L10 Fluorescence and Optical Sensors · Biosensors and Instrumentation Stewart Smith Beijing University of Posts and Telecommunications 2019 Summary • Fluorescence techniques •

Stewart SmithBiosensors and InstrumentationBeijing University of Posts and

Telecommunications 2019

Fluorescence and Optical Sensors

Lecture 10

1



Summary

• Fluorescence techniques

• Optical sensor Technology

• Optical detectors

• Case study - Label free DNA detection

2



Fluorescence• Fluorescent materials

absorb photons which excite electrons

• When they decay to a ground state they can re-emit photons

• These will have a lower energy

Excited State

Ground State

hν

Blue LightHigh Energy

hν

Green LightLower Energy

3



Fluorescence• The shift in λ is called

the “Stokes shift”

• The delay between excitation and emission is lifetime

• Quantum yield is ratio of photons out against number of photons in

Excited State

Ground State

hν

Blue LightHigh Energy

hν

Green LightLower Energy

4



Fluorescence• Fluorescence is at the

heart of biological measurement

• Fluorescent spectroscopy of endothelial cells

• Autofluorescence or labelling?

5



Fluorescence in Biology

• Some cells express fluorescent proteins like GFP or have other natural fluorescence

• Otherwise they need to be tagged or labelled with a fluorophore

• This is a fluorescent dye, or maybe a quantum dot that is selectively attached

6



Fluorescence in Biology• Not just used at a cellular level, most

important use is in microarrays

• Arrays of spots of probes, specific lengths of DNA, proteins or antibodies

• Target molecules from the tested sample are labelled with fluorophores

• They selectively attach to the probes

7



Fluorescence in Biology• Not just used at a cellular level, most

important use is in microarrays

• Arrays of spots of probes, specific lengths of DNA, proteins or antibodies

• Target molecules from the tested sample are labelled with fluorophores

• They selectively attach to the probes

7



Fluorescence Activated Cell Sorting (FACS)

• Type of flow cytometry

• Cells labelled with specific fluorophores

• Cells in suspension are sprayed in droplets

• Sorted depending on fluorescent label

Laser

Type 1

Flow

ChargedDeflectionPlates

+/−DropletCharging

Computer Control

Camera/Detector

Labelled cells

Waste Type 2

Sheath

8



Fluorescence Microscopy Techniques

• Spectroscopy - measure different emission wavelengths

• FLIM - lifetime imaging, excite fluorophore with a pulse and measure decay

• TIRF - Total internal reflection fluorescence microscopy, uses near-field effects

• FRET - Förster resonance energy transfer

9



Reducing Fluorescence• Photobleaching is a process through which a

fluorophore is photochemically altered

• Constant exposure to excitation wavelength eventually reduces fluorescence

• Quenching is reduction of fluorescence through chemical or other stimuli

• Exploited in a number of sensors

10



Summary





11



Optical Fibre Sensors• Enables use of well developed optical techniques

for bio/chemical sensing

• Optical fibres are low attenuation so can allow remote measurement (not wireless)

• Do not require electrical connection which may be essential in some environments

• Low interference, high sensitivity/bandwidth

12



Optode and Fibre Sensors• The optode (or sometimes optrode) is a chemical

transducer that is read optically

• They often use an optical fibre or another type of wave guide

• Two types:

‣ Extrinsic - the fibre guides light to/from sensor

‣ Intrinsic - the fibre itself is part of the sensor

13



Extrinsic Optical Fibre Sensors

• Optical fibres guide light to and from the sensor, which could be chemical or physical

• Detection method could depend on reflectance, absorbance, fluorescence etc.

Sensor

Light Source

LightDetector

Optical Fibres

14



Oxygen Optode Fibre Sensor

• Ruthenium based fluorophores are quenched by oxygen

• Immobilise the fluorophore in a matrix at the tip of an optical fibre

• Coat with oxygen permeable membrane (PTFE/Teflon)

• Multimode fibres to carry both excitation and emission wavelengths

Fibre

15



Oxygen Sensor• Results from sensor

using phase modulation of light

• Designed for in-vivo pO2 measurement

• Intensity and phase dependent on O2 concentration

P.A.S. Jorge et al. / Sensors and Actuators B 103 (2004) 290–299 297

difference is expected to be maximum around 188 kHz,which is the ideal modulation frequency [5]. This way, an in-crease in modulation frequency is expected to improve sen-sor performance. In Fig. 5(a) and (b) the phase and the fluo-rescence intensity response of the sensing system to O2/N2saturation cycles can be observed. The phase signal showssome instability indicating the need for SNR improvement.In order to demonstrate the phase insensitivity to opticalpower drift a simple test was performed. With the sensinghead in a 21% O2 atmosphere, the optical power injectedinto the fibre system was changed up to 25%. Fig. 6 showsthe consequence of this variation in the intensity and phaseof the fluorescence signal. Although a significant change in

