Miniature Ambient Ionization Mass Spectrometry System For Analysis of Microorganisms

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1 Miniature Ambient Ionization Mass Spectrometry System For Analysis of Microorganisms R. Graham Cooks 1 and Zheng Ouyang 2 1 Department of Chemistry and 2 Weldon School of Biomedical Engineering Purdue University, West Lafayette, IN 4796

2 Mass Spectrometry Analysis Instrument Systems and Analytical Procedures Procedure Instrument Automation Sample Prep Varies significantly, dependent on the sample matrix and target analyst Multiple devices Poor GC/LC Relativestandard, analyte dependent Integrated system/expensive/ Large Highly automated MS Highly standard,analyte dependent Integrated system/expensive/ Large Highly automated MScanbesmall Example of a handheld Mini 11 mass spectrometer Purdue University L. Gaoet al. Anal. Chem. 28 doi: 1.121/ac81275x

3 Miniature Mass Spectrometer Pumping technology Mini 11: Weight: 1 lbs Size: 1" 6" 5" Power Consumption: <W Mass Range: 8/15 Atmospheric pressure ionization Mini mass spectrometers are maturing quickly However, mass spectrometer itself does not perform analysis independently

4 Approach for Autonomous Analysis: Ambient Ionization + Mini MS Example: blood analysis Target Analysis Mini 12: Complete Analysis System W12.5 x H16 x D22.5 Weight lbs Power: 65W average Therapeutic Drug Monitoring Amitriptyline in whole blood LOQ: 7.5 ng/ml RSD: < 1% Analysis Time: < S Solvent: < 2µL

5 Possible Embodiment for Aerosol/Bacteria Analysis Collection of Aerosols Air Flow Consumable Sample Cartridge With Paper Substrate Paper Spray MS Analysis DC 4 kv Spray Solvent MS Inlet

6 Mini Ambient MS Approach: Tier 1, Tier 2 or Tier 3? Ambient Ionization Methods 24-25: two 213: More than Flowprobe LESA (Liquid Extraction Surface Analysis) Commercial Products DESI (Desorption Electrospray Ionization) DART (Direct Analysis in Real Time) LAESI (Laser Ablation Electrospray Ionization )

7 Mini Ambient MS Approach: Tier 1, Tier 2 or Tier 3? Miniature MS (-ters or systems) Many mini MS capable of analysis of gas samples Systems with Atmospheric Pressure Interfaces Mini 12 PS-MS Griffin DESI-MS Purdue University Microsaic Systems plc

8 Mini Ambient MS Approach: Tier 1, Tier 2 or Tier 3? Find biomarkers that Can be efficiently sampled and ionized by ambient ionization methods Can be used for high specificity bacteria classification/identification Lipid profiles can be used to characterize disease state Cancer margin diagnosis J. S. Wiseman, et al. Angew. Chem. Int. Ed.26 45, Tumor grade and cancer cell concentration Eberlin, L. S. et al. Cancer Res 212, Can lipid profiles be used for bacteria classification? YES, Lipid profiles have been used for microorganism ID by ambient ionization (but not the methods of TS and PS used here): Cooks, R. G., et al. Faraday Discuss.211, 149, Song, Y., et al. Chem. Commun.27, Zhang, J. I., et al. Int. J. Mass. Spectrom.211, 31,

9 Real-Time Microorganism Analysis by DESI DESI-MS of freshly harvested cells dried on PTFE In vivo recognition of Bacillus subtilis by DESI-MS (a) E. coli (b) P. aeruginosa Bacillus subtilis as a biofilm growing on agar nutrient: simple, high quality mass spectra dominated in both the positive and negative ion modes by signals due to the cyclic lipopeptide, Surfactin. J. Mass Spectrom. 25,, Analyst. 29, 134,

10 DESI-MS of Microorganisms: Statistical Analysis PCA plot and loading plot of three replicate measurements, collected in the negative ion mode over a period of 3 days of analyses and combined No derivatization, high reproducibility and in situ identification including sub-species differentiation bacteria (positive ion mode) Inter-day evaluation J. Mass Spectrom. 27, 42, PCA score plot of three strains of Escherichia coli and two strains of Salmonella typhimurium Chem. Commun. 27, 1, 61-63

11 Touch Spray for Direct Microorganism Detection Relative Abundance Citrobacter farmeri (-) TS-MS Single Scan Citrobacter farmeri (-) TS-MS 2 min average Current LOD ca. 1 4 cfu Ahmed Hamid 11

12 (-) TS-MS Relative Abundance Staphylococcus aureus Gram Positive Bacteria Citrobacter farmeri Gram Negative Bacteria Candida albicans Yeast

