RESUMEN
Acute stroke is the third leading cause of mortality and disability worldwide. Administration of appropriate therapy for acute stroke is critically dependent on timely classification into either ischemic or hemorrhagic subtypes, which have divergent treatment pathways. The current classification method is based on neuroimaging, which generally requires the transport of the patient to a hospital-based facility unless a mobile stroke unit is available. Plasma glial fibrillary acidic protein (GFAP) level has been identified as a useful blood-based biomarker to differentiate stroke subtypes. However, its conventional immunoassay methods are laboratory-based and time-consuming. Novel approaches for rapid stroke classification near the patients are urgently needed. Here, we report the development and testing of a microfluidic-based magnetoimpedance biosensor platform for measuring GFAP levels. The platform consists of a microfluidic chip for GFAP extraction from a blood sample and a magnetoimpedance (MI) biosensor that employs Dynabeads as a magnetic label to capture the GFAP molecules. We demonstrated the detection of recombinant GFAP protein in phosphate-buffered saline (PBS) and in mouse blood samples (detection limit 0.01 ng/mL) and of physiological GFAP in blood and plasma samples (detection limit 1.0 ng/mL) obtained from acute stroke patients. This detection level is within the range of cut-off levels required for clinical stroke subtype differentiation. This platform has the potential to be incorporated into a small device with further development to assist in the classification of acute stroke patients and clinical decision-making at the point-of-care.
Asunto(s)
Técnicas Biosensibles , Isquemia Encefálica , Accidente Cerebrovascular , Animales , Biomarcadores , Isquemia Encefálica/diagnóstico , Hemorragia Cerebral/diagnóstico , Hemorragia Cerebral/terapia , Proteína Ácida Fibrilar de la Glía , Humanos , Ratones , Microfluídica , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/terapiaRESUMEN
The magnetic beads detection-based immunoassay, also called magneto-immunoassay, has potential applications in point-of-care testing (POCT) due to its unique advantage of minimal background interference from the biological sample and associated reagents. While magnetic field detection technologies are well established for numerous applications in the military, as well as in geology, archaeology, mining, spacecraft, and mobile phones, adaptation into magneto-immunoassay is yet to be explored. The magnetic field biosensors under development tend to be multilayered and require an expensive fabrication process. A low-cost and affordable biosensing platform is required for an effective point-of-care diagnosis in a resource-limited environment. Therefore, we evaluated a single-layered magnetic biosensor in this study to overcome this limitation. The shape-induced magnetic anisotropy-based planar hall effect sensor was recently developed to detect a low-level magnetic field, but was not explored for medical application. In this study, the elliptical-shaped planar hall effect (EPHE) sensor was designed, fabricated, characterized, and optimized for the magneto-immunoassay, specifically. Nine sensor variants were designed and fabricated. A customized measurement setup incorporating a lock-in amplifier was used to quantify 4.5 µm magnetic beads in a droplet. The result indicated that the single-domain behaviour of the magnetic film and larger sensing area with a thinner magnetic film had the highest sensitivity. The developed sensor was tested with a range of magnetic bead concentrations, demonstrating a limit of detection of 200 beads/µL. The sensor performance encourages employing magneto-immunoassay towards developing a low-cost POCT device in the future.
Asunto(s)
Técnicas Biosensibles , Técnicas Biosensibles/instrumentación , Diseño de Equipo , Inmunoensayo/instrumentación , Separación Inmunomagnética , MagnetismoRESUMEN
Thin-film magneto-impedance (MI) biosensors have attracted significant attention due to their high sensitivity and easy miniaturization. However, further improvement is required to detect weak biomagnetic signals. Here, we report a meander thin-film biosensor preparation to investigate the fabrication parameters influencing the MI effect. Specifically, we hypothesized that an optimal film thickness and sensing area size ratio could be achieved to obtain a maximum MI ratio. A meander multilayer MI biosensor based on a NiFe/Cu/NiFe thin-film was designed and fabricated into 3-, 6-, and 9-turn models with film thicknesses of 3 µm and 6 µm. The 9-turn biosensor resembled the largest sensing area, while the 3- and 6-turn biosensors were designed with identical sensing areas. The results indicated that the NiFe film thickness of 6 µm with a sensing area size of 14.4 mm2 resembling a 9-turn MI biosensor is the optimal ratio yielding the maximum MI ratio of 238%, which is 70% larger than the 3- and 6-turn structures. The 3- and 6-turn MI biosensors exhibited similar characteristics where the MI ratio peaked at a similar value. Our results suggest that the MI ratio can be increased by increasing the sensing area size and film thickness rather than the number of turns. We showed that an optimal film thickness to sensing area size ratio is required to obtain a high MI ratio. Our findings will be useful for designing highly sensitive MI biosensors capable of detecting low biomagnetic signals.
Asunto(s)
Técnicas Biosensibles , Impedancia EléctricaRESUMEN
HLA-B*15:02 screening before administering carbamazepine is recommended to prevent life-threatening hypersensitivity. However, the unavailability of a point-of-care device impedes this screening process. Our research group previously developed a two-step HLA-B*15:02 detection technique utilizing loop-mediated isothermal amplification (LAMP) on the tube, which requires two-stage device development to translate into a portable platform. Here, we report a heater-integrated lab-on-a-chip device for the LAMP amplification, which can rapidly detect HLA-B alleles colorimetrically. A gold-patterned micro-sized heater was integrated into a 3D-printed chip, allowing microfluidic pumping, valving, and incubation. The performance of the chip was tested with color dye. Then LAMP assay was conducted with human genomic DNA samples of known HLA-B genotypes in the LAMP-chip parallel with the tube assay. The LAMP-on-chip results showed a complete match with the LAMP-on-tube assay, demonstrating the detection system's concurrence.
Asunto(s)
Hipersensibilidad a las Drogas , Antígenos HLA-B , Dispositivos Laboratorio en un Chip , Alelos , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido NucleicoRESUMEN
Biosensors based on magneto-impedance (MI) effect are powerful tools for biomedical applications as they are highly sensitive, stable, exhibit fast response, small in size, and have low hysteresis and power consumption. However, the performance of these biosensors is influenced by a variety of factors, including the design, geometry, materials and fabrication procedures. Other less appreciated factors influencing the MI effect include measuring circuit implementation, the material used for construction, geometry of the thin film sensing element, and patterning shapes compatible with the interface microelectronic circuitry. The type magnetic (ferrofluid, Dynabeads, and nanoparticles) and size of the particles, the magnetic particle concentration, magnetic field strength and stray magnetic fields can also affect the sensor sensitivity. Based on these considerations it is proposed that ideal MI biosensor sensitivity could be achieved when the sensor is constructed in sandwich thick magnetic layers with large sensing area in a meander shape, measured with circuitry that provides the lowest possible external inductance at high frequencies, enclosed by a protective layer between magnetic particles and sensing element, and perpendicularly magnetized when detecting high-concentration of magnetic particles.
Asunto(s)
Técnicas Biosensibles , Nanopartículas , Impedancia Eléctrica , Campos Magnéticos , MagnetismoRESUMEN
Outbreaks of foodborne diseases have become a global health concern; hence, many improvements and developments have been made to reduce the risk of food contamination. We developed a centrifugal microfluidic automatic wireless endpoint detection system integrated with loop mediated isothermal amplification (LAMP) for monoplex pathogen detection. Six identical sets were designed on the microfluidic compact disc (CD) to perform 30 genetic analyses of three different species of foodborne pathogens. The consecutive loading, mixing, and aliquoting of the LAMP primers/reagents and DNA sample solutions were accomplished using an optimized square-wave microchannel, metering chambers and revulsion per minute (RPM) control. We tested 24 strains of pathogenic bacteria (Escherichia coli, Salmonella spp and Vibrio cholerae), with 8 strains of each bacterium, and performed DNA amplification on the microfluidic CD for 60min. Then, the amplicons of the LAMP reaction were detected using the calcein colorimetric method and further analysed via the developed electronic system interfaced with Bluetooth wireless technology to transmit the results to a smartphone. The system showed a limit of detection (LOD) of 3 × 10-5ngµL-1 DNA by analysing the colour change when tested with chicken meat spiked with the three pathogenic bacteria. Since the entire process was performed in a fully automated way and was easy to use, our microdevice is suitable for point-of-care (POC) testing with high simplicity, providing affordability and accessibility even to poor, resource-limited settings.
Asunto(s)
Colorimetría/instrumentación , Escherichia coli/aislamiento & purificación , Enfermedades Transmitidas por los Alimentos/microbiología , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Salmonella/aislamiento & purificación , Vibrio cholerae/aislamiento & purificación , Técnicas Biosensibles/economía , Técnicas Biosensibles/instrumentación , Colorimetría/economía , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Diseño de Equipo , Escherichia coli/genética , Microbiología de Alimentos , Dispositivos Laboratorio en un Chip , Límite de Detección , Técnicas Analíticas Microfluídicas/economía , Técnicas de Amplificación de Ácido Nucleico/economía , Salmonella/genética , Vibrio cholerae/genéticaRESUMEN
In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10(-3) ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment.