Dielectrophoretic characterization of antibiotic-treated Mycobacterium tuberculosis complex cells.

Multi-drug resistant tuberculosis (MDR-TB) has become a serious concern for proper treatment of patients. As a phenotypic method, dielectrophoresis can be useful but is yet to be attempted to evaluate Mycobacterium tuberculosis complex cells. This paper investigates the dielectrophoretic behavior of Mycobacterium bovis (Bacillus Calmette-Guérin, BCG) cells that are treated with heat or antibiotics rifampin (RIF) or isoniazid (INH). The experimental parameters are designed on the basis of our sensitivity analysis. The medium conductivity (σ(m)) and the frequency (f) for a crossover frequency (f(xo1)) test are decided to detect the change of σ(m)-f(xo1) in conjunction with the drug mechanism. Statistical modeling is conducted to estimate the distributions of viable and nonviable cells from the discrete measurement of f (xo1). Finally, the parameters of the electrophysiology of BCG cells, C(envelope) and σ(cyto), are extracted through a sampling algorithm. This is the first evaluation of the dielectrophoresis (DEP) approach as a means to assess the effects of antimicrobial drugs on M. tuberculosis complex cells.

Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis.

Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low cost detection of Mycobacterium Tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on microtip surface is linearly changed with increasing immersion depth. Using a reference microtip, the immersion depth is compensated for a sensing microtip. On the microtip surface, target bacteria are concentrated and organized by a coffee ring effect, which amplifies the electric current. To enhance the signal-to-noise ratio, both the sample processing- and rinsing steps are presented with use of deionized water as a medium for the amperometric measurement. When applied to cultured MTB cells spiked into human sputum, the detection limit was 100 CFU/mL, comparable to a more labor-intensive fluorescence detection method reported previously.

Semi-automated, occupationally safe immunofluorescence microtip sensor for rapid detection of Mycobacterium cells in sputum.

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.

Nanotips for single-step preparation of DNA for qPCR analysis.

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.

Electrolyte-free Amperometric Immunosensor using a Dendritic Nanotip.

Electric detection using a nanocomponent may lead to platforms for rapid and simple biosensing. Sensors composed of nanotips or nanodots have been described for highly sensitive amperometry enabled by confined geometry. However, both fabrication and use of nanostructured sensors remain challenging. This paper describes a dendritic nanotip used as an amperometric biosensor for highly sensitive detection of target bacteria. A dendritic nanotip is structured by Si nanowires coated with single-walled carbon nanotubes (SWCNTs) for generation of a high electric field. For reliable measurement using the dendritic structure, Si nanowires were uniformly fabricated by ultraviolet (UV) lithography and etching. The dendritic structure effectively increased the electric current density near the terminal end of the nanotip according to numerical computation. The electrical characteristics of a dendritic nanotip with additional protein layers was studied by cyclic voltammetry and I-V measurement in deionized (DI) water. When the target bacteria dielectrophoretically captured onto a nanotip were bound with fluorescence antibodies, the electric current through DI water decreased. Measurement results were consistent with fluorescence- and electron microscopy. The sensitivity of the amperometry was 10 cfu/sample volume (103 cfu/mL), which was equivalent to the more laborious fluorescence measurement method. The simple configuration of a dendritic nanotip can potentially offer an electrolyte-free detection platform for sensitive and rapid biosensors.

Nanotip analysis for dielectrophoretic concentration of nanosized viral particles.

Rapid and sensitive detection of low-abundance viral particles is strongly demanded in health care, environmental control, military defense, and homeland security. Current detection methods, however, lack either assay speed or sensitivity, mainly due to the nanosized viral particles. In this paper, we compare a dendritic, multi-terminal nanotip ('dendritic nanotip') with a single terminal nanotip ('single nanotip') for dielectrophoretic (DEP) concentration of viral particles. The numerical computation studies the concentration efficiency of viral particles ranging from 25 to 100 nm in radius for both nanotips. With DEP and Brownian motion considered, when the particle radius decreases by two times, the concentration time for both nanotips increases by 4-5 times. In the computational study, a dendritic nanotip shows about 1.5 times faster concentration than a single nanotip for the viral particles because the dendritic structure increases the DEP-effective area to overcome the Brownian motion. For the qualitative support of the numerical results, the comparison experiment of a dendritic nanotip and a single nanotip is conducted. Under 1 min of concentration time, a dendritic nanotip shows a higher sensitivity than a single nanotip. When the concentration time is 5 min, the sensitivity of a dendritic nanotip for T7 phage is 10(4) particles ml(-1). The dendritic nanotip-based concentrator has the potential for rapid identification of viral particles.

Electric field guided assembly of one-dimensional nanostructures for high performance sensors.

Various nanowire or nanotube-based devices have been demonstrated to fulfill the anticipated future demands on sensors. To fabricate such devices, electric field-based methods have demonstrated a great potential to integrate one-dimensional nanostructures into various forms. This review paper discusses theoretical and experimental aspects of the working principles, the assembled structures, and the unique functions associated with electric field-based assembly. The challenges and opportunities of the assembly methods are addressed in conjunction with future directions toward high performance sensors.