development of a point-of-care rapid and sensitive

© 2012 Fraunhofer CMI

Development of a point-of-care rapid and sensitive bacteremia diagnostic

Alexis Sauer-Budge, PhD

Email: [email protected]

Anna Boardman, PhD – poster #44


Fraunhofer is Europe’s Largest Applied R&D Organization

n  20,000 Employees Worldwide

n  €1.8+ Billion in Annual Projects

n  Non-Profit Organization

n  60+ Locations

n  Founded 1949


Fraunhofer Center for Manufacturing Innovation (CMI): Next Generation, High-Precision Automation Systems

Biotechnology

Semiconductor

Photonics

Other High Precision Applications


Current Antibiotic Susceptibility Methods: Traditional n  Culture-based methods

–  Pros §  Gold standard §  Minimal sample prep §  Phenotypic method (doesn’t require known genomic

markers or known antibiotic mechanisms of action) §  Sensitive to emerging strains

–  Cons §  Too long requiring physicians to treat without

identification or susceptibility information §  Requires growth à long times §  May not be sensitive to fastidious organisms


Our Approach n  Develop new tests that do not rely on growth or

nucleic acid amplification to identify antibiotic susceptibility

–  Rapid enough to inform initial drug treatment –  Automatable, including sample preparation –  Extendable to emerging threats –  Phenotypic

n  Surface Enhanced Raman Spectroscopy/Microscopy (Poster #44)

n  Microfluidic platform for stress-induced antibiotic susceptibility (Poster #42)


SEMs of the in-situ grown Au nanoparticle covered SERS (SiO2) substrate: a metal ion doped sol-gel procedure

←" ~500 nm "→

x10

Clusters of 1 – 15 ~80 nm Au particles are evident on the surface of the SiO2

SERS substrate.

•  Enhancement factor @ 785 nm for glycine is ~5 x 107

•  substrate shelf life is 5 months (important for point-of-care and portability use)

A scanning electron micrograph of a two-cell chain of B. anthracis Sterne on a Au nano-particle covered SiO2 SERS chip. Note the relative size scales of the bacterium and the nano-structured surface roughness.

Dr. Ranjith Premasiri, Prof. Larry Ziegler et al. J Phys Chem B. 2005;109(1):312-20.


SERS spectra of some bacterial species (5 genera)

n  Each species exhibits unique vibrational “fingerprint” w/ excellent S/N

n  No features >1700 cm-1

n  Species arranged top-to bottom by phylogenic proximity - only moderate correlation w/ this lineage seen.

n  G(+) vs.G(–) diff. not obvious n  However, regions of

homology: BC, BA, BT 1250 -1650 cm-1

n  Other common spectral features: 735, 965, 1030, 1320 cm-1

Single scans; 2mW @ 785 nm; 10 sec collection Renishaw Raman microscope (RM-2000); 10 - 30 cells

G(+) G(+) G(+) G(+) G(-) G(-) G(-)

Dr. Ranjith Premasiri, Prof. Larry Ziegler et al. J Phys Chem B. 2005;109(1):312-20.


Major Challenge : Sample Preparation for SERS Detection n  How do we get ~100 CFU concentrated and isolated from billions of blood cells, and

deposited onto SERS substrate??

n  Cell ratio analogy – 1 person in Texas (1:20,000,000)

n  Volume ratio analogy – Jupiter (1E15 km3) to Mercury (6E10 km3) (5 orders of magnitude)


System Overview : How can we develop a stat test for bacteremia?

10 mL Bacteremic

Blood

Micro-evaporator

Bacterial Concentrator

Portable SERS Instrument

Portable SERS instrument

Lee Sauer-Budge Klapperich Ziegler


Introduction To Bacterial Adhesion n  Bacteria prefer to grow on solid surfaces than in liquid

n  Adhesion is a two phase process –  Phase One: Initial, instantaneous, and reversible physical phase

§  Physicochemical interactions dominate –  Phase Two: Time dependent, irreversible molecular and cellular phase

§  Molecular and cellular interactions §  Selective bridging of bacterial surface polymers

§  Capsules, fimbriae, pili, slime

Katsikogianni, M. and Y. F. Missirlis (2004). Deupree, S. M. and M. H. Schoenfisch (2008). Gristina, A. G. (1987).