0 60 120 180 240 300 360 420 480-20

-18

-16

-14

-12

-10

-8(a)

80%

100%

39,1%

60%

20,6%

12,3%8%

0,4%

φ d (d

egre

es)

time (s)

0 60 120 180 240 300 360 420 4800,03

0,04

0,05

0,06

0,07

0,08

0,09

0,10

0,11(b)

100%80%

60%

39,1%

20,6%

12,3%8%

0,4%

Fluo

resc

ence

inte

nsity

(mV)

time (s)

Fig. 8. System response to step variations of O2 concentration level: (a) phase response; (b) fluorescence intensity response.

the intensity response occurs, the phase response remainsessentially unchanged. This confirms the ability of thephase detection scheme to avoid power fluctuations inducederrors.The Stern–Volmer plots obtained from phase measure-

ments with all configurations are clearly non-linear, indicat-ing that the dopant is not homogeneously distributed withinthe silica matrix. Instead, the ruthenium complex occupiesenvironments with different oxygen accessibilities. In thissituation, the standard Stern–Volmer equation (Eq. (1)),based on a single exponential decay, no longer describesaccurately the quenching behavior. Alternatively a dual ex-ponential model, corresponding to two different dominant

16



Oxygen Sensor• Results from sensor

using phase modulation of light

• Designed for in-vivo pO2 measurement

• Intensity and phase dependent on O2 concentration

P.A.S. Jorge et al. / Sensors and Actuators B 103 (2004) 290–299 297

difference is expected to be maximum around 188 kHz,which is the ideal modulation frequency [5]. This way, an in-crease in modulation frequency is expected to improve sen-sor performance. In Fig. 5(a) and (b) the phase and the fluo-rescence intensity response of the sensing system to O2/N2saturation cycles can be observed. The phase signal showssome instability indicating the need for SNR improvement.In order to demonstrate the phase insensitivity to opticalpower drift a simple test was performed. With the sensinghead in a 21% O2 atmosphere, the optical power injectedinto the fibre system was changed up to 25%. Fig. 6 showsthe consequence of this variation in the intensity and phaseof the fluorescence signal. Although a significant change in

0 60 120 180 240 300 360 420 480-20

-18

-16

-14

-12

-10

-8(a)

80%

100%

39,1%

60%

20,6%

12,3%8%

0,4%

φ d (d

egre

es)

time (s)

0 60 120 180 240 300 360 420 4800,03

0,04

0,05

0,06

0,07

0,08

0,09

0,10

0,11(b)

100%80%

60%

39,1%

20,6%

12,3%8%

0,4%

Fluo

resc

ence

inte

nsity

(mV)

time (s)

Fig. 8. System response to step variations of O2 concentration level: (a) phase response; (b) fluorescence intensity response.

the intensity response occurs, the phase response remainsessentially unchanged. This confirms the ability of thephase detection scheme to avoid power fluctuations inducederrors.The Stern–Volmer plots obtained from phase measure-

ments with all configurations are clearly non-linear, indicat-ing that the dopant is not homogeneously distributed withinthe silica matrix. Instead, the ruthenium complex occupiesenvironments with different oxygen accessibilities. In thissituation, the standard Stern–Volmer equation (Eq. (1)),based on a single exponential decay, no longer describesaccurately the quenching behavior. Alternatively a dual ex-ponential model, corresponding to two different dominant

16



Intrinsic Optical Sensors

• Optical fibres work through total internal reflection at the core-cladding interface

• Evanescent wave extends beyond this by some small distance, typically less than λ

• This can excite fluorescence in a sensing material coating the fibre

17



Intrinsic Optical Sensors• Sensing can be distributed along the fibre

• Penetration depth is low so fouling can be a serious problem depending on environment

Sensing Material

Fibre

Sensing zone

18



Surface Plasmon Resonance

• Evanescent wave can excite plasmons

• These are coherent electron oscillations in a thin metal layer (Au)

• Reflected beam contains information about attachments

19



Integrated SPR Biosensor 20



Summary





21



Light Dependent Resistor

• High resistance semiconductor with metal contacts

• Incident light creates electron hole pairs

• This reduces the resistivity of the material

22



Photomultiplier Tube

• Photons generate electrons at photocathode which are accelerated towards dynode

• Impact excites more electrodes so there is amplification at each dynode

Glass Vacuum Tube

Photon

Electron

Dynodes (electron multipliers)

