Study of Electrokinetic Response for Drug Susceptibility of Mycobacterium Tuberculosis Complex Cells
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The alarming increase of multi-drug resistant tuberculosis (MDR-TB) has become a serious issue, especially with TB affecting nearly a third of the global population and claiming over a million lives each year. Drug resistance emerges from gene mutations and improper treatment regimens of the drug-susceptible patients, which is transmitted to others when left undetected and untreated. Testing every patient for drug-resistance would be ideal; however, the additional time and resources available for current supplementary tests for drug-resistance are severely limited. The main challenge of drug susceptibility testing is in discovering a simple and inexpensive method. Drug-susceptibility tests can be differentiated into two categories of phenotypic or genotypic methods. Genotypic methods such as INNO-LiPA Rif, GenoType MTBDR, and Gene Xpert MTB/RIF are very rapid with high sensitivity and specificity, but suffer from the trade-off of high cost, power supply, and requirement of skilled trained personnel. It is also challenging in that the target drug-resistance sequences should be identified prior to running the tests. Phenotypic drug-susceptibility tests such as MGIT, NRA, and MODS are based on detection of bacterial growth in the presence of antibiotics. Although very reliable and mostly cheap, it requires trained personnel and relatively slow, taking weeks for results. Thus low-cost yet rapid identification of patients with MDR-TB is crucial for increasing chances of survival and controlling the possibility of transmission. To address the need for a low-cost and direct detection of TB, we developed a sputum sample preparation protocol and a point-of-care TB diagnostic device based on the previous microtip platform. The newly developed sputum protocol was biosafe while retaining the integrity of the antigens so that it can be used in BSL 1 and 2 labs. The biosafe sputum samples were used in the microtip immunofluorescence sensor, where it combines fluid flow circulation and an AC electric field to concentrate target cells in a 1 mL volume to the microtip surface. The surface of the microtip was decorated with IgY antibodies specific to Mycobacterium to capture these cells. Once captured, the microtip sensor was labeled and washed for immunofluorescent detection. The device was automated from the beginning of cell capture to the washing of the fluorescent antibodies to minimize operational errors and manual labor. The automated immunofluorescent microtip sensor takes 30 minutes for detection with a detection limit of 100 CFU/mL. To study electrokinetic effects of viable and non-viable Mycobacterium cells, control and heat-killed Bacillus Calmette-Guérin (BCG) cells were tested on planar electrodes with frequencies ranging from 1 kHz to 10 MHz to determine if the microtip assay will be capable of being implemented as a drug-susceptibility test. Electrophysiology of cells changes upon cell death, especially when the cell wall and membrane is compromised, changing the electric properties. By analyzing the AC electroosmotic and dielectrophoreic forces, the frequency of 5 MHz was chosen to differentiate viable and non-viable BCG. At the chosen frequency, BCG cells treated with temperatures of 50ºC ~ 80ºC were tested to determine the temperature influence on cell integrity. A transition temperature to differentiate viable and non-viable BCG cells was found to be 60ºC by dielectrophoresis (DEP). Over the transition temperature the lipid cell wall becomes more permeable, which reduces the DEP force the cell experiences. Heat-killed cells treated at 85ºC were shown to be able to be differentiated from control. The cells were further analyzed by characterizing their dielectric properties by matching the experimental crossover frequencies at various medium conductivities. This showed heat-killing decreased the cell envelope capacitance and cytoplasm conductivity, which was shown to be deformed under SEM. To study drug-effects on DEP, first line TB drugs of rifampin (RIF) and isoniazid (INH) were used to treat BCG cells and compared with untreated BCG cells on planar electrodes. RIF and INH were chosen due to their significance in treatment of TB and their different treatment mechanism. RIF inhibits RNA transcription with no direct interaction with the cell wall whereas the INH inhibits synthesis of mycolic acids in the cell wall structure. Frequency of 5 MHz was employed to differentiate drug-treated and non-treated BCG cells. Cell count of attracted cells showed RIF with similar responses to control, whereas INH-treated cells could be differentiated by the 4th day. RIF-treated cells are only differentiated from control by comparing the aggregation and clusters of cells measuring the fluorescence intensity. Properties of drug-treated cells were also characterized by matching the crossover frequencies at various medium conductivities. RIF-treated cells showed partial change in the cytoplasm conductivity, while INH-treated cells showed the largest change in the cell envelope capacitance as well as the cytoplasm conductivity. The DEP response correlated with the expected drug mechanism of the administered antibiotic. The future work will entail evaluation of the microtip and nanotip biosensor as drug-susceptibility tests.
- Mechanical engineering