Factors That Influence Bacterial Adhesion In Phase 1 n  Bacteria type

–  Different bacterial surface properties, adhesion molecules, lipid composition, etc.

n  Physical forces move/keep bacteria to/at surface

–  Brownian motion –  Van der Waals attraction –  Gravitational forces –  Surface electrostatic charge effects –  Hydrophobic interactions

n  Long range (non-specific, >50nm, mutual forces):function of distance and free energy

n  Short-range interactions (<5nm) –  chemical bonds (e.g. hydrogen bonding) –  Ionic/dipole interactions –  Hydrophobic interactions

n  Bacterial movement –  Chemotaxis (concentration gradients of

chemical factors)/haptotaxis (surface bound)

Katsikogianni, M. and Y. F. Missirlis (2004). Gottenbos, B., H. J. Busscher, et al. (2002).


Material Surface Characteristics n  Chemical composition/Hydrophobicity

–  Hydrophilic surfaces that entrain water molecules resist non-specific binding

n  Surface charge – Most bacteria carry a slight negative

charge, thus negatively charged surfaces are best

–  Influenced by solution properties (pH, ionic strength, blocking agents, etc.)

n  Surface roughness –  Rough surfaces can trap bacteria –  Edwards paper: Calculated highest

adhesion to irregularities that conform to the bacteria’s size (here 0.32um bacteria radius)

–  Shape of irregularity contributes as well

Katsikogianni, M. and Y. F. Missirlis (2004).

Edwards, K. J. and A. D. Rutenberg (2001).


Material Surface Characteristics: Surface Roughness Shape n  Shape of surface

topography can interact differently with different types/shapes of bacteria

n  Measured by AFM

S. aureus P. aeruginosa

Whitehead, K. A., D. Rogers, et al. (2006).


What does this mean for us? n  Keeping in mind that we need to be able to isolate a variety of types of

bacteria (which may have different surface properties) and that we cannot control the variations in blood (hemocrit, fat content, etc.), we must focus on what we can control and make compromises

n  Material surface properties: –  Hydrophilic and negatively charged –  Ideal surface roughness <1um Ra

n  Suspending buffer –  Keep in mind blood components: cellular and soluble (plasma) –  Lyse, dilute, digest, buffer


Three Stage Prototype with a preferential lysis procedure n  The 3 stages are designed to seamlessly integrate with each other

for ease of use.

Stage 1 Stage 2 Stage 3

Anna Boardman, Sandy Allison, Holger Wirz, Doug Foss, Alexis Sauer-Budge


Processing steps



Percent Recovery Summary by Input Concentration


n  Data not released for print


Optimizing performance for different bacteria n  Data not released for print



SERS spectra of S. aureus from infected rats after processing n  Data not released for print

Anna Boardman, Sandy Allison, Alexis Sauer-Budge, Jean Lee, Linhui Wang, Ranjith Premasiri, Larry Ziegler


Summary n  Concentration and purification of

bacteria from initial sample is a challenging problem that must be addressed for rapid downstream diagnostics

n  Our approach combines preferential lysis of mammalian cells with a series of centrifugation steps

n  Device design, surface properties, and solution chemistry are key parameters to control


Acknowledgements n  Fraunhofer Bacterial

Concentrator Team – Dr. Anna Boardman – Sandy Allison – Holger Wirz – Doug Foss – Prof Andre Sharon – Bettina Sabban – Felix Schmid – Jasmine Loeder – Michael Zeiss – Ulrich Schlegel

n  Boston University SERS – Prof Larry Ziegler – Dr. Ranjith Premasiri

n  Boston University Microfluidic Evaporator

–  Prof Catherine Klapperich –  Jared Saffie

n  Brigham and Women’s Hospital/ HMS Animal Models

–  Prof Jean Lee –  Dr. Linhui Wang

n  Funding sources: – BU-Fraunhofer Alliance for

Medical Devices, Instrumentation, and Diagnostics

– NIH NIAID R01AI090815

development of a point-of-care rapid and sensitive

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