Anode

Photocathode

Current

Output

23



Photodiode• p-n or p-i-n junction diode

operated in reverse bias

• Photons absorbed in the depletion region create electron-hole pairs

• These are swept apart by bias voltage leading to a light dependent current

p−type

intrinsic semiconductor

n−type semiconductor

Anode

Cathode

Lig

ht

24



Charge Coupled Device• Originally developed as a

shift register memory

• CCD is the basis for many digital cameras

• Charge induced by light exposure is stored in potential wells

• Read out by moving charge along a row

25



CMOS Camera• Alternative to CCD cameras

• Common in mobile phones

• Arrays of photodiodes with CMOS addressing

• STMicroelectronics imaging division based in Edinburgh

• Originally spun-out from UoE

26



Single Photon Avalanche Diodes (SPADS)

• Solid-state replacement for PMT

- 50 ps jitter

- 30 ns dead time

- 6 Hz dark count rate

- 20 μm pitch

- 20 V breakdown

- 0.35 μm CMOS

27



SPAD - Geiger Mode

IntegrationAv

alan

che Geiger

Reverse Bias Voltage (V)

Curre

nt (A

)

(1) AvalancheBreakdown(2) Quenching

(3) Reset

SPADhν

VD

RL

28



SPAD Outputs

Low Intensity Medium Intensity

High Intensity

0V

3.3V

29



Micro-Fluorimeter

CMOS Backplane

CMOS SPAD Detector Array

Microarray Spots

GaN µLED Array

Filter

30



Microfluidic Cytometer 31



Summary





32



Case Study - DNA Based Biosensor

• Sensor using artificial DNA “switches”

• Output mechanism uses FRET

• Control through electrochemical release of ions

33



Resonant Energy Transfer

• FRET involves energy transfer between two fluorophores with overlapping spectra

• Excitation of the donor leads to emission from the acceptor

Light spectrum

Excitation Emission

Donor Acceptor

Emission Excitation

34




• The energy transfer is a near field process

• Förster length is between 50 and 100Å

• The acceptor quenches the donor fluorescence

5nm

480 nm

436 nm

CFP YFP

35




• The energy transfer is a near field process

• Förster length is between 50 and 100Å

• The acceptor quenches the donor fluorescence

5nm

480 nm

436 nm

CFP YFP

5nm

535 nm

FRET

436 nm

480 nm

CFP YFP

35



Holliday Junction

• Named for Robin Holliday

• 4-way junction between DNA strands

• Artificial but similar to a structure that occurs in replication

36



‘OPEN’ Conformation

Holliday Junction

• HJs can switch between two shapes

• This happens when exposed to cations

• These screen phosphate charges leading to collapse

37



Holliday Junction

• HJs can switch between two shapes

• This happens when exposed to cations

• These screen phosphate charges leading to collapse

‘CLOSED’ Conformation

37



Sensor Concept• Attach donor and

acceptor fluoro-phores to HJ arms

• Switching detected through observing the FRET signal

• This measures presence of ions Open

No FRETClosed FRET

38



Sensor Concept

• The emission spectra of the FRET changes with HJ shape

• Emission peak of the donor drops and the acceptor increases when the HJ closes

Low salt No FRET

fluor

esce

nce

wavelength / nm

λ1 λ2

D AA

‘OPEN’

470 nm 517 nm

39



Sensor Concept

High salt FRET occurs

fluor

esce

nce

wavelength / nm

λ3λ1

D A

‘CLOSED’

470 nm 584 nm• The emission spectra

of the FRET changes with HJ shape

• Emission peak of the donor drops and the acceptor increases when the HJ closes

40



HJ FRET Biosensor

• Probe is an incomplete, three strand HJ with fluorophores attached

• Completed with complementary oligonucleotide and switching observed

41



HJ Actuation Microsystem

Ag/AgCl Reference Electrode

Ion Switching Film

Interdigitated electrodes for electrochemical control of switching ion (Mg2+) conc.

42



HJ Actuation Microsystem

• Pro-HJ probes spotted onto glass cover slips

• Fixed over micro-electrodes with spacer to form fluid chambers

• Complementary and non-complementary oligos in solution

43



FRET Measurements

• Measurements made using fluorescence scanner designed for imaging microarrays

• Excitation of the donor and imaging at both the donor and acceptor emission wavelength

• Extraction of intensity data from spots and calculation of FRET ratio (Iacceptor/Idonor)

44



FRET Image• Wetted area in centre of

image

• 2 types of spot:

‣ HJ with donors and acceptors

‣ HJs with only donor dye

45



Initial Results

• Mg2+ ions switched into and out of solution0.0000

1.5000

3.0000

4.5000

6.0000

Mg- Mg+ Mg- Mg+ Mg- Mg+ Mg- Mg+

46



Case Study Summary• Switching of HJ molecular conformation can be

measured with FRET

• Completion of an HJ with a complementary DNA strand can be detected

• Label free biosensing of DNA with standard microarray measurement

• Switching controlled with microelectrodes

47

Documents

L10 Fluorescence and Optical Sensors · Biosensors and Instrumentation Stewart Smith Beijing University of Posts and Telecommunications 2019 Summary • Fluorescence techniques •