13 (±) TS-MS Gram Negative Bacteria Escherichia coli Relative Abundance PG PE PE dimers

14 (-) TS-MS Gram Positive Bacteria Relative Abundance PG (3:) PG (31:) PG (32:) PG (33:) PG (34:) PG (33:1) Staphylococcus aureus Staphylococcus epidermidis Staphylococcus hominis

15 (-) TS-MS Gram Positive Bacteria Staphylococcus capitis Relative Abundance Staphylococcus lugdenensis Staphylococcus saprophyticus

16 R e l a t i v e A b u n d a n c e (-) MS PG (3:1) (-) MS PS (26:) PG (32:1) PG (34:1) TS-MS Gram Negative Bacteria PG (33:1) PG (36:2) Citrobacter farmeri Citrobacter freundii R e l a t i v e A b u n d a n c e [PE(33:1)+H + ] 8[PE (32:1)+H + ] [PE (32:1)+Na + ] [PE (32:1)+Na + ] [PE(33:1)+Na + ] [PE (34:1)+Na + ] 7.5 (+) MS (+) MS PE dimers PE dimers

17 TS-MS Gram Negative Bacteria 1 (-) MS Proteus penneri (+) MS R e l a t i v e A b u n d a n c e (-) MS R e l a t i v e A b u n d a n c e Proteus vulgaris PE PE (+) MS PE dimers PE dimers

18 Positive ion Touch Spray-MS (+) Touch Spray-MS Single touch 3D PLS-DA Normalized to TIC -1 o Separation of Fungi & Bacteria o Separation between Gram Negative and Gram Positive Bacteria o Separation of Gram Negative Species Fungi- Candida Gram Negative- Proteobacteria Gram Positive- Firmicutes 18

19 R elative A bundance Transfer from Touch Spray to Paper Spray (-) Escherchia coli PG (32:1) PG (34:1) PG (3:1) PG (33:1) PG (31:1) Methanol Solvent PG (35:1) Methanol Solvent Touch Spray Single Scan (.3 s) Intensity: 5x1 4 Linear Trap Quad mass spectrometer Single touch from the colony (.1 of the colony size) to the teasing probe Paper Spray Single Scan (.3 s) Intensity: 1x1 3 Linear Trap Quad mass spectrometer Single colony to the filter paper (.2 µm pore size)

20 Relative Abundance [PE (32:1)+H + ] Transfer from Touch Spray to Paper Spray (+) Escherchia coli [PE (33:1)+H + ] [PE(32:1)+Na + ] [PE (32:1)+K + ] [PE(33:1)+Na + ] [PE(31:1)+Na + ] [PE (34:1)+Na + ] 7.5 Touch Spray Single Scan (.6s) Intensity: 4x1 4 Linear Triple Quad mass spectrometer Single touch from the colony (.1 of the colony size) to the teasing probe Methanol Solvent Paper Spray Single Scan (.6s) Intensity: 2x1 4 Linear Triple Quad mass spectrometer Single colony to the filter paper (.2 µm pore size) Methanol Solvent

21 (-) PS-MS Gram Positive Bacteria Staphylococcus capitis R elative A bundance Staphylococcus saprophyticus

22 (±) PS-MS Gram Negative Bacteria: Broth Filration 1ml of bacteria in TSB paper spray_broth_ecoli_3 # RT: AV: 32 NL: 5.12E3 F: ITMS - p ESI Full ms [.-1.] Escherichia coli (-) PS-MS (.2 µm) Relative Abundance HV Solvent Sample.2µm Paper Charged droplets with analyte To Mass Analyzer F: ITMS + p ESI Full ms [2.-2.] Relative Abundance (+) PS-MS

23 Automated Library Search and Multivariate Statistics Sample MS Data Identification Microorganism ID T: ITMS - p ESI Full ms [15.-2.] R e l a t i v e A b u n d a n c e TS Spectrum Neg. Mode E. Coli Fungi Gram Negative Microorganism ID: E. coli Gram Positive Probability Match: 99.9% Statistic Analysis Mini Ambient Ionization MS Standard MS Data

24 Miniature Ambient Ionization MS: to get ready Develop and optimization of data analysis Full characterization of bacteria analysis using ambient ionization Sampling ionization condition Matrix effects Multiple microorganisms in same sample Detection limits Development of mini ambient MS 24

25 Acknowledgements Data and interpretation Ahmed Hamed and Alan Jarmusch Funding from Institut biomèrieux, Lyon, France Collaboration and technical assistance Gaspard Gervasi, biomerieux(marcy l Etoile, France) Samples and technical assistance from David Pincus, biomerieux (St Louis, MO)

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