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Dielectrophoresis (DEP) can be an effective tool to show the physiological change of bacterial cells. The behavior of bacterial cells under an electric field is complicated due to the combined effects of electrokinetic phenomena. This paper presents the study of the electrokinetic behavior of heat-treated Mycobacterium bovis Bacillus Calmette-Guérin (BCG) cells for a cell counting method. Through numerical and experimental study, heat-treated BCG cells are compared with control BCG cells. At various frequencies with the medium conductivity of 0.07 S/m, the equilibrium positions of both control- and heat-treated cells are analyzed in the combined fields of DEP and AC electroosmosis (ACEO). As DEP changes from negative to positive in electroosmotic flow, the equilibrium position of cells is bifurcated from the upper center between two electrodes onto the edges of both electrodes. It was found that the cells floating on electrodes should not be counted as attracted cells because the floating was resulted from the combined effect of the negative DEP and ACEO. According to the analysis, an optimum frequency is proposed to differentiate control cells from heat-treated cells using a cell counting method. The presented study will offer physical insight for the cell counting to differentiate live and dead Mycobacterium bovis BCG cells treated with heat and drugs.

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-Guerin, 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.

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.

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.

Tuberculosis (TB) is an epidemic affecting one-third of the world’s population, mostly in developing and low-resource settings. People having active pulmonary TB are considered highly infectious; therefore, it is critical to identify and treat these patients rapidly before spreading to others. However, the most reliable TB diagnostic methods of bacterial culture or nucleic acid amplification are time-consuming and expensive. The challenge of TB diagnosis lies in highly sensitive and specific screening with low cost. Here, we present an LNA-modified microtip-sensor, which is capable of selectively detecting low-abundance DNA from bacteria. When genomic DNA of Bacillus Calmette-Guerin (BCG, a surrogate marker of Mycobacterium bovis), and genomic DNA of Staphylococcus epidermidis (S. epi) are used, the microtip-sensor yields the detection limit of 1,000 copies/mL within 20 minutes. The high sensitivity and specificity approaching nucleic acid amplification methods can potentially overcome the current challenges for rapid TB screening.Copyright © 2011 by ASME

Rapid detection of tuberculosis (TB) has been critically demanded over the last century. To detect TB, numerous methods screening Mycobacterium tuberculosis (MTB) have been developed. However, the methods still have challenges of rapid and specific enrichment of MTB. In this study, we present a novel specific enrichment method of MTB using a microfabricated tip. Through our simulation study, a wavy-shaped microtip is designed to enhance capturing efficiency of bacteria. Using an optimized tip, bacteria are attracted by dielectrophoresis and captured by affinity binding and capillary action. In experiment, a surrogate marker of MTB, Bacilli Calmette-Guerin (BCG), is enriched with the micromachined tip. When a microtip decorated with capturing antibodies is used, BCG cells spiked in saliva are detected at the concentration of 5×105 CFU/mL within 20 minutes. The tip enrichment system demonstrates the potential for rapid, culture-free detection of MTB in a raw sample.

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.

Successful long-term preservation of Mycobacterium tuberculosis (MTB) cells or sputum specimens is very important for sample transport, biobanking, research and the development of new drugs, vaccines, biomarkers, and diagnostics. In this work, M . bovis Bacillus Calmette–Guerin (BCG) and M. tuberculosis H37Ra were used as models of MTB complex strains to study cryopreservation of MTB complex cells in diverse sample matrices (phosphate buffer saline (PBS), Middlebrook 7H9 medium with or without added glycerol, and human sputum). Efficacy of cryopreservation was quantified by microbiological culture. Results showed that among the variables tested, slow cooling rate was the most critical factor for the successful cryopreservation of Mycobacterium tuberculosis complex cells. Preincubation of frozen/thawed MTB cells in 7H9 broth before culturing did not help the cells repair cryoinjury. Cell inactivation by fast cooling was not associated with a compromised cell envelope, as indicated by the comparison results of microbiological culture and the BacLight live/dead staining. After cryopreservation, the MTB cell surface antigen can still function well for the MTB diagnosis as biomarker. For the Point-of-Care (POC) diagnosis of MTB, an occupationally safe (biosafe) sputum liquefaction protocol and a semi-automated antibody-based microtip immunofluorescence sensor were developed. Sputum was treated with a synergistic chemical–thermal protocol that included moderate concentrations of NaOH and detergent at 60 °C for 5–10 min. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. The biosafe sputum liquefaction method, cryopreservation protocol, and the microtip device make it possible to diagnose the tuberculosis with high feasibility, sensitivity and accuracy.

A long-sought goal for infectious disease care is a rapid and accurate diagnostic tool that is compatible with the needs of low-resource settings. To identify target biomarkers of infectious diseases, immunoassays utilizing the binding affinity between antigen and antibody have been widely used. In immunoassays, the interaction between antigen and antibody on sensor surfaces should be precisely controlled for specific identification of targets. This paper studies the specific capturing mechanisms of target bacteria onto sensor surfaces through investigation of combined effects of capillary action and binding affinity. As a model system, cells of both Escherichia coli and the Bacillus Calmette-Guerin strain of Mycobacterium bovis were used to study specific and nonspecific capturing mechanisms onto a microtip sensor. The capillary action was observed to arrange the concentrated cells onto the two-dimensional sensor surface. Due to the capillary-induced organization of target cells on the antibody-functionalized sensor surface, the number of the captured target cells was three times greater than that of the non-targeted cells. The capturing and detection capabilities varied with the width of a microtip. The specific capturing mechanism can be used to enhance the sensitivity and specificity of an immunoassay.