Dr. A. Peter Snyder and Dr. Rabih E. Jabbour Private Citizens June 26, 2013

Transcription

1 State of the Art for Autonomous Detection Systems using Mass Spectrometry White Paper for the Department of Homeland Security and Institute of Medicine Dr. A. Peter Snyder and Dr. Rabih E. Jabbour Private Citizens June 26, 2013

2 Mass Spectrometry Separates gas phase ions according to their m/z (mass to charge ratio) values. Utilizes different sources of ionization to generate ions in gas phase, i.e. chemical ionization, electron impact, electrospray, API, etc. Utilizes various analyzers such as time-of-flight, quadrupole, ion trap, or a combination of both, to move the ions from the region where they are created, to a detector, where they produce an amplified signal. Can detect down to low attomoles of substances.

3 TIER 1: Tier 1: Two MS based systems: Hamilton Sundstrand CB Mass Spectrometer (HSMS) Single Particle Aerosol Mass Spectrometer (SPAMS)

4 Hamilton Sundstrand Mass Spectrometer (HSMS): Uses liquid expendable. The liquid reagent tetramethylammoniumhydroxide (TMAH) is deposited on the aerosol spot. It is heated (pyrolyzed) in a ballistic fashion to about o C within 14 seconds

5 Hamilton Sundstrand CB Mass Spectrometer derivatized fatty acids (FAMES). Vibration tested Humvee. Vacuum system operates at about 35 W. 5.8 cubic feet and average of 500 W power. Two stage virtual impaction: 300 L/min to 1 L/min with 50-90% efficiency (2-10 microns). The 330 L/min aerosol input flow rate with a 2 minute sampling time and 50 agent containing particles per liter of air (ACPLA) equates to 33,000 particles collected which produces a S/N = 5. This is laboratory data.

6 Hamilton Sundstrand CB Mass Spectrometer Outdoor Joint Field Trials-6 (JFT-6) at Defence Research Establishment Suffield (DRES), Medicine Hat, Alberta, Canada during August 2000 had Bacillus globigii (BG) spore and Erwinia herbicola (EH) aerosol releases at the 30 ACPLA level. S/N of the JFT-6 bacterial FAME spectra were between S/N = No real quantitative figures of merit have been done with respect to false positive (FP) and probability of FP (Pfp).

7 Bioaerosol Mass Spectrometer (BAMS): No liquid expendable. Much of the success is due to the multivariate data analysis methods that delineate the simultaneously captured positive and negative ion mass spectra, because two separate TOF-MS tubes emanate 180 o from the ion source. Typical mass spectra for a BG spore showed positive ions < 150 Da and negative ions < 200 Da.

8 Single Particle Aerosol MS (SPAMS) 10,000 particles per second. Prior to the MS system: The particle s position and velocity predicted when it passes through laser beam regions of the instrument. After the sizing region, the particle is interrogated by a laser-induced fluorescence (LIF) region to determine the presence of UV fluorescence which indicates biological nature.

9 Single Particle Aerosol MS (SPAMS) San Francisco, CA airport (SFO). Approximately one million particles were tracked and recorded over a 7 week period. NO FALSE NEGATIVES.

10 Single Particle Aerosol MS (SPAMS) Disadvantages: Very large size and high cost. Operating costs are very low. A comprehensive set of ROC curves have been performed to detail the Pfp and detection of false negatives (Pfn) figures of merit.

11 TIER 2: Tier 2 systems cannot begin to address the DHS analytical figures of merit. Current systems have not addressed any final or mature numbers because of the very preliminary designs of the basic research laboratory apparatus. BUT, there is promise that if appropriate engineering is applied, a robust system may be attained for autonomous outdoor and field testing.

12 Microfluidics Sample Handling Microfluidics handles liquid in the µm-nm volumes, concentrate pathogens in a relatively small amount of liquid. Microfluidics is economical with much less reagent consumption, suitable for a large scale deployment, and field application.

13 Micro-Total Analysis Systems (utas): Sample Refinement and Processing Characterized by a microfabricated lab-on-achip and pressurized to allow the flow of liquid through the system. Biochips have been manufactured to include micro-lc separation columns and electrospray ionization (ESI).

14 Proteomics-on-a-chip: Microfluidic Proteomic Reactor Performance The protein sample is digested efficiently in small volume well (10-50 µl) Proteomic reactor on a polymeric chip does not contaminate the HPLC-ESI-MS-MS with residual chemical from the interferences and enzyme cleavage residues.

15 Genome to--proteome An organism s genome is transformed into its theoretical proteome or protein equivalent. Theoretical proteins are translated into theoretical peptides. Experimental proteins are cleaved into peptides and identified by LC-ESI-MS-MS. Experimentally identified peptides are then matched to the complete theoretical list of the entire bacterial database.

16 One-pot Bacteria Mixture Purification Analyzed by LC-MS-MS with ABOID algorithm, patented by ECBC. ABOID compares the experimental unique peptides to a constructed proteome database of bacterial genus, species, and strains entries using taxonomic classification. ABOID showed capability of non-restrictive identification of known, emerging, and mixture of microbes in real world samples in various double blind tests LOD of bacteria and viruses varied from 10E7 to 3.3 x 10E3 cfu/ml.

17 ID of Double Blind Microbial Mixtures by ABOID Exp. Actual

18 Bacterial Mixture ID by ABOID

19 Genus Level Identification of Unsequenced and Emerging Biothreats Suppose bacteria is not resident in the database. Can still identify to the next highest taxonomic level. No false positives were observed for any of the blind samples that were analyzed, including the blank sample.

20 Advantages of Proteomics-MS No prior information about the sample is required for analysis. No specific reagents are needed in the analysis process Can classify an organism when a primer/probe set is not available Proteomics MS can provide a presumptive identification of a true unknown organism by mapping its phylogenetic relationship with other, known pathogens.

21 TIER 3: Beyond 2020 There is a market gap in this field, although there are speculations about the renaissance of commercial techniques, based on nucleic acid sequencing, since the Abbott PLEX-ID product was discontinued in September 2012.

22 TIER 3 Interface PCR with MS Interface antigen-antibody with MS.