Adaptive Focused Acoustics

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Adaptive Focused Acoustics
Adaptive Focused Acoustics for Improved Sample
Preparation Sample Control Sample Processing
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Overview

• Current Issues
• Technology
• Disruption homogenization
• Dissociation extraction
• Dissolution solution control
• Examples data
• Summary

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Current Issues
Reproducibility Recovery Scale low /
high Contamination Ease of use
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Analytical chemistry - analogy

• Distribution of Errors Generated During Sample
  Analysis
• R.E. Majors, An Overview of Sample Preparation
  LC-GC, vol. 9, No. 1, 1991

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Current processes

• Mortar/pestle, bead beater
• Size limit e.g., 100 micron size limit with
  mortar/pestle
• Poor recovery
• Autogizer blender
• Contact
• Sonicator
• Low frequency/unfocused leads to standing waves gt
  hot spots
• Not reproducible
• Contact

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Sonics
Sonicator wave length 10cm
Acoustic wave length 1mm Operates at 200 - 800khz
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Technology
Non contact Closed tube Isothermal Evolved out
of the Lithotripsy kidney stone disruption Very
robust and industrialised Previously only used in
RNA extraction Rosetta etc.
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Video natural product
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Video instrumentation
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Process disruption
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Skeletal muscle larger mass
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Disruption mechanical recovery

• Tomtec Autogizer (polytron), then analyzed by
  LC/MS/MS quantitation for extracted small
  molecule drug and normalized for comparison.
• Tissue masses 100-150mg
• triplicates
• Similar for brain and liver
• Higher for skeletal and cardiac

acoustic
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Small molecule recovery

• Tissue extraction from in vivo animal dose
• Higher Covaris process vs. a rotary polytron
  process
• C14 tagged molecule
• Total CPM 100 is supernatant and pellet.
• Marked improvement in precision with Covaris at
  level of the assay-based error?

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Automated pulverization
Hard, fibrous, or large mass tissue

• Impact level
• Variable, controlled
• One-button activation
• Safety-interface on sample lid
• Safety interlocks
• Closed impact zone
• Sound attenuation

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CryoPrep video
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Solution control enzyme reaction

• BSA 0.5 ug/ul trypsin digest
• 200 ul
• NUNC 96 pp
• 50 mM HEPES, 2 nM CaCl, pH 8.0
• Point transducer vs. line transducer systems

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Metabolic profiling

• CONTROL
• Yeast w/o Covaris 100 methanol 70C
  extraction internal standard at 28.47

• COVARIS
• Yeast stationary phase extracted with Covaris,
  followed by methanol 50C extraction internal
  standard at 28.32

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RBC infected with Plasmodium

• dissolution following Covaris process
• good reproducibility of Covaris and GC-MS

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Covaris

• Overview
• Data
• Disruption - homogenization
• Dissociation extraction
• Dissolution solution control
• Summary

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Dissolution serum proteins

• Ciphergen - Mass spec ProteinChip
• Number of known peaks
• Control 33
• Covaris 44
• Intensity
• Covaris gt control
• Covaris
• _at_ 5degC _at_ pH 8.0
• pH 6.0 gt no effect
• more complexity and higher recovery

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Solution control dissolution

• L-Tyrosine
• Powder 1.5 mg
• Distilled water 2 ml
• Temperature
• Covaris 25 /- 1 oC
• Stir bar 27 /- 1 oC
• Time course at constant energy
• Covaris 3 runs
• Control stir bar 2 runs
• Treat gt filter through 0.45um gt dilute gt read ABS
  _at_ 280nm

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Solution control dissolution

• Clozapine
• DMSO lyophilized pellet
• P-propylene 96 well plate
• 50 MeOH / 50 water
• Time course at constant energy
• LC integrated peak area analysis
• Time
• 2.5 seconds of Covaris energy better than 180
  seconds of microplate sonicator energy

Covaris process
Microplate sonicator
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Conclusion

• AFA
• Enables
• High Quality
• Non Contact
• Isothermal
• Reproducible
• Closed vessel
• Sample Prep

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Application areas

• High-throughput screening
• Cell Lysis Mammalian, Insect, E-coli, plant
• Compound Dissolution and Re-solution
• Continuous flow processing
• High Throughput Chemistry
• Drug Metabolism and Pharmacokinetics
• RNA extraction - Homogenisation of biological
  tissue
• Tissue homogenisation for Proteomic studies

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Questions
Thanks