Week 2 : Optical biosensors

Question 1

What is the difference between background fluorescence and autofluorescence?

Answer 1

autofluorescence originate from endogenous sample constituent in which naturally fluorescent molecules are present.

background fluorescence originates from unbound or nonspecifically bound probes

Question 2

how to increase fluorescence detection sensitivity and decrease Background Fluorescence and Autofluorescence?

Answer 2

(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

Question 3

Give an example of common autofluorescent molecules in cell.

Answer 3

DNA, Folic acid, retinol.

Question 4

What are the sources of fluorescent molecules?

Answer 4

(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.

Question 5

How can you use Fluorescent Small Molecules in biosensing?

Answer 5

(1) can be designed to bind preferentially to cancer biomarkers (2) can be attached to receptors/probes to target them

Question 6

How can you use Fluorescent nanomaterials in biosensing?

Answer 6

require surface to be functionalized with
receptors to confer biomarker specificity.

Question 6

How can you use Fluorescent nanomaterials in biosensing?

Answer 6

by functionalizing the surface with receptors to confer biomarker specificity.

Question 7

Give an example of fluorescent nanomaterilas.

Answer 7

Carbon dots, Quantum dots, metal nanoclusters, polymer NPS

Question 8

What are the types of fluorescent small molecule-based probes, in terms of mechanisms?

Answer 8

1) Labels
2) Molecular Beacons
3) Intercalator Dyes
4) Molecular Rotors

Question 9

Explain Molecular Beacons

Answer 9

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.

Question 10

How do Molecular beacons function?

Answer 10

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

Question 11

Explain Intercalating Dye.

Answer 11

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.

Question 12

Give an example of Intercalating Dye

Answer 12

(1) SYBR Green (SG)
(2) picoGreen (PG)
(3) SYBR Gold
(4) YOYO dyes

Question 13

Give an example of Intercalating Dye

Answer 13

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

Question 14

Explain Molecular Rotors

Answer 14

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

Question 15

How do Molecular Rotors function?

Answer 15

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.

Question 16

Give an example (type) of fluorescent nanomaterials

Answer 16

1) Quantum Dots (QDs)
2) Gold Nanoparticles (AuNPs)

Question 17

Describe Quantum dots

Answer 17

• 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

Question 18

What are the advantages of NP probes?

Answer 18

(1) high quantum yeild
(2) more photostable
(3) longer fluorescence lifetime
(4) have broader absorption spectra

Question 19

Explain gold NPs.

Answer 19

1) diameter of 1 to 100nm
2) rely on fluorescence quenching
3) emission spectra varies based on size

Question 20

Give an example of using gold NPS.

Answer 20

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.

Question 21

Draw and explain Jablonski Diagram

Answer 21

-

Question 22

Why Fluorescence-based sensors are common? (Features)?

Answer 22

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

Question 23

What Challenges are there for Fluorescence-based sensors

Answer 23

1) Background autofluorescence
2) photobleaching

Question 24

What are the Principles of Fluorescence? (how it works)

Answer 24

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)

Question 25

Describe Jablocki Diagram

Answer 25

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)

Question 26

Define the energy of photons

Answer 26

E =hf, where h (plank constant)=6.62607015 × 10^(-34) m^(2)kg/s & f=c/y where c=3^(8)m/s

Question 27

Following absorption, how many decay pathway are there?

Answer 27

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

Question 28

Which is greater, wavelength of emission or absorption?

Answer 28

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

Question 29

Why is phosphorescence lifetime longer?

Answer 29

1) Delayed emission of absorbed photons 2) Electron pathway transitions: S1 -> T1 -> S0 casing spin reverse

Question 30

What are the Characteristics of ideal fluorophore for readout?

Answer 30

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

Question 31

What is the challange of using multiple fluorophores

Answer 31

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 )

Question 32

What is photobleaching?

Answer 32

to all processes which lead fluorescence to fade permanently overtime

Question 33

How to counteract photobleaching?

Answer 33

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

Question 34

What defines a fluorophores brightness?

Answer 34

1) Molar extinction coefficient (ε) = A=Log(I0/I)=εlc 2) Fluorescence quantum yield (ɸ)

Question 35

Mathematically define Fluorescence quantum yield (ɸ)

Answer 35

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.

Question 36

When Fluorescence lifetime (τ) occure?

Answer 36

When the intensity I = 1/e ≈ 63%

Question 37

How to draw a Jablonski Diagram?

Answer 37

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)

Question 38

What is FRET?

Answer 38

Förster Resonance Energy Transfer (FRET)=mechanism of energy transfer between two chromophores

Question 39

What are the Conditions of FRET?

Answer 39

(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.

Question 40

Fluorescence-based sensors can rely on three different types of fluorophores, what are they?

Answer 40

(1) Add fluorescent labels (2) use fluorescent probes (3) use enzyme substrates

Question 41

Types of Colorimetric Biosensors

Answer 41

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)

Question 42

Advantages of Colorimetric Biosensors

Answer 42

allow rapid, portable, and cost-effective analyte measurements, promising point-of-care testing