TL -Spectrometry and Applications in Biomedical Research and Diagnostics

Mladen FrankoUniversity of Nova Gorica

INTERNATIONAL SCHOOL OF QUANTUM ELECTRONICS -57th Course

ERICE - SICILY: October 19th -26th, 2016

2

Transmission techniques -how to improve sensitivity?

Sample

I 0 I - transmission

A=-logT=-log(I/I0)

A=εLC≅ (I0-I)/2.303I0

Emission

Radiatioless deexcitation

Molecular energy diagram and related excitation/deexcitation processes

3

• A, R, F: absorbance, resonant and nonresonant fluorescence

• I, P: intersystem crossing, phoshorescence

• C, B: internal conversion, vibrational relaxation

Nonradiative modes of relaxation (C, B)Nonradiative modes of relaxation (C, B)

Basics of thermal lens effect

• During non-radiative relaxation of excited species temperature in the sample increases (10-4 - 10-3 K)

• a temperature gradient is generated with maximum temperature at the axis of the excitation beam

• the resulting refractive index gradient acts as a lens (mostly: dn/dT< 0, diverging lens)

• laser beam is defocused (single beam or pump/probe configuration)

• beam radius and its intensity at the beam axis changes • relative change in the beam intensity is proportional to

the absorbance of the sample and to the power of the excitation beam.

4

SAMPLE

TLS – how to make good use of something not desired in laser technology?

∆I

Signal = ∆I/I

8

Nonsteady thermal diffusion

• T(r,t)….. temperature• D………thermal diffusivity• ρ………. density• cp……….heat capacity• Q(r,t)……source term (“heat”)• vx………..velocity of the medium in x direction

By solving nonsteady the thermal diffusion equation, changes in refractive index and related TLS signal can be calculated for different beam geometries and excitation regimes (pulsed, cw)

),(),(

),(),(

trQC

1

x

trTvtrTD

t

trT

px

2

ρ+

∂∂−∇=

∂∂

9

Pulsed and cw excitation with a Gaussian beam

• Pulsed:

• cw:

E0 ….pulse energy a…..pump laser beam radius

t0 …..pulse width Pav…cw laser average power

α…...absorbance (cm-1) ω…..modulation frequency

Q(r,t) = 2αE0

πa2t0

exp −2(x2 + y2) /a2[ ]

Q(r,t) = 2αPav

πa2 exp −2(x2 + y2) /a2[ ]{ }× (1+ cosωt)

10

Thermal lens signal

• w2(0)….radius of an unperturbed probe beam at the detector site

• w2(t)…..time dependent radius of a probe beam perturbed by the thermal lens

• w0…….radius of the probe beam at its waist

)0(w

)0(w)t(w)t(s

22

22

22 −=

−++

−=

2

21212

0

2

220

22 )t(f

zzzz

z

1

)t(f

z1w)t(w

11

Simplifications for usual far field experimental configuration

• z2>>z1, z2>>z0 = πw02/λ

• f(t)>>z1, f(t)>>z0

• @ t=0, f(0)=∞– λ …..probe beam wavelength

– z0…..confocal distance

)t(f

z2)t(s 1−=

Refractive index change and focal distance of thermal lens

• n0….. unperturbed refractive index at ambient temperature TA

collinear: transversal:

• f…..thermal lens focal length

• ℓ….interaction length

)t,y,x(TT

nn)t,y,x(n

AT0 ×

∂∂+=

dyx

T

T

n

f

12

2

∫∞

∞−

∂∂

∂∂−=

∂∂

∂∂−=

2

2

r

T

T

n

f

1lℓ

13

TLS signal for collinear configuration

• Pulsed: (t0→0)

• cw:

– tc….time constant = a2ρcp/4k=4a2D

– k….thermal conductivity of the sample

2cc

210

)t/t21(

1

tka

)T/n(zAE4)t(s

+π∂∂−=

)t2/t1(

1

ka

)T/n(APz2)t(s

c2

1

+π∂∂−=

lα=A ℓ

TLS signalfor transversal configuration

• Pulsed: (t0→0)

• cw:2/3

cc

10

)t/t21(

1

kat2

)T/n(zE2)t(s

+π∂∂α−=

2/1c

1

)t2/t1(

1

ka2

)T/n(Pz2)t(s

+π∂∂α−=

TLS signal form

Time/ms scale

sign

al

sign

al

Excitation Excitation

Pulsed CW

Time/ms scale

16

TLS signalin a single beam experiment

• P/a2 changes with increasing z1

• the signal maximum is found at z1=z0

(parabolic model) z0 = πa02/λ

• or at z1=z0√3 (aberrant model)

)t2/t1(

1

k

)T/n(AP)t(s

c+λ∂∂−=

+λ∂∂−= −

3)t/t1(

1tan

k

)T/n(AP)t(s

c

1

E - Enhancement factor in TLS

EA31arctgλk

AdTdn 2.303PI∆I 2.303=

−=

Thermo-optical properties of solvents for TLS measurements

TLS - advantages

• High sensitivity

• signal proportional to excitation laser power

• absorbances as low as 10-7 can be measured

• Enables On-line detection

• fast response of TLS signal (on µs to ms time scale)

• Capability of measuring small samples

• sub-pL volumes can be probed

• detection in microfluidic systems

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TLS – drawbacks and solutions

• Sensitivity still needs improvement– Higher laser power? (photo-labile compounds)

– Modify solvents

• Limited availability of laser sources– Coloring reactions, indirect detection

• Poor selectivity– Single wavelength measurements

– Coupling to separation techniques (HPLC, IC, CE)

• Photodegradation– Measure in flowing systems

20

21

Dual beam TLS spectrometer for detection in FIA, HPLC and bioassays

photodiode

pump

sample cell

filter

chopper Lock-in

PC

photodiode

pump

lens

sample cell

filter

pump laser, e.g.Ar, Kr, NdYAG, CO, Ti-safire240 – 5000 nm,up to few 100 mW

probe laserlow power, He-Ne, diode

Ref.

