Integrated microfluidic enzyme reactor mass spectrometry platform for detection of anthrax lethal factor.
In this work, we have developed a coupled microfluidic enzyme reactor mass spectrometry platform for the detection of protein toxins such as anthrax lethal factor. The lethal toxin produced during Bacillus anthracis infection is a complex protective antigen, which localizes the toxin to the cell receptor and lethal factor (LF). We have demonstrated, in this work, the applicability of a microfluidic reactor for the capture and concentration of enzyme reaction solid-phase. The reaction solid-phase consists of anti-LF monoclonal antibodies immobilized on magnetic protein G beads for the capture of LF. The captured LF, on exposure to optimized peptide substrate, hydrolyzes into two smaller peptide products. These cleavage products were then analyzed by mass spectrometer coupled to the microfluidic reactor. This resulted in efficient sample preparation, high sensitivity, larger reaction sites, less reagents consumption and shorter analysis time. We have showed here reproducible detection of anthrax lethal factor in concentration range of 40 to 0.5 ng/mL with a detection limit of 1 ng/mL. The enzymatic reaction and the analysis were performed in less than 15 minutes, indicating a rapid diagnostic tool for early anthrax prognosis.
Aravamudhan
Shyam
S
Nanotechnology Research Center, Georgia Institute of Technology, Atlanta, GA 30332 USA. saravamu@gatech.edu
Joseph
Paul J
PJ
Kuklenyik
Zsuzsanna
Z
Boyer
Anne E
AE
Barr
John R
JR
D000942:Antigens, Bacterial / Q000032:analysis* / Q000276:immunology; D001427:Bacterial Toxins / Q000032:analysis* / Q000276:immunology; D010450:Endopeptidases / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D018189:Immunomagnetic Separation / Q000295:instrumentation*; D013058:Mass Spectrometry; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000295:instrumentation*; D018511:Systems Integration
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin; D010450:Endopeptidases