Optical Detection of Foodborne Bacterial Pathogens using Nanosensors

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Characterisation of Nanoparticle Conjugates

Nanoparticles were functionalised with antibodies and lectins to allow for binding to foodborne bacterial pathogens. Belowis a list of the nanoparticle conjugates which were prepared and characterised:

1)Silver nanoparticles (AgNP) functionalised with a Raman reporter, PEG635 and antibody (Ab)

-AgNP + MGITC + PEG635 + E. Coli Ab

-AgNP + DACITC + PEG635 + S. Typh Ab

-AgNP + PPY + PEG635 + MRSA Ab

-AgNP + FITC + PEG635 + MSSA Ab

-Antibodies used:

  • E. Coli Ab - Escherichia coli Ab
  • S. Typh Ab – Salmonella typhimurium Ab
  • MRSA Ab - Methicillin-resistant Staphylococcus aureus Ab
  • MSSA Ab - Methicillin-sensitive Staphylococcus aureus Ab

-Raman reporters used:

  • MGITC – Malachite Green isothiocyanate
  • DACITC – Dimethylamino-methylcoumariisothiocyanate
  • PPY – Pyrazolpyridine
  • FITC – Fluorescein isothiocyanate

Stages of optimisation with associated data:

i)Raman reporter concentration studies

ii)PEG635 concentration study

iii)EDC/sNHS conditions tested – cross-coupling chemistry for functionalising Ab to AgNP

iv)Ab concentration study (S. Typh used)

v)AgNP – Ab Optimised Conditions

The AgNPconjugates were characterised using Extinction Spectroscopy, size and zeta potential and SERS analysis. The SERS analysis was conducted using a Snowy Range Instrument with a 532 nm laser excitation. The SERS spectra were obtained in the .spc format. Spectra were acquired using Peak (version V.2.1) software. The data was pre-processed using Grams AI software (version 7.0), before being exported to Excel were it was processed.The Extinction Spectroscopy, size and zeta potential data was processed in Excel.

2)Silver coated magnetic nanoparticles (Ag@MNP) functionalised with PEG5000 and a lectin

-Ag@MNP + PEG5000 + Con A

-Lectin used:

  • Concanavalin A

Stages of optimisation with associated data:

i)PEG5000 concentration study

ii)Con A concentration study

iii)Ag@MNP – Con A Optimised Conditions

The Ag@MNP conjugates were characterised using Extinction Spectroscopy, size and zeta potential. The Extinction Spectroscopy, size and zeta potential data was processed in Excel.

Fixed Bacterial Imaging – Renishaw Instrument with 633 nm laser excitation

Single bacterial imaging and mapping was conducted to ensure the NP conjugates were binding efficiently to the surface of the bacteria. Belowis a list of the bacterial pathogens plus the nanoparticle conjugates which were characterised:

-E. Coli Bacteria + E. Coli Conjugates

-E. Coli Bacteria + S. Typh Conjugates (cross reactivity study)

-S. Typh Bacteria + S. Typh Conjugates

-S. Ent Bacteria + S. Ent Conjugates

-The conjugates consist of:

  • AgNP + Raman reporter + PEG635 + Ab (Ab is specific for bacterial strain)
  • Ag@MNP + PEG5000 + Con A

Dark field images were obtained in the .jpg format. The maps were acquired and pre-processed using Renishaw WIRE (version 4.2) software. The spectra were obtained in the .wdf or .spc format. After pre-processing the data was exported to Excel to enable the Raman spectra to be processed.

Solution Assay - Renishaw Instrument with 532 nm laser excitation

The SERS assay was performed initially with single bacterial pathogen detection for each of the bacterial strains individually but then additional experiments were conducted to investigate the multiplexing capabilities of this assay. In addition, an E. Coli concentration study and cross reactivity experiments wereconducted. Below is a list of the experiments with assay specific information:

-Single Pathogen Detection Assays with 10 million and 100,000 colony forming units (CFUs):

  • E. Coli Bacteria + E. Coli Conjugates
  • S. Typh Bacteria + S. Typh Conjugates
  • MRSA Bacteria + MRSA Conjugates
  • MSSA Bacteria + MSSA Conjugates

-Cross-Reactivity Assays with 100,000 colony forming units (CFUs):

  • E. Coli Bacteria + S. Typh Conjugates
  • E. Coli Bacteria + S. Aureus Conjugates
  • S. Typh Bacteria + E. Coli Conjugates
  • S. Typh Bacteria + S. Aureus Conjugates
  • MRSA Bacteria + S. Typh Conjugates
  • MRSA Bacteria + E. Coli Conjugates

-E. Coli – CFU Concentration Study:

  • E. Coli Bacteria (various concentrations) + E. Coli Conjugates
  • Various concentrations used : 10 million to 10 CFUs

-Multiple Pathogen Detection Assays with 1000 colony forming units (CFUs):

  • Duplex:
  • E. Coli and S. TyphBacteria + E. Coli Conjugates and S. Typh Conjugates
  • E. Coli and MRSA Bacteria + E. Coli Conjugates and S. Aureus Conjugates
  • S. Typh and MRSA Bacteria + S. Typh Conjugates and S. Aureus Conjugates
  • Triplex:
  • E. Coli, S. Typh and MRSA Bacteria + All Conjugates (E. Coli, S. Typh and S. Aureus conjugates)

-The conjugates consist of:

  • AgNP + Raman reporter + PEG635 + Ab (Ab is specific for bacterial strain)
  • Ag@MNP + PEG5000 + Con A

The spectra were acquiredusing Renishaw WIRE (version 4.2) software. The spectra were obtained in the .wdf or .spc format. The data was pre-processed using Grams AI software (version 7.0), before being exported to Excel were it was processed and then transferred to Powerpoint for comparison.