Adjustable beam size/position TLM

23

Temperature dependent TLS signal in water

100 200 300 400 500 600 700 800 900 1000

150

200

250

300

350

400

- 5.87 deg. C - 2.80 deg. C + 0.02 deg. C + 3.26 deg. C + 7.58 deg. C + 20.00 deg. C

Pro

be b

eam

inte

nsity

(m

V)

Time (ms)

contribution of dn/dc !

24

The effect of photosensitivity onTLS signal (case of Cr-DPC)

0.00 0.07 0.14 0.21 0.28 0.350.42

0.48

0.54

0.60

0.66

0.72

0.78

0.84

0.90

0.96 Blank Experimental data Fitting using Eq. (4)

Ce = (1.00 ± 0.01)x10-7 Mol/LK

T = (8.0 ± 0.1) s-1

θ´ = (1.9 ± 0.01)x106 L/MolC

0 = 4x10-7 Mol/L

tc = 0.004 sT

rans

ient

sig

nal (

au)

Time (s)

b

a

c

Cr(VI) Experimental data Fitting using Eq. (11)

Simulation using Eq. (4)

t1/2=80 ms

Pedreira et al.: J. Appl. Phys., 100 (2006)Chem. Phys. Lett. 396(2004)221

HPLC-TLS degtermination of carotenoids in blood plasma

∆I∝λ

26

The role of BTL in the transport of antioxidants across the cellular wall

0

0,2

0,4

0,6

0,8

1

1,2

0 10 20 30 40 50 60 70 80 90

Time (s)

Rel

ativ

eb

iliru

nin

con

cen

trat

ion

Glucouse Lactate Ethanol

Lactate + specific antibody

0

0,2

0,4

0,6

0,8

1

1,2

0 10 20 30 40 50 60 70 80 90

Time (s)

Lactate

Low cytosolic NADH/NAD+

High cytosolic NADH/NAD+

Improvement of selectivity by separation techniques (HPLC, IC)

Martelanc M., Žiberna L., Passamonti S., Franko M.:Anal. Chim. Acta 809, 2014, 174–182.

LOD: 90 pM

LOQ: 250 pM

Free bilirubin in blood serum samples

Simultaneous determination of bilirubin and biliverdin

Time (s)

TLS

sig

na

l (m

V)

BR

BV

M.F.: 67 % MeOH

M.F.: 97 % MeOH (1.5 nM)

(5 nM)

154(2016)92–98

First detection and modulation of bilirubin in vascular endothelial cels

Scientific Reports 6:29240 DOI: 10.1038/srep29240

0 5 10 15 20 25

time (min)

Addition of poly-APS (10 mg/L)

Double dual beam TLS

UV -Vis spectrophotometer

0 5 10 15 20 25

time (min)

Advantages of TLS: extremely high sensitivity, small sample capability(100 – 1000 times lower LOD than SF)

0 10 20 30 40 50 60 70

0

100

200

300

400

1 ml sampleof iceberg lettuce

1 ml sample of onion

28.59

42.77

49.22

lock

-in s

igna

l/mV

time/min

Bioanalytical FIA system

AChECOLUMN

TLS DETECTION CELL

substrate

Valve1

sample

PUMP

Carrier buffer

Enzymeregenerator

0a

aA x

R = n

n-1

Valve2

L. POGAČNIK, M. FRANKO, Biosens. Bioelectron.. 2003, 18, 1-9

32

33

FIA -ELISA -TLS Detection of Food Allergens

Beta-lactoglobulin

• TLS signal proportional to the amount of allergen retained on the immunocolumn

• Analysis time < 8 min

LOD for beta-lactoglobulin (BLG) =2.3 pg/ 100 µL (190 pg by ELISA – Bethyl)LOD for ovalbumin (OVA) =1 ng/ 100 µL (1 µg by ELISA – Abcam)

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Determination of BLG and OVA by FIA -ELISA -TLS

Determination of NGAL -a biomarker of acute kidney injury

LOD= 1.5 pg/mL TLS signals for replicate injections of two aliquots of 500-times diluted blood plasma sample (217 pg/mL)

Pump beam: 476.5 nmSpl. Injection: 0.5 µLFlow rate: 5 µL/min

LOD= 10 pg/mL

Detection of NGAL in blood plasma of patients after percutaneous coronary

angiography (injection of contrast agents) by Tecanreader and µFIA -TLM

2

Basic literature on TLS

• S.E. Bialkowski, Photothermal Spectroscopy Methods for Chemical Analysis, John Whiley & Sons, Inc., New York, 1996.

• C.D. Tran , M. Franko, "Thermal Lens Spectroscopy" in: Encyclopedia of Analytical Chemistