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.
Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America.
Shinnosuke
S
Inoue
Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, United States of America.
Annie L
AL
Becker
Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America.
Jong-Hoon
JH
Kim
Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America.
Zhiquan
Z
Shu
Departments of Medicine-Division of Medical Genetics and Genome Sciences, University of Washington, Seattle, Washington, United States of America.
Scott D
SD
Soelberg
Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, United States of America.
Kris M
KM
Weigel
Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America.
Morgan
M
Hiraiwa
Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America.
Andrew
A
Cairns
Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America.
Hyun-Boo
HB
Lee
Departments of Medicine-Division of Medical Genetics and Genome Sciences, University of Washington, Seattle, Washington, United States of America.
Clement E
CE
Furlong
NanoFacture, Inc., Bellevue, Washington, United States of America.
Kieseok
K
Oh
NanoFacture, Inc., Bellevue, Washington, United States of America.
Kyong-Hoon
KH
Lee
Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America.
Dayong
D
Gao
Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America.
Jae-Hyun
JH
Chung
Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, United States of America.
Gerard A
GA
Cangelosi
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000295:instrumentation / Q000379:methods*; D054884:Host-Pathogen Interactions; D006801:Humans; D056656:Lab-On-A-Chip Devices; D046208:Microchip Analytical Procedures / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000166:cytology* / Q000502:physiology; D016272:Occupational Health; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*; D013997:Time Factors; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology