Shinnosuke Inoue

Immunosensor towards low-cost, rapid diagnosis of tuberculosis

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.

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 106-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.

Specific capture of target bacteria onto sensor surfaces for infectious disease diagnosis

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.

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.