nanoparticles for the rapid detection of Staphylococcus aureus - - PowerPoint PPT Presentation

Aptamer-functionalized gold nanoparticles for the rapid detection

• f Staphylococcus aureus

Riquelme, M.V., Leng, W., Pruden, A., Vikesland, P.

Department of Civil and Environmental Engineering Virginia Tech, Blacksburg, VA.

Staph loves humans!

• Carried by ~30% of healthy individuals
• Nosocomial and community acquired infections
• 40-60% resistant to multiple antibiotics (MRSA)
• Current detection methods are culture dependent

Annals of Clinical Microbiology and Antimicrobials 2006, 5:2 doi:10.1186/1476-0711-5-2

Environmental reservoirs of S. aureus and MRSA:

– Public surfaces (Currency, public transport, etc.) – Food and food environments – Animals/wildlife – Beaches – Drinking water supplies worldwide – Wastewater

Need fast, simple, reliable detection mechanisms

Staph in the environment

• Detect environmental pathogens and contaminants of concern
• Minimize sample volume, target concentration, and detection time
• Increase efficiency, sensitivity
• Enable in vivo and in situ detection
• Minimize sample preparation and perturbation required
• Decrease material use and waste volumes

Detection system:

• Sensor: Aptamer + Raman active molecule
• Transducer: Raman Spectroscopy

Nanosensors

Aptamer Raman spectroscopy Raman-active molecule

Benefits and sustainability:

Aptamers

Nature Protocols 5, 1169 – 1185 doi:10.1038/nprot.2010.66

• Nucleic acid molecules

that have been selectively enriched based on their binding affinity to a particular target (e.g., bacterial cell, virus, molecule, etc.)

• Enrichment process:

Systematic Evolution of Ligands by Exponential Enrichment (SELEX)

Cao et al. 2008

Raman & SERS

Lou et al., 2011 – DOI: 10.1007/s00216-011-5067-3 http://www.doitpoms.ac.uk/tlplib/raman/method.php

• Rule & Vileskand, 2009 - DOI: 10.1021/es801531t

Colorimetric detection

(red-shift of surface plasmons)

• SERS-based

detection

Pathogen detectio n ARG detection

Aggregation-based target detection

200 nm

Rule & Vileskand, 2009 - DOI: 10.1021/es801531t

Apt-AuNP functionalization

Target detection assay

Bacterial culture (S. aureus or negative controls) Apt-AuNPs Mixture incubated @ 37 oC for 1 hr

• S. aureus-specific detection

• S. aureus-specific detection
• S. aureus + Sa43_AuNPs
• S. intermedius + Sa31_AuNPs

• S. aureus quantitation

S . a u r eu s S .ep i d e r m i di s

• E. faecium

E.coli P . a e r u gin

• s

a control 500 1000 1500 2000 2500 3000 3500

• S. aureus
• S. epidermidis

100 1k 10k

Counts 30,000 cells 300 cells

3000 cells

Counts

Different Apt-AuNPs…

Acknowledgements