Structural sensing Flashcards
What do we mean by structural sensing?
detection of a threat by use of the structural elements of the threat, usually from binding to part of these elements with something that is easy to detect
eg an agglutination test
What are the five bio-analysis methods?
Culture
Microscopy
Immunoassay
PCR
Chemical Assays
Describe Method of Bioanalysis Culture
grow agents in conditions that specifically relate certain types of bacteria/fungi etc ie rose Bengal agar is a growing media that inhibits bacteria and allows the identification of the presence of yeast and moulds
Describe Method of Bioanalysis Microscopy
Morphological identification of particles
Describe Method of Bioanalysis Immunoassay
particles with specific epitopes matching the assay antibodies
particles with parts of the allergen that are identified by the body (epitodes) that in this case match the antibodies present in the assay test.
Describe Method of Bioanalysis PCR
DNA matching test
Describe Method of Bioanalysis chemical assays
identify the biomass of specific chemicals eg ATP
Limitations of bioanalysis method culture
underestimates concentration of all organisms
nonculturable organisms are invisible
non culturable are classed as noninfective
Limitations of bioanalysis method microscopy
limited to groups of organisms, not specific strains
Limitations of bioanalysis method immunoassay
limited to organisms that the assay is designed for, therefore only binary results
Cross reactivity is common
Limitations of bioanalysis method PCR
limited to organisms that the assay is designed for, therefore only binary results
highly specific
highly sensitive
Limitations of bioanalysis method Chemical analysis
only an indicator for large quantities of organisms
what is meant by direct binding event?
direct binding of the target to a specific molecular recognition element
how does direct binding event works?
a reversable ‘lock and key’ event like and antibody with a viral protein
9 molecular recognition elements for biosensing
single strand DNA
Antibody
Peptide
Enzyme
Lectine
Receptor
Aptamer
Small molecule
imprinted molecule
What is an antibody?
also known as an immunoglobulin Ig is a large, Y shaped protein produced mainly by plasma cells that is used by the immune system to neutralize pathogens such as pathogenic bacteria and viruses.
How do immunoassay ticket systems work?
liquid sample added to test strip and other reagents added if required
target molecules wick through the ticket, bind to immobilized agents and detection molecules in a ‘sandwich’ format
wick usually travels through a testing area before reaching a control line, if no line shows up on control after allotted time, then the test needs repeating.
Define SELEX?
Systematic Evolution of Ligands by EXponential Enrichment
a method of increasing the number of DNA/RNA strands required for testing and use them to sequence and characterise DNA found in the environment
The starting single stranded DNA or RNA library
(10 14 ~10 16 random oligonucleotides) is composed of sequences 20~100 nucleotides in length with a random region in the middle flanked by fixed primer sequences.
After incubation with the target of interest, the bound
oligonucleotides are partitioned from unbound
sequences and amplified by PCR . repeated 2-15 times before used as biomarker identification tools.
The resulting enriched DNA pool is used for the next round of selection.
What is SELEX short for?
Systematic Evolution of Ligands by EXponential Enrichment
Dog’s nose; how does this compare with current detection systems
6 high level approaches for structural sensing
Magnetic
optical
electrochemical
mass
acoustic/piezoelectric
MEMS
Types of optical
Fluorescence
Absorbance
SPR/RM
Luminescence
RAMAN
Types of magnetic
magneto-elastic
giant magneto-resistance (GMR)
types of MEMS
MEOMS
cantilevers
microfluids
microcalorimetry
types of acoustic/piezo-electric
surface wave acoustic
quartz microbalance
types of mass
time of flight ion trap
ion mobility (MS/MS)
types of electrochemical
amperometry
potentiometric
conductimetric
molecular electronics
What is a molecular recognition element?
Molecular recognition event is typically a specific interaction that is reversible, analogous to the interaction between a lock and a key, although in many cases the binding would more accurately be described as induced fit, during which the recognition element changes shape upon binding.
What does a direct binding event rely on?
Affinity of the target for the molecular recognition elements
Non-specific binding of extraneous material at the binding site
Sensitivity of detection
If attomolar (10 18 ) detection levels are required then high affinity molecular recognition elements with minimal non specific binding required
considerations for structure based sensing?
Sample collection,
Sample concentration,
Binding of the target to the molecular recognition element,
Possible addition and removal of “reporter” groups,
Detection of target molecular recognition element complex,
Analysis of the output signal,
Renewal of the sensor surface for repeated monitoring.
target inhibitor for single strand DNA?
complementary sequence of DNA
target inhibitor for Antibody
proteins, carbohydrates, small organic molecules etc
target inhibitor for Peptide
proteins, carbohydrates, small organic molecules etc
target inhibitor for enzyme
substrate such as biochemicals like glucose, acetic acid
target inhibitor for lectin
carbohydrate
target inhibitor for receptor
proteins, carbohydrates, small organic molecules
target inhibitor for aptamer
proteins, carbohydrates, small organic molecules etc
target inhibitor for small molecules
proteins, cells etc
target inhibitor for imprinted moelcules
proteins, small organics molecules, whole cells etc
target inhibitor for
8 SELEX methods
IP-SELEX
Capture-SELEX
Cell-SELEX
CE-SELEX
M-SELEX
AFM-SELEX
AEGIS-SELEX
Animal-SELEX
Key aspects of IP-SELEX
includes immunoprecipitation
Key aspects of Capture-SELEX
oligonucleotide library is immobilized on a support instead of the targets to identify aptamers against small soluble molecules
Key aspects of Call-SELEX
utilizes whole live cells as targets for selection of aptamers
Key aspects of CE-SELEX
involes separation of ions based on electrophoretic mobility
Key aspects of M-SELEX
combines SELEX with a microfluid system
Key aspects of AFM-SELEX
employs AFM to create a 3D image of the sample surface
Key aspects of AEGIS-SELEX
utilizes libraries with the artificially expanded genetic code
Key aspects of animal-SELEX
aptamers are selected directly within live animal models
advantages of IP-SELEX
selects aptamers against proteins under normal physiological conditions
increased affinity and specificity
advantages of Capture-SELEX
suitable for the selection of aptamers against small molecules
immobilization of the target not required
used for discovery of structure switching aptamers
advantages of Cell-SELEX
Prior knowledge of the target not required
Aptamers are selected against molecules in their native state
Many potential targets available on the cell surface
Protein purification not required
advantages of CE-SELEX
Fast
only 1-4 rounds of selection required
reduced non specific binding
target immobilization not required
advantages of M-SELEX
Rapid
Very effective (small amounts of reagents required)
Applicable to small molecules
Automatable
advantages of AFM-SELEX
able to isolate high affinity aptamers
Fast 3-4 rounds
advantages of AEGIS-SELEX
high specificity of the selected aptamers
advantages of animal-SELEX
selected aptamers bind the targets in their natural environment
Prior knowledge of target not required
Minimal optimization needed
advantages of -SELEX
disadvantages of IP-SELEX
more time consuming than standard SELEX
disadvantages of Capture-SELEX
some oligonucleotides from the library might not be released/selected
disadvantages of Cell-SELEX
suitable for cell surface targets
Requires high level of technical expertise
Costly
Time consuming
Post SELEX identification of target required
disadvantages of CE-SELEX
not suitable for small targets
expensive equipment
disadvantages of M-SELEX
Not suitable low purity/recovery of aptamers
target immobilization required
disadvantages of AFM-SELEX
expensive equipment required
Immobilization of target aptamers required
disadvantages of AEGIS-SELEX
Poor recognition of the unnatural bases by natural DNA polymerases
disadvantages of Animal-SELEX
time consuming (man rounds required)
Dog’s nose; how does this compare with current detection systems?
test kits in the range of 1g-1ug (visible to particles)
Field instruments in the range of 1ug-1pg (particles/vapour)
Dogs in the range of 1fg-1ag (vapour)
Dog density of olfactory epithelium?
170cm^2
(humans have 10cm^2)
How does the dog receptors mean it has a better sense of smell?
It is the interactions of odour molecules with specific receptor proteins that give specificity.
Each olfactory odour receptor neuron has only one functional odour receptor; if the odour molecule interacts with the receptor then the nerve cell will respond.
