"... we were able to successfully construct the SPR imaging device and acquire both an SPR and fluorescent molecular image of similar regions of a bacterial sample on the biosensor."

Peptide array biosensor for high throughput and multiplexed detection of foodborne pathogens

Investigators: Kinam Park, James F. Leary

Project Report 2007 - 2008

» Download Project Report 2007 - 2008

Project Rationale

The increased incidence of pathogen-contaminated food places a new emphasis on the rapid detection and quantification of foodborne pathogens. Therefore, we are developing a surface plasmon resonance (SPR) imaging biosensor for the rapid, label-free, and high throughput detection of foodborne pathogens. This device integrates an SPR imaging system with a biosensor array immobilized onto a sample surface containing specific short peptide ligands. A group of short peptides specific to certain pathogenic bacteria will be microcontact-printed on a gold chip in linear patterns. This peptide-imprinted gold chip functions as a biosensor array for the specific detection of unknown foodborne pathogens. To determine what fraction of pathogenic bacteria are live or dead and to confirm the SPR results, we have created a novel hybrid SPR/molecular imaging portable system.

The device would offer a commercial advantage to the food processing industry. It is miniaturized, has fewer components, and is easier to use compared to the current detection systems. This biosensor would detect foodborne pathogens present in <100 CFU/g of contaminated food within ten minutes.

Project Objectives

  • Synthesize and characterize peptides.
  • Fabricate and characterize a peptide biosensor array and microfluidic flow cell.
  • Design and assemble a compact SPR imaging device.
  • Utilize SPR imaging for real-time detection of foodborne pathogens.
  • Optimize the device for high throughput and multiplexed detection.

Project Highlights

We constructed a SPR imaging device using the Kretschmann configuration. The main components of this device are a light source, polarizer, a prism, and a detector. A diode laser with a coupled beam expander is our light source. The incident light is t-polarized using a rotating polarizer. A 60°-60°-60° BK7 prism is mounted on a goiniometer, while a BK7 coverslide with a 4x4 array of 50 nm thick gold dots is placed on the top surface of the prism. The purpose of the prism is to cause total internal reflection that will create the evanescent field necessary for surface plasmon resonance. To capture the reflectance image, a charge-coupled device (CCD) camera is fitted with a long working distance 4X microscope objective. The CCD is connected to a laptop computer and PixelScope Professional™ software is used to capture the image from the CCD.

Next we captured the SPR image of a fluorescent sample and validated the SPR image using fluorescence microscopy. To do this we stained E. coli O157:H7 cultures with the LIVE/DEAD BacLight™ Bacterial Viability Kit. The fluorescently labeled E. coli was deposited onto a sensor chip containing a 50 nm thick layer of gold that had been functionalized with an antibody specific to E. coli O157:H7. The bacterial sample was allowed to air dry and SPR images were taken immediately afterward. The slide was then imaged fluorescently using an inverted Nikon Diaphot fluorescent microscope fitted with the appropriate optical filters. We fabricated a microfluidic cell by pouring liquid PDMS onto a stereolithographically fabricated photoresist template and then peeling off the PDMS mold. The PDMS flow cell was placed on top of the peptide functionalized gold chip and placed on the SPR prism. The capture ligand patterns were generated by microcontact printing.

Our most important accomplishment is that we were able to successfully construct the SPR imaging device and acquire both an SPR and fluorescent molecular image of similar regions of a bacterial sample on the biosensor.

Annual Reports

Investigators

  • James Leary
  • Kinam Park