A Fibreoptic Biosensor for Detection of Microbial Contamination

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A Fibre-optic Biosensor for Detection of
Microbial Contamination

• Reviewed by David Jason Leggat

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Get to the Point!

• A review of the fundamental design criteria for
  development of nucleic acid biosensors.
• Reports preliminary exploration of the use of
  biosensors for the detection of microbial
  contamination.
• Identifies specific genetic sequences that denote
  specific microbial presence like E. coli.

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Why Do Such a Thing?

• Methods for identifying specific bacteria
  contamination have great potential
• Useful with quality control of foodstuff, water
  contamination, human infections, etc.
• Classical methods of nucleic acid hybridization
  assays are too time consuming
• Many labs require faster methods for practical
  results

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What We Want

• An assay that is an improvement to those
  currently used.
• Reversible
• Reusable
• More Sensitive (requires small quantities)
• More Selective (identifies specific organism)
• And Still Easy to Use

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Considerations

• Environmental effects on the binding capacity of
  immobilized selective molecular recognition.

• Probe density and length.

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Immobilizing Probes

• Immobilization of oligonucleotides.
• Fused to silica optical fibre substrates
• Using a silane reagent to modify the optical
  fibre substrates.
• Followed by oligonucleotide synthesis protocols.
• Allowed synthesis of oligonucleotides at
  controlled densities of substrate surface.

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Interfacial environments vs. bulk solution

• Interfacial environments
• Show 2-4 times lower melting temperature
• Improved selectivity of hybridization

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Optimizing fibre-optic biosensor analytical
function

• The Densities of the immobilized ssDNA can affect
  selectivity.
• The accidental formation of fully complementary
  dsDNA before hybridization.
• Controlling ssDNA density, solution ionic
  strength, and temperature improves selectivity by
  a power of two compared to bulk solutions.

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Hybridization Percentage

• Bulk solution yields a maximum 30 of fully
  matched dsDNA.
• Immobilized ssDNA system yields a maximum 55 of
  fully matched dsDNA.
• The presence of genomic DNA did not significantly
  affect the rate or percentage of hybridization
  experiments.

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Classical Methods of Detection

• Coliforms can be indicators of bacterial
  pathogenic contamination.
• PCR methods use LacZ, lamB, and uid genes as
  targets for primers for coliform detection.
• Can yield false results
• Can require several hours to examine

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A fibre-optic biosensor for E. coli

• Detects oligonucleotides to identify E. coli
• 25mer sequence of LacZ gene.
• ssDNA is fused to silica optical fibre.
• Hybridization detection by fluorescent
  intercalating dye (ethidium bromide).
• Can detect very small amounts of cDNA and/or
  genomic DNA
• Analysis requires less than 1 min. sensor is
  reusable.

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Future Desire

• Development of a self-contained biosensor
• Uses attachment of intercalating fluorescent dye
  to probe by molecular tether.
• Dye SYBR 101
• Reduces background fluorescent
• Allows internal standardization
• Reduces toxic exposure to operator

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Optic-fibre Spectrofluorimeter

• A special spectrometer used to measure
  fluorescent dye SYBR 101
• Modified with a fluid handling system

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How Its Done!

• (Procedure)

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Attachment of SYBR 101 to ssDNA probe

• The ssDNA is first attached to a linker.
• The ssDNA-linker is then attached to the SYBR 101
  dye.

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Preparation for Hybridization

• Optical fibres prepared (p.342)
• Linkage of DMT-HEG to ssDNA-linker-dye substrates
  (p.343)
• Synthesis of oligonucleotides (p.343)
• PE-ABI 391-EP DNA synthesizer
• Teflon synthesis column

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E. Coli and Salmon Sperm

• E. coli and salmon sperm DNA was isolated,
  purified, and concentrated.
• E. coli DNA
• Desired experimental DNA to test hybridization
• Salmon sperm DNA
• Used as non-complementary DNA to test specific
  hybridization

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Hybridization

• Sensors are cleaned to remove contaminants
• Sensors are activated
• Three cycles of thermal denaturaton and
  reanneling
• Exposure to cDNA in PBS
• Hybridization assays for optical sensors with
  cDNA and ethidium bromide.
• Removal of bound DNA.
• Salmon sperm DNA hybridizations served as a
  control
• Non-complementary, no hybridization
• L-DNA probe hybridization in bulk solution
• All hybridizations were done in triplicate

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Results and Discussion
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The Point Again

• To develop a biosensor that is rapid and
  sensitive.
• Detection of coliforms as indicators
• Using immobilized oligonucleotides as recognition
  elements for bioassays and biosensors.

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Speedy Unhindered Results

• Hybridization resulting in fluorescence is
  achieved quickly even at low concentrations of
  DNA.
• Less than 20 sec.
• The presence of other DNA, as in environmental
  samples, does not block the biosensor from
  functioning.

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Improved Fluorescence

• Fluorescence is strongest furthest from the
  biosensor surface.

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Spectrofluorimetric scan of SYBR 101-LacZ probe

• LacZ cDNA shows practically no florescence.
• Due to a lack of hybridization
• The presence of SP-DNA shows very low amounts of
  fluorescence.
• Due to a small amount of hybridization
• Uncomplimentary DNA

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Spectrofluorimetric scan of SYBR 101-LacZ probe

• The presence of
• L-DNA increases the fluorescence due to
  increased hybridization.
• The addition of
• SP-DNA causes minor inhibition of fluorescence
  due to undesired hybridization.

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Spectrofluorimetric scan of SYBR 101-LacZ probe

• Detection of other environmental DNA is possible
  based on intensity.

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Enhanced Nucleotide Coupling

• Immobilized oligonucleotides on solid supports
  linked to substrate.
• Results in increased hybridization efficiency.
• This probe is attached to HEG linker to increase
  placement onto the solid support.

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Improvements

• Fluorescence is maximized with a 11
  stoichiometrical ratio of dye to duplex
  structure.
• Increasing the PBS concentration three times
  results in increased stability of dsDNA and
  eliminates non-selective absorption by
  Spectrofluorometer.

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Conclusions
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Was It Worth It?

• Improvements were found for a biosensor that can
  lead to a biosensor that is
• Easy to use
• Reversible
• Selective
• Sensitive
• Reusable

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Time Well Spent!

• The article has produced significant results that
  show the potential for both future investigation
  and industrial use.
• The procedures demonstrated a fair amount of
  consideration for detail.
• Results were produced after several repetitions
  of the procedure.
• Statistical investigation was used to determine
  acceptable data within standard deviations.

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Future Ramifications?

• These improvements can lead to
• Reusable biosensors
• These can lead to improved identification and
  analysis of microbes.
• Use with understanding biofilms
• Use with improving waste processes
• Use with improving food and water processing

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The End!

• Eat Quiznos Subs!