Biosensors Flashcards
What is a biosensor
An analytical device that incorporates a biological component to detect an analyte concentration (or some other measurable function) and transduces into a measurable signal (ex. pH, electrical, heat, light)
Examples of biological components in biosensors
Antibodies (specifically bind analyte)
Enzyme (catalyse reaction with analyte)
Microorganisms (engineered bacteria since sensitive to pollutants, toxins, etc)
Nucleic acids (gene sensors based on base pairings)
Organelles, cells or tissues (more complex analysis systems ex. mitochondria detects changes in ATP levels)
What is a genetically encoded biosensor
Engineered proteins that can be expressed in a biological system (introduce the DNA/plasmid/RNA that encodes) to directly measure an analyte concentration or biological function within that system.
Typically the sensors incorporate intrinsically fluorescent or bioluminescent proteins for sensor output since easy to measure. Changes in the biological effect measured causes changes in the intensity or wavelength (sensors that can naturally change their light emission profile in response) of light
What can genetically encoded biosensors measure?
Thousands of different types of genetically encoded light emitting biosensors
Can now develop them to measure almost any sort of function in a living cell:
Intracellular environment (pH, membrane potential, etc)
Second messenger concentrations (cAMP, IP3, Ca2+ and just about all others)
Metabolite concentrations (glucose, APT, ADP, GTP)
Enzyme activity (kinases, proteases)
Protein-protein interactions (dimerisation, oligomerisation)
Protein conformation changes (receptor activation)
and more/whatever else you want if your creative enough to design appropriate biosensor
General predominant application of genetically encoded biosensors
Tool for biological and biomedical research to define what is happening in a cell/tissue/organism
Traditional method to measure things in cells and advantages of biosensors in comparison
At individual time points for each set of samples, slice cell to collect lysate and quantify analyte (western blot, immunoassay etc) in snapshots of individual timepoints
Biosensors:
Measuring in living cells in real-time allows for dynamic measurements which provides spatial measurements and improved resolution in temporal changes
Ex.
Can see how individual cells respond at different time points compared to others
Can see subcellular dynamics (where in the cell things are happening)
Downsides of biosensors
- Expressing in desired cell type may be difficult (immune cells don’t take up and express foreign material well since designed not to and or primary cells like neurons).
+ Instead of plasmids use viruses to transfect since better at getting DNA into cells. Or use genome editing
- Expressing the sensor may influence the biology being studied
- Biosensor measure relative not absolute quantities and can’t be converted (ex. with immunoassay can plot molar concentration of analyte measured, not RFU)
GFP development from organism to biosensor in lab
Originally identified in Aequorea victoria jelly fish in 1960s
27kDa, 11 strand beta barrel around a helix containing the fluorescent moiety
Wild type does not fold well at 37 degrees so point mutations were incorporated to produce eGFP with improved brightness and folding in mammalian cells
Variants form an internal chromophore without requiring additional cofactors so can be successfully expressed in cells that don’t come from jellyfish
Further optimised to produce various colour variants and now we have full pallet from IF to blue light emitting. Nobel prize won in 2008 by Tsien for development of GFP
Now there’s a database (FPbase) of the properties of the different FPs, accounting for multiple factors like brightness, lifetime, and stability to select the best one for your application
What is bioluminescent
The production and emission of light by a living organism through a chemical reaction
Luciferase development from organism to biosensor in lab and applications
Luciferase is a term first used by Raphael Dubois for a class of oxidative enzymes that produce bioluminescence (ex. in fireflies oxidases luciferin to oxyluciferin with light by-product).
Reaction uses ATP (also magnesium ions) so it’s bioluminescence is dependent on ATP conc. and alters it. As a biosensor, measuring things in a cell that’s dependent on ATP would be problematic.
Firefly luciferase genetically encoded biosensor therefore is not used to study living cells/activity assays,
Used in:
- reporter assays for transcriptional activity in cells (put luciferase under control of promoter of interest, lyse cells and measure total conc. of luciferase)
- with biotinylation can detect levels of ATP in cell viability or for kinase activity assays
Development beyond firefly luciferase of others from organism to biosensor in lab and applications
Bioluminescent protein in Renilla reniformis, a sea pansy, deep sea organism expressing Renilla luciferase; RLuc (ex. oxidises resin to produce blue light by-product)
Requires no cofactors so useful over firefly luciferase in cell activity assays.
Luciferase in deep sea shrimp Oplophorus gracilirostris catalyses coelenterazine oxidation (no ATP required) with blue light by-product
Optimised version (NanoLuc) are much smaller and ~100x brighter than RLuc and FLuc
Pros and Cons of fluorescence or bioluminescence technologies (5)
Fluorescence:
Is brighter (better suited to microscopy/imaging, particularly to obtain high spatial resolution of subcellular localisation)
Larger number of FPs with diverse properties to choose from (more flexibility in sensor design)
Bioluminescence:
- Less bright and requires specialised equipment for imaging. Better for non imaging approaches like high throughput assay with a microplate reader
Does not require external light source (better for photosensitive cells like retina as excitation light for FPs will change what’s happening in cell)
No phototoxicity or photobleaching from shining a bright light (better for long-term measurements)
Very low background as little autofluorescence
Technologies that use biosensors and brief overview of mechanism
Intrinsically context sensitive sensors:
Spectral property (intensity of light emission, excitation/emission properties) changes in response to measured analyte change.
Resonance energy transfer sensors (FRET and BRET):
Measures relative distance between two light emitting proteins (donor and acceptor) with suitable spectral properties. In close proximity (<10 nm) the donor’s emitted light energy will directly transfer to acceptor. Measure relative wavelengths of light emitted by acceptor and donor at the 2 wavelengths (ratio) to assess their proximity.
Fragment complementation sensors:
Split light emitting protein into two inactive halves that activate in close proximity. Fuse fragments to protein/s to measure proximity by changes in emitted light intensity.
Pros and Cons of Intrinsically context sensitive sensors
- Hard to rationally design protein to be sensitive to analyte. Difficult to predict how/if it will work (trial and error)
+ (Albeit a limited) number fluorescent/bioluminescent proteins naturally have contact sensitive to an analyte of interest and so easy to implement
+ Small and simple since only one protein expressed.
Pros and Cons of Resonance energy transfer sensors (FRET and BRET)
+ Dynamic measurements as acceptor and donor become closer/further
+ Small changes in proximity induces large changes in amount of RET (very sensitive)
+ Measuring ratio corrects a lot of experimental variability (cell number, etc)
- In FRET (FÖrster/Fluorescence) hard to selectively only excite donor without directly exciting acceptor (excitation crosstalk). Not in BRET since chemical not light is used to activate donor to emit light. Need to consider which wavelength to use; minimising excitation of acceptor but allowing sufficient excitation of donor (why FRET is less commonly used in plate based assays since hard to fine tune)
- In FRET and BRET there’s almost always emission crosstalk. Since the emission spectra of the donor is often broad, emission from donor and acceptor will be emitted at the measured wavelength
- Where to attach protein without affecting function (trial and error)
Pros and Cons of Fragment complementation sensors
+ Luciferase is fast and reversible allowing for dynamic measurements
+ Real-time spatial and temporal information
+ Often better dynamic ranges than RET
+/- Unlike RET, measures large changes in light intensity not small changes in ratio of two wavelengths emitted. Depending on context may be useful
+ Only need to measure one wavelength so depending on setup may be faster measurements (no need to switch optics)
- Where to attach protein without affecting function (trial and error)
- Measuring light intensity requires more care taken into technical things like ensuring same cell number and other variabilities
- Split fluorescence is not suitable for most sensors
Examples of biosensors implemented in intrinsically context sensitive sensors
Natural context sensitive Ca2+ biosensor: Aequrin
Aequrin bioluminescent protein in Aequorea victoria catalyses oxidation of coelenterazine luciferin in an oxidation reaction dependent on Ca2+ concentration (when a Ca2+ is bound to each of the 3 Ca2+ binding sites the reaction occurs)
Engineered context sensitive Ca2+ biosensor: Pericam
Fusion protein of:
- Calmodulin (binds myosin light chain when Ca2+ is present for muscle contraction)
- Circularly permutated eGFP (different N and C terminal position but essentially the same overall structure). One of the two peptides in the center of barrel structure is fused to
- M13 helix of myosin light chain
Hypothesis: When Ca2+ is present, calmodulin will bind M13 inducing a conformational change leading to change in fluorescent properties of cpEGFP
Reality: Structural information shows calmodulin binding to M13 closes solvent access to the GFP chromophore causing large increase in fluorescence
Trial and error of rearranging order of fusion protein to obtain conformation responsive to Ca2+
Examples of how biosensors may be implemented in resonance energy transfer sensors and pros/cons
In FRET commonly eCFP (mCerulean) and eYFP (mVenus) due to strong spectral overlap
In BRET commonly RLuc and NanoLuc (blue/cyan) paired with yellow or green FP
Examples:
Intermolecular sensor
Donor fused to protein A and Acceptor fused to protein B to measure protein-protein interaction
+ Often easiest to rationally design
- Requires expression of 2 proteins
Intramolecular sensor
Donor and Acceptor fused to different regions of Protein A to measure conformational changes
Donor and Acceptor fused to synthetic peptide to measure protease activity on cleaving the peptide
Donor and Acceptor fused to synthetic Proteins A and B respectively. Protein A and B are connected through a linker and measure changes in Protein A and B interactions/binding under particular conditions
+ Only express one protein
+ Ratiometric measure
- Rational design can be more challenging
Examples of biosensors implemented in resonance energy transfer based technologies
PKA based cAMP sensor (intermolecular sensor):
Fusion of the PKA dimer regulatory subunits with 2 CFP and the PKA dimer catalytic subunits with 2 YFP.
cAMP binding regulatory domains of PKA leads to separation of regulator and catalytic domains and decrease in FRET.
Downside: Biosensor (PKA) activation has lots of downstream targets for phosphorylation, so changes cell function when introduced and activated, also expressing multiple subunits is problematic and more complicated than expressing one protein.
EPAC based cAMP sensor (intramolecular sensor):
Structure revealed cAMP binding domains in cAMP binding proteins undergo significant conformational change when bound to cAMP. In EPAC there’s a large hinge movement in which the structure folds over.
Fuse CFP and YFP (or other light emitting proteins) to either end of EPAC. When cAMP is bound the conformational change in EPAC separates the FPs so they no longer undergo RET.
Considerations selecting RET protein pairs
FRET:
Spectral overlap between donor emission and acceptor excitation
High quantum yield of donor (good at emitting light)
High excitation coefficient of acceptor (measure of efficiency in accepting energy)
BRET:
Spectral overlap between donor emission and acceptor excitation
Bioluminescent donor brightness (good at emitting light)
High excitation coefficient of acceptor (measure of efficiency in accepting energy)
Fewer BRET pairs than FRET pairs
Examples of biosensors implemented in protein complementation based technologies
FPs: Used to study protein-protein interactions. FPs recombine irreversibly since chromophore forms through covalent bonds (can’t measure both increases and decreases, not used to study dynamic changes)
More typically BiLC (RLuc or NanoLuc) is used. Similar design to inter/intra molecular RET sensor.
EPAC based cAMP BiLC sensor:
In EPAC there’s a large conformational change when cAMP binds of a hinge movement in which the structure folds over.
Fuse two halves of RLuc to either end of EPAC. When cAMP is bound the conformational change in EPAC separates the halve so they no longer fluoresce.
