The overall goal of the following experiment is to design a label free optical biosensor for rapid bacteria detection based on a nano structured porous silicon. This is achieved by constructing oxidized porous silicon to be used as the optical transducer element of the biosensor. As a second step specific capture probes, such as antibodies are immobilized onto the porous surface to provide the active component of the biosensor.
Next, the biosensor is exposed to the target bacteria in order to directly capture the bacteria cells onto the antibody. Modified oxidized porous silicon surface results are obtained that show intensity changes in the thin film optical interference spectrum of the biosensor. Due to bacteria capture light microscopy is used in order to confirm the presence of the bacteria on the biosensor surface.
The main advantage of a, our technique over existing methods like conventional culture and techniques, is that our method detects the presence of bacteria in a very rapid manner in less than an hour. This method can help answer key challenges in environmental monitoring of specific microorganisms, as well as ensuring water and food safety. It also allows detection of specific bacteria species in real time and in point of care settings To begin etch silicon wafers in a three to one solution of aqueous hydrogen fluoride and ethanol for 30 seconds at a constant current density of 385 milliamps per square centimeter.
Please note that hydrogen fluoride is a highly corrosive liquid and it should be handled with extreme care. Rinse the surface of the resulting poor silicon film with ethanol several times. Then dry the films under a dry nitrogen gas next oxidize the freshly etched porous silicon samples in a tube furnace at 800 degrees Celsius for one hour in ambient air.
To bio functionalize the oxidized porous silicon sample first incubate the film for one hour and 95%MPTS solution. After rinsing with toluene, methanol and acetone, dry the sine treated oxidized porous silicon sample under a dry nitrogen gas. Next, incubate the sine modified sample in one milliliter of 100 millimolar PEOI oto acetyl biotin solution for 30 minutes.
Rinse the resulting biotin treated sample with 0.1 molar phosphate buffered saline or PBS solution several times. Next, incubate the sample for 30 minutes in one milliliter of 100 micrograms per milliliter strep evidence solution before rinsing it again with the PBS solution several times. Then incubate the streptavidin modified oxidized porous silicon sample for 30 minutes in one milliliter of 100 micrograms per milliliter, biotinylated e coli, monoclonal IgG antibody, or with 100 micrograms per milliliter.
Biotinylated rabbit IgG to fluorescently labeled the scaffold. Incubate the IgG modified surfaces with 15 micrograms per milliliter. Fluorescein tagged anti rabbit IgG for 40 minutes.
Incubate with 15 micrograms per milliliter. Fluorescein tagged anti-US IgG as a control. Examine the samples under a fluorescence microscope after rinsing the modified samples with the PBS solution several times.
Cultivate e coli K 12 bacteria in a 10 milliliter tube with five milliliters of luria britani or LB medium. Incubate the bacteria overnight shaking at 37 degrees Celsius after overnight growth. In LB medium, read the optical density at a wavelength of 600 nanometers or OD 600 using a spectrophotometer to determine the bacterial concentration.
The number of cells is directly proportional to the OD 600 measurement.Place. The IgG modified oxidized porous silicon scaffolds and the control. Neat oxidized porous silicon samples in a custom made plexiglass flow cell.
Fix the flow cell to ensure that the sample reflectivity is measured at the same spot during all the measurements. Incubate the samples with the Coli K 12 suspension for 30 minutes at room temperature. Then remove the bacteria suspension by flushing the cell with saline, 0.85%weight per volume for 30 minutes.
Monitor the changes in the reflectivity data throughout the experiment on A CCD spectrometer. Carry out all optical measurements in an aqueous surrounding after collecting spectra on A CCD spectrometer analyzed by applying fast four ear transform. Finally, confirm the presence of the bacteria on the biosensor surface by observation of the samples under an upright light microscope immediately after the biosensing experiment.
Shown here is a high resolution scanning electron micrograph of the porous silicon films after thermal oxidation. The oxidized porous silicon layer is characterized by well-defined cylindrical pores with a diameter in the range of 30 to 80 nanometers. The attachment of the antibodies to the oxidized porous silicon surface is confirmed by fluorescent labeling, followed by observation of the surface under a fluorescence microscope.
In addition, fluorescent studies allow characterization of the activity and antigenic specificity of the surface immobilized antibodies by binding of fluorescently tagged anti rabbit IgG and anti mouse IgG as a control. Also shown is the control experiment with no IgG to demonstrate bacteria biosensing changes in the amplitude or intensity of the light reflected from the oxidized porous silicon nanostructure are monitored a fast for your transform spectrum of the biosensor before and after the introduction of the e coli bacteria is shown here Following exposure to e coli bacteria. An intensity decrease of seven plus or minus 1%is recorded while insignificant changes are observed for the unmodified oxidized porous silicon surface.
Moreover, in order to confirm the presence of captured cells onto the oxidized porous silicon surface, the biosensors are observed under a light microscope immediately after the completion of the biosensing experiment displayed. Here are immobilized bacteria cells on the biosensor surface. While no cells are observed on the unmodified control surfaces Once mastered, this technique can be done in less than an hour if performed properly.
After watching this video, you should have a good understanding of how to plan a specific optical bio sensor for monitoring a target analyte.
