functionalities. In this paper, the utility of the analyzer has been demonstrated with the execution of a simple, consecutive, lateral flow assay of a model biological system and the test results were detected with up converting phosphor labels that are excited at infrared frequencies and emit in the visible spectrum.