Micro- and Nanostructured Immuno-Tip Sensors for Point-of-Care Diagnosis of Tuberculosis.

摘要

Point-of-care(POC) diagnosis of tuberculosis (TB), one of the most widely spread diseases in the globe today, has been critically demanded over a century. To date, many diagnostic methods have been developed for detection of Mycobacterium tuberculosis (MTB); polymerase chain reaction latex agglutination, enzyme-linked immunosorbent assay, radiometric detection, genprobe amplified MTB direct test, TB rapid cultivation detection technique, and flowcytometry. The methods are more sensitive and rapid than the traditional microbial culture-based methods; however, the performance should be further improved. In addition, due to the large volumes and the low analyte concentration of actual sputum samples, a cumbersome concentration step is required. Currently available concentration methods employ centrifugation, microfiltration, or magnetic beads. However, the methods are limited in cumbersome preparation steps, low yield, and low throughput. To address the challenges, microtip- and nanotip immunosensors are studied for fluorescence- and electrical measurements, respectively.;For a microtip immunofluorescence sensor, concentration mechanisms based on flow circulation and electric field are combined at different scales to concentrate target bacteria in 1 mL sputum samples onto the surfaces of microscale tips. Genus-specific antibodies are immobilized on the microtip surface for specific capturing. Experimentally, the concentration mechanism is studied. Then, the performance of conentration mechanism is validated by using an immunofluorescence. To understand specific capturing mechanism of target bacteria on a microtip surface, the bacterial behavior on the microtip surface during evaporation is investigated as a function of the substrate properties, liquid composition, and environmental conditions. The novel concentration mechanism using a microtip can detect MTB complex cells in sputum within 25 minutes. The detection limit in sputum is 200 CFU/mL with a success rate of 96 %, which is comparable to PCR. The presented microtip based immunosensor shows great potential for POC diagnostic tool.;For simpler POC diagnosis, an electrical detection method using a dendritic nanotip is presented. The dendritic nanotip is composed of SWCNTs and Si nanowires, which is designed to maximize the surface area for high sensitivity. Upon the binding of target cells on to SWCNTs of a nanotip, the gating effect is induced to increase the resistance of the sensor system. The working principles are studied for each binding layer by cyclic voltammetry. A SWCNT-based dendritic nanotip is utilized to concentrate target bacteria by using an electric field and capillary effect. The captured targets on the nanotip are detected by measuring the resistance change in deionized water. The detection results are verified by fluorescence- and scanning electron microscopes. In the experimental results, Bacillus Calmette-Guérin (BCG) cells strain of Mycobacterium bovis can be detected within 15 minutes using both electrical and fluorescent detection. The sensitivity is 1,000 CFU/mL for both methods.;As a future plan, the surface phenomena for specific detection of target bacteria will be studied to understand the specific capturing mechanism on microtip surface. The detection mechanism using a nanotip will be studied further to clarify the detection mechanism.