ABSTRACT
An occupationally safe (biosafe) sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with M. tuberculosis complex cells showed approximately 10(6)-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between Mycobacterium species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for M. tuberculosis, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure.
Subject(s)
Biosensing Techniques/methods , Fluorescent Antibody Technique/methods , Microchip Analytical Procedures/methods , Mycobacterium tuberculosis/cytology , Sputum/microbiology , Tuberculosis/diagnosis , Biosensing Techniques/instrumentation , Fluorescent Antibody Technique/instrumentation , Host-Pathogen Interactions , Humans , Lab-On-A-Chip Devices , Microscopy, Fluorescence/methods , Mycobacterium tuberculosis/physiology , Occupational Health , Reproducibility of Results , Sensitivity and Specificity , Time Factors , Tuberculosis/microbiologyABSTRACT
A single-step concentration and elution method is developed for detection of DNA in buffer, saliva, and blood. A nanotip capturing DNA using an electric field and capillary action is directly dissolved in buffer for qPCR analysis. The concentration yield and the relative parameters are compared with those of a commercial kit.
Subject(s)
Analytic Sample Preparation Methods/methods , DNA/genetics , DNA/isolation & purification , Nanotechnology/methods , Polymerase Chain Reaction/methods , Bacteriophage lambda/genetics , Carbon Compounds, Inorganic/chemistry , DNA/analysis , DNA/blood , DNA, Viral/analysis , DNA, Viral/genetics , Genome, Human/genetics , Humans , Nanowires/chemistry , Saliva/chemistry , Silicon Compounds/chemistry , Time FactorsABSTRACT
A rapid, accurate tuberculosis diagnostic tool that is compatible with the needs of tuberculosis-endemic settings is a long-sought goal. An immunofluorescence microtip sensor is described that detects Mycobacterium tuberculosis complex cells in sputum in 25 minutes. Concentration mechanisms based on flow circulation and electric field are combined at different scales to concentrate target bacteria in 1 mL samples onto the surfaces of microscale tips. Specificity is conferred by genus-specific antibodies on the microtip surface. Immunofluorescence is then used to detect the captured cells on the microtip. The detection limit in sputum is 200 CFU mL(-1) with a success rate of 96%, which is comparable to PCR.
Subject(s)
Fluorescent Antibody Technique/instrumentation , Microfluidic Analytical Techniques/instrumentation , Mycobacterium tuberculosis/isolation & purification , Sputum/microbiology , Tuberculosis/diagnosis , Biosensing Techniques/economics , Biosensing Techniques/instrumentation , Equipment Design , Fluorescent Antibody Technique/economics , Humans , Limit of Detection , Microfluidic Analytical Techniques/economics , Time FactorsABSTRACT
Rapid, low cost screening of tuberculosis requires an effective enrichment method of Mycobacterium tuberculosis (MTB) cells. Currently, microfiltration and centrifugation steps are frequently used for sample preparation, which are cumbersome and time-consuming. In this study, the size-selective capturing mechanism of a microtip-sensor is presented to directly enrich MTB cells from a sample mixture. When a microtip is withdrawn from a spherical suspension in the radial direction, the cells that are concentrated by AC electroosmosis are selectively enriched to the tip due to capillary- and viscous forces. The size-selectivity is characterized by using polystyrene microspheres, which is then applied to size-selective capture of MTB from a sample mixture. Our approach yields a detection limit of 800 cells mL(-1), one of the highest-sensitivity immunosensors to date.
Subject(s)
Cell Separation/methods , Mycobacterium tuberculosis/isolation & purification , Animals , Bacteriological Techniques , Cell Line , Cell Separation/instrumentation , Drosophila/cytology , Fluorescent Antibody Technique/methods , Microscopy, Electron, Scanning , Microspheres , Mycobacterium tuberculosis/cytology , Particle Size , Polystyrenes , Sensitivity and Specificity , Tuberculosis/diagnosis , Tuberculosis/microbiology , ViscosityABSTRACT
Various nanowire (or nanotube)-based devices have been investigated to fulfill future demands on semiconducting devices, nanoscale electromechanical systems, and biosensors. To fabricate such devices, an electric field-induced assembly method has demonstrated a great potential for one dimensional assembly. In this paper, our novel approaches for fabricating hybrid nanofibrils are presented to enhance the multiple functionalities, the production rate and the fibril length. These approaches offer unique opportunities for fabricating hybrid nanofibrils composed of silicon carbide (SiC) nanowires and single walled carbon nanotubes (SWCNTs).
ABSTRACT
This paper presents a bio-mimetic microfluidic device that mimics the high compliance and the beating frequency of biological cilia in order to achieve bio-compatible manipulation of microfluidics. Because the highly compliant cilia can easily collapse due to interaction energy and surface tension, the major challenge in developing a bio-mimetic device is the manufacturing of highly compliant cilia. An underwater fabrication method is developed to avoid the cilia collapse by lowering the surface energy of the cilia. Another challenge is to mimic the low beating frequency (10-100 Hz) of biological cilia. The proposed microfluidic device is excited by a piezo actuator to resonate the cilia in water. Due to the highly compliant nature of the silicone cilia, the resulting actuation frequency is in the beating frequency range of biological cilia. Simulations and experiments are presented to demonstrate microfluidic manipulation by resonance of the assembled cilia array.
Subject(s)
Biomimetic Materials/chemistry , Cilia , Microfluidic Analytical Techniques/instrumentation , Silicones/chemistry , Computer Simulation , Dimethylpolysiloxanes/chemistry , Elastic Modulus , Equipment Design , Microspheres , Vibration , WaterABSTRACT
The dielectrophoretic assembly of silicon carbide (SiC) nanowires in a microfluidic flow is shown to enhance the orientation and deposition yield of nanowires. The fluid flow delivers and orients the nanowires in the vicinity of a gap, and they are attracted and deposited by a dielectrophoretic force. Depending upon their lengths, the nanowires are selectively attracted to the gap because the dielectrophoretic force is largest when the lengths are comparable to the gap size. Precise control over the fluid flow and dielectrophoresis shows various interesting phenomena such as landing, shifting, and uniform spacing of nanowires during the assembly process. As a result, the precise control enables the selective positioning of nanowires only at the gap where the fluid direction is consistent with the electric field orientation.
