Xianbo Qiu

An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids

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 isothermal amplification reactor with an integrated isolation membrane for point-of-care detection of infectious diseases

A simple, point of care, inexpensive, disposable cassette for the detection of nucleic acids extracted from pathogens was designed, constructed, and tested. The cassette utilizes a single reaction chamber for isothermal amplification of nucleic acids. The chamber is equipped with an integrated, flow-through, Flinders Technology Associates (Whatman FTA®) membrane for the isolation, concentration, and purification of DNA and/or RNA. The nucleic acids captured by the membrane are used directly as templates for amplification without elution, thus simplifying the cassettes flow control. The FTA membrane also serves another critical role-enabling the removal of inhibitors that dramatically reduce detection sensitivity. Thermal control is provided with a thin film heater external to the cassette. The amplification process was monitored in real time with a portable, compact fluorescent reader. The utility of the integrated, single-chamber cassette was demonstrated by detecting the presence of HIV-1 in oral fluids. The HIV RNA was reverse transcribed and subjected to loop-mediated, isothermal amplification (LAMP). A detection limit of less than 10 HIV particles was demonstrated. The cassette is particularly suitable for resource poor regions, where funds and trained personnel are in short supply. The cassette can be readily modified to detect nucleic acids associated with other pathogens borne in saliva, urine, and other body fluids as well as in water and food.

A timer-actuated immunoassay cassette for detecting molecular markers in oral 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.

Finger-actuated, self-contained immunoassay cassettes

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.

Point-of-care oral-based diagnostics

Many of the target molecules that reside in blood are also present in oral fluids, albeit at lower concentrations. Oral fluids are, however, relatively easy and safe to collect without the need for specialized equipment and training. Thus, oral fluids provide convenient samples for medical diagnostics. Recent advances in lab-on-a-chip technologies have made minute, fully integrated diagnostic systems practical for an assortment of point-of-care tests. Such systems can perform either immunoassays or molecular diagnostics outside centralized laboratories within time periods ranging from minutes to an hour. The article briefly reviews recent advances in devices for point-of-care testing with a focus on work that has been carried out by the authors as part of a NIH program.

A PCR reactor with an integrated alumina membrane for nucleic acid isolation

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 chambers 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 membranes high resistance to liquid flow, a specialized flow control system was devised to facilitate sample introduction and membrane washing.

A portable, integrated analyzer for microfluidic – based molecular analysis

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.

Microfluidic Paper-Based Sample Concentration Using Ion Concentration Polarization with Smartphone Detection

A simple method for microfluidic paper-based sample concentration using ion concentration polarization (ICP) with smartphone detection is developed. The concise and low-cost microfluidic paper-based ICP analytical device, which consists of a black backing layer, a nitrocellulose membrane, and two absorbent pads, is fabricated with the simple lamination method which is widely used for lateral flow strips. Sample concentration on the nitrocellulose membrane is monitored in real time by a smartphone whose camera is used to collect the fluorescence images from the ICP device. A custom image processing algorithm running on the smartphone is used to track the concentrated sample and obtain its fluorescence signal intensity for quantitative analysis. Two different methods for Nafion coating are evaluated and their performances are compared. The characteristics of the ICP analytical device especially with intentionally adjusted physical properties are fully evaluated to optimize its performance as well as to extend its potential applications. Experimental results show that significant concentration enhancement with fluorescence dye sample is obtained with the developed ICP device when a fast depletion of fluorescent dye is observed. The platform based on the simply laminated ICP device with smartphone detection is desired for point-of-care testing in settings with poor resources.

POINT-OF-CARE TEST FOR C-REACTIVE PROTEIN BY A FLUORESCENCE-BASED LATERAL FLOW IMMUNOASSAY

An innovative, portable fluorescence reader was developed for the determination of C-reactive protein based on a lateral flow immunoassay. The C-reactive protein concentration was proportional to the intensity of the test line which was calibrated relative to the control line. To quantify the fluorescence intensity of the lateral flow strip, a custom illumination module, which concentrated the excitation beam from an ultraviolet light-emitting diode, was developed for strip scanning. Accordingly, a high sensitive photodiode with a preamplifier was chosen as the detector for fluorescence. For good repeatability, the strip scanning resolution was set to 5 μm between data points by controlling a linear stage actuated by a stepper motor. Four double-logistic calibration models were compared. The sensitivity for C-reactive protein was 0.1 mg/L and the linear dynamic range extended to 400 mg/L. The optical reader provides a new and simple approach for the determination of C-reactive protein and may be modified for other similar biomarkers.

Investigation of thiolysis of NBD amines for the development of H2S probes and evaluating the stability of NBD dyes

In order to evaluate the thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) amines for development of H2S probes, herein we investigated the reactivity and selectivity of a series of NBD amines for the first time. The piperazinyl- and piperidyl-based NBD probes could react efficiently with micromolar H2S in buffer (pH 7.4), while such NBD(S) (nitrobenzothiadiazole) derivatives showed much slow thiolysis even in the presence of millimolar H2S. Low reactivity was also observed for thiolysis of these ethylamino-, ethanolamino- and anilino-based NBD probes. Therefore, almost all NBD amines used in bioimaging should be stable, in consideration of the presence of only micromolar endogenous H2S in vivo. Moreover, the piperazinyl-NBD derivatives could be efficient in the development of fluorescent H2S probes and for directly visualizing H2S by paper-based detection.