RESUMEN
Herpes simplex virus (HSV) is one of the most prevalent viruses, with acute and recurrent infections in humans. The current gold standard for the diagnosis of HSV is viral culture which takes 2-14 days and has low sensitivity. In contrast, DNA amplification by polymerase chain reaction (PCR) can be performed within 1-2 h. We here describe a multiparameter PCR assay to simultaneously detect HSV-1 and HSV-2 DNA templates, together with integrated positive and negative controls, with product detection by melting curve analysis (MCA), in an array of semi-solid polyacrylamide gel posts. Each gel post is 0.67 µL in volume, and polymerized with all the components required for PCR. Both PCR and MCA can currently be performed in one hour and 20 min. Unprocessed genital swabs collected in universal transport medium were directly added to the reagents before or after polymerization, diffusing from atop the gel posts. The gel post platform detects HSV templates in as little as 2.5 nL of raw sample. In this study, 45 genital swab specimens were tested blindly as a preliminary validation of this platform. The concordance of PCR on gel posts with conventional PCR was 91%. The primer sequestration method introduced here (wherein different primers are placed in different sets of posts) enables the simultaneous detection of multiple pathogens for the same sample, together with positive and negative controls, on a single chip. This platform accepts unprocessed samples and is readily adaptable to detection of multiple different pathogens or biomarkers for point-of-care diagnostics.
Asunto(s)
ADN Viral/análisis , Genitales/virología , Herpesvirus Humano 1/aislamiento & purificación , Herpesvirus Humano 2/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Cartilla de ADN , Diseño de Equipo , Herpes Genital/diagnóstico , Herpes Genital/virología , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/genética , Humanos , Límite de Detección , Desnaturalización de Ácido Nucleico , Reacción en Cadena de la Polimerasa/instrumentación , Proyectos de Investigación , Espectrometría de Fluorescencia , TemperaturaRESUMEN
This work describes the use of polyacrylamide gel and PCR reagents photopolymerized in a mold to create an array of semisolid posts that serve as reaction vessels for parallel PCR amplification of an externally added template. DNA amplification occurred in a cylindrical, self-standing 9 × 9 array of gel posts each less than 1 µL in volume. Photopolymerization of the gel with an intercalating dye added prior to polymerization permitted acquisition of real-time PCR data and melting curve analysis data without the need for any type of post-PCR staining procedures. PCR was equally efficient and reproducible when template DNA was polymerized within the gel or when exogenous template was added atop precast gel posts. PCR amplification occurred with template from purified DNA or from raw urine of patients with BK viruria. Multiple primer sets can be utilized per gel post array with no detectable cross contamination. As few as 34 BK virus templates were consistently detected by PCR in an individual gel post. Amplification of HPA1 and FGFR2 genes in human genomic DNA (gDNA) required as little as 2-5 ng of gDNA template/gel post. The device prototype includes a Peltier element for PCR thermal cycling and a CCD camera to capture fluorescence for product detection. Our technology is amenable to integration in point of care microdevices.
Asunto(s)
Virus BK/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Resinas Acrílicas/química , Antígenos de Plaqueta Humana/genética , Virus BK/genética , ADN Viral/orina , Genotipo , Humanos , Integrina beta3 , Desnaturalización de Ácido Nucleico , Transición de Fase , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genéticaRESUMEN
We present a prototype microfluidic device developed for the continuous dielectrophoretic (DEP) fractionation and purification of sample suspensions of biological cells. The device integrates three fully functional and distinct units consisting of an injector, a fractionation region, and two outlets. In the sheath and sample injection ports, the cell sample are hydrodynamically focused into a stream of controlled width; in the DEP fractionation region, a specially shaped nonuniform (isomotive) electric field is synthesized and employed to facilitate the separation, and the sorted cells are then delivered to two sample collection ports. The microfluidic behavior of the injector region was simulated and then experimentally verified. The operation and performance of the device was evaluated using yeast cells as model biological particles. Issues relating to the fabrication and operation of the device are discussed in detail. Such a device takes a significant step towards an integrated lab-on-a-chip device, which could interface/integrate to a number of other on-chip components for the device to undertake the whole laboratory procedure.
Asunto(s)
Separación Celular/métodos , Electroquímica/métodos , Electroforesis/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Levaduras/citología , Electrodos , Electroforesis/instrumentación , Electroforesis por Microchip , Diseño de Equipo , Microquímica , Microelectrodos , Modelos Químicos , Modelos EstadísticosRESUMEN
The transition of MEMS technology to nano fabrication is a solution to the growing demand for smaller and high-density feature sizes in the nanometer scale. Nanoimprint lithography (NIL) techniques for fabricating micro- and nano-features are discussed including hot embossing lithography (HEL), UV Molding (UVM) and micro contact printing (microCP). Recent results in micro and nano-pattern transfer are presented where features ranged from < 100 nm to several centimeters. We also present a comparative study between standard glass microfluidic chips and their HEL counterparts by metrology. Hot-embossed microfluidic chips are shown to be faithful replicates of their parent stamps. NIL is presented as a promising avenue for low-cost, high throughput micro and nano-device fabrication.
Asunto(s)
Diseño de Equipo/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Nanoestructuras/química , Nanotecnología/métodos , Evaluación de la Tecnología Biomédica , Análisis de Falla de Equipo , Ensayo de Materiales , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/tendencias , Miniaturización , Nanoestructuras/ultraestructura , Nanotecnología/tendencias , Propiedades de SuperficieRESUMEN
As microfluidic chips come to integrate the higher levels of functionality required for the implementation of advanced bioanalytical protocols, a crucial factor is that of cost. Although glass chips provide advantages in multilayer integrations, their cost is far higher than that of polymer chips. However, a simple and effective rejuvenation protocol for glass microchips may enable higher levels of integration and functionality on glass microchips. Here we present a method to rejuvenate glass microchips that had been used for capillary electrophoresis to the extent that their performance was degraded. This degradation was due to one of the two mechanisms: (i) a deterioration of the polymer coating on the inner surface of the microchannel or (ii) an aging of the glass substrate. Using the method presented here, we have rejuvenated more than 50 such "aged" microchips. The performance of these microchips was fully restored after the rejuvenation and lasted for hundreds of DNA separation runs. Our experiments indicate that the loss of resolution in microchip separations was not associated with glass aging, but was due to the degradation of the polymer coating on the inner surface of microchannels. This suggests that it is possible to extend the microchip lifetime "forever" using the rejuvenation protocol and that the exploration of higher levels of integration and functionality on glass microchips (or of hybrid structures involving materials capable of withstanding the reagents and elevated temperatures used) is feasible.
Asunto(s)
Acrilamidas/química , Electroforesis por Microchip/instrumentación , Vidrio/química , Técnicas Analíticas MicrofluídicasRESUMEN
We present an improvement of the field inversion electrophoresis (FIE) method in which the passage of sample such as DNA back and forth within a short length of a microchannel can provide a similar resolution to that of a significantly longer microchannel. In constant field FIE the application of an alternating potential (e.g., +/- V) over short periods of time (e.g., several Hz) can provide enhanced separations of DNA fragments. In contrast, the present method consists of a series of separations, each of much longer duration, under high and low fields in such a way that the resolution is enhanced. This method is readily modeled and allows improved resolution to be obtained from extremely short microchannels (e.g., 8 mm) while requiring relatively low applied voltages (e.g., less than 600 V). An additional advantage is that this method can allow for the same equipment to be used in a rapid, low-resolution mode or in a slower, high-resolution mode through what might be referred to as an automated "zoom" capability. We believe that this method may facilitate the integration of microfluidic devices and microelectronic devices by allowing these devices to be of a similar small scale (< 1 cm).
Asunto(s)
Electroforesis en Gel de Campo Pulsado/instrumentación , Microfluídica/instrumentación , ADN/aislamiento & purificación , Electroforesis Capilar/instrumentación , Electroforesis Capilar/métodos , Electroforesis Capilar/normas , Electroforesis en Gel de Campo Pulsado/normas , MiniaturizaciónRESUMEN
We have developed a high-throughput microfabricated, reusable glass chip for the functional integration of reverse transcription (RT) and polymerase chain reaction (PCR) in a continuous-flow mode. The chip allows for selection of the number of amplification cycles. A single microchannel network was etched that defines four distinct zones, one for RT and three for PCR (denaturation, annealing, extension). The zone temperatures were controlled by placing the chip over four heating blocks. Samples and reagents for RT and PCR were pumped continuously through appropriate access holes. Outlet channels were etched after cycles 20, 25, 30, 35, and 40 for product collection. The surface-to-volume ratio for the PCR channel is 57 mm(-1) and the channel depth is 55 microm, both of which allow very rapid heat transfer. As a result, we were able to collect PCR product after 30 amplification cycles in only 6 min. Products were collected in 0.2-mL tubes and analyzed by agarose gel electrophoresis and ethidium bromide staining. We studied DNA and RNA amplification as a function of cycle number. The effect of the number of the initial DNA and RNA input molecules was studied in the range of 2.5 x 10(6) - 1.6 x 10(8) and 6.2 x 10(6) - 2 x 10(8), respectively. Successful amplification of a single-copy gene (beta-globin) from human genomic DNA was carried out. Furthermore, PCR was performed on three samples of DNA of different lengths (each of 2-microL reaction volume) flowing simultaneously in the chip, and the products were collected after various numbers of cycles. Reverse transcription was also carried out on four RNA samples (0.7-microL reaction volume) flowing simultaneously in the chip, followed by PCR amplification. Finally, we have demonstrated the concept of manually pumped injection and transport of the reaction mixture in continuous-flow PCR for the rapid generation of amplification products with minimal instrumentation. To our knowledge, this is the first report of a monolithic microdevice that integrates continuous-flow RT and PCR with cycle number selection.