Week 2 : Optical biosensors Flashcards
What is the difference between background fluorescence and autofluorescence?
autofluorescence originate from endogenous sample constituent in which naturally fluorescent molecules are present.
background fluorescence originates from unbound or nonspecifically bound probes
how to increase fluorescence detection sensitivity and decrease Background Fluorescence and Autofluorescence?
(1) Minimize this signal distortion by probes excited at longer wavelengths (>500nm) e.g. Alexa 647
(2) Extract the biomarkers from their original environment prior to analysis. Note: this extra sample processing step can result in error and variability, dependent on extraction efficiency, and biomarker purity
(3) Fluorescence lifetime imaging (FLIM): the chances of your signal and sample autofluorescence having the same lifetime signal are smaller than the chances of spectral overlap
Give an example of common autofluorescent molecules in cell.
DNA, Folic acid, retinol.
What are the sources of fluorescent molecules?
(1) Naturally occurring fluorescent small molecules or proteins. DISADVANTAGES: low fluorescence quantum yield & low excitation/emission wavelengths.
(2) There are also synthetic fluorescent molecules:
engineered small molecules or engineered nanoparticles.
How can you use Fluorescent Small Molecules in biosensing?
(1) can be designed to bind preferentially to cancer biomarkers (2) can be attached to receptors/probes to target them
How can you use Fluorescent nanomaterials in biosensing?
require surface to be functionalized with
receptors to confer biomarker specificity.
How can you use Fluorescent nanomaterials in biosensing?
by functionalizing the surface with receptors to confer biomarker specificity.
Give an example of fluorescent nanomaterilas.
Carbon dots, Quantum dots, metal nanoclusters, polymer NPS
What are the types of fluorescent small molecule-based probes, in terms of mechanisms?
1) Labels
2) Molecular Beacons
3) Intercalator Dyes
4) Molecular Rotors
Explain Molecular Beacons
Typically single-stranded oligonucleotide hybridization probes that can adopt a stable stem-and-loop structure in solution.
•loop contains a probe sequence that is complementary to that of the nucleic acid biomarker, and stem is formed by annealing of two complementary “arm” sequences located on either side of the probe sequence (i.e., 3’ and 5’ ends).
• fluorophore is covalently linked to the end of one arm and a quencher is covalently linked to the end of the other arm.
How do Molecular beacons function?
MBs do not fluoresce when they are free in solution, but when they hybridize to a target sequence they undergo a conformational change that enables them to fluoresce brightly
Explain Intercalating Dye.
small molecules that can intercalate between Watson-Crick
base pairs of double-stranded DNA and emit fluorescence of intensity orders of magnitude greater than when the dye is free in solution.
Give an example of Intercalating Dye
(1) SYBR Green (SG)
(2) picoGreen (PG)
(3) SYBR Gold
(4) YOYO dyes
Give an example of Intercalating Dye
DAPI
(1) binds to A-T regions (Major groove)
(2) used in fluorescence microscopy
(3) can be used in live and fixed cells
(4) passes through the membrane less efficiently in live
cells and therefore provides a marker for membrane viability
Explain Molecular Rotors
probes known to form twisted intramolecular charge transfer (TICT) complexes in the excited state producing a fluorescence quantum yield that is dependent on the surrounding environment
How do Molecular Rotors function?
Following photoexcitation, this motif can relax via fluorescence emission or internal nonradiative process that involves molecular rotation between the donor and the acceptor
• When rotation is hindered (e.g., because dye is intercalated between base pairs), relaxation occurs via an increased fluorescence emission
• When the dye is in solution, relaxation proceeds mainly via a nonradiative pathway, meaning no (or lower) emission of fluorescence.
Give an example (type) of fluorescent nanomaterials
1) Quantum Dots (QDs)
2) Gold Nanoparticles (AuNPs)
Describe Quantum dots
• Composed of a semiconductor core capped with a shell for stability
• surface can be coated with hydrophilic, hydrophobic, or amphiphilic ligands which can be further linked with proteins, drugs, antibodies, and other bioreceptors
• emission spectra can be tuned by adjusting the size
What are the advantages of NP probes?
(1) high quantum yeild
(2) more photostable
(3) longer fluorescence lifetime
(4) have broader absorption spectra
Explain gold NPs.
1) diameter of 1 to 100nm
2) rely on fluorescence quenching
3) emission spectra varies based on size
Give an example of using gold NPS.
Nanoflare; functionalized AuNPs with ssDNA that are complemntry to the target sequence and a reporter sequnce. when the target is not presented, the reporter sequnce contianng the flurofre is quenched.
Draw and explain Jablonski Diagram
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Why Fluorescence-based sensors are common? (Features)?
1) dynamic range,
2) high sensitivity, multiplexing capabilities
3) ability to measure multiple fluorescence properties (e.g. fluorescence intensity and fluorescence lifetime).
4) allows the detection single molecules
What Challenges are there for Fluorescence-based sensors
1) Background autofluorescence
2) photobleaching
What are the Principles of Fluorescence? (how it works)
1) Excitation light (absorption energy) by a molecule at an exact wavelength
2) Fluorescence emission takes place to realease that energy
(better to draw it)
Describe Jablocki Diagram
1) a representation of fluorescence excitation and emission
2) LEFT side displays the singlet states (S0, S1, .., Sn-1) from Low energy to high energy
3)RIGHT side displays the triplet states (reversal spin happens here)
Define the energy of photons
E =hf, where h (plank constant)=6.62607015 × 10^(-34) m^(2)kg/s & f=c/y where c=3^(8)m/s
Following absorption, how many decay pathway are there?
1) non-radiative: horizontal internal conversion & vertical vibrational relaxation (lost of energy by direct transfer to nearby molecule (e.g. water). Both brings molecules to S1
Which is greater, wavelength of emission or absorption?
absorption happens at a higher energy while emission happens to release that unwanted extra energy. Through this process some non-radiative decay (internal conversions & vibrational relaxation) might occur to bring down molecules to S1 which results in some energy lost. Therefore, photons emitted have lower energy that absorbed photons. energy and wavelength are inversely proportional such that high energy photons have the shortest wavelengths. thereby, absorption happens at shorter wavelengths
Why is phosphorescence lifetime longer?
1) Delayed emission of absorbed photons
2) Electron pathway transitions: S1 -> T1 -> S0 casing spin reverse
What are the Characteristics of ideal fluorophore for readout?
1) Large Stoke’s shift (minimal overlap between excitation and absorption spectra and greater separtion)
2) High photostability (resistant to photobleaching)
3) High “brightness”: High quantum yield & High molar extinction coefficient
What is the challange of using multiple fluorophores
Too much overlap can result in bleed through or crosstalk between different fluorophores and thus loss or misperception of the signal information.
(Green is still detected in yellow )
What is photobleaching?
to all processes which lead fluorescence to fade permanently overtime
How to counteract photobleaching?
1) Choose the right fluorophore or “label” in you biosensor, synthetic dyes work better
2) Scan for shorter times
3) Reduce excitation light intensity
4) (in microscopy) Use high magnification (high NA objective lens)
5) Use “antifade” reagents (not compatible with live viable cells) (anti-oxidants) -> becuase inter crossing and triplet state happens with oxygen
What defines a fluorophores brightness?
1) Molar extinction coefficient (ε) = A=Log(I0/I)=εlc
2) Fluorescence quantum yield (ɸ)
Mathematically define Fluorescence quantum yield (ɸ)
N emitted/ N absorbed = kr /(kr+krn)
radiative transition rate (kr) = radiative (light emitting) processes e.g. fluorescence and phosphorescence
sum of non-radiative rates (knr), = processes such as internal conversion, intersystem crossing, and energy transfer.
When Fluorescence lifetime (τ) occure?
When the intensity I = 1/e ≈ 63%
How to draw a Jablonski Diagram?
1) Draw electronic states (Singlet and Triplet)
2) Draw Excitation arrow
3) Draw internal conversion (vibrational relaxation)
4) Draw 3 pathways to S0
5) Non-radiative relaxation
6) Radiative (Fluorescence)
7) Radiative (Phosphorescence)
What is FRET?
Förster Resonance Energy Transfer (FRET)=mechanism of energy transfer between two chromophores
What are the Conditions of FRET?
(1) that there is a significant overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor
(2) that both the donor and the acceptor molecules are in close proximity to each other.
Fluorescence-based sensors can rely on three different types of fluorophores, what are they?
(1) Add fluorescent labels (2) use fluorescent probes (3) use enzyme substrates
Types of Colorimetric Biosensors
Classified into two types based on the driving forces of color change:
(1) ”Plasmonic Colorimetric Sensors” based on phenomenon of Localised Surface Plasmon Resonance (LSPR) resulting from aggregation or deaggregation of materials. e.g. AuNP color changes based on particle diameter
(2) “Enzymatic Colorimetric Sensor” resulting from catalysis by enzyme-mimicking materials e.g. NP can decompose H2O2 which can oxidize a chromogenic substrate (e.g. TMB)
Advantages of Colorimetric Biosensors
allow rapid, portable, and cost-effective analyte measurements, promising point-of-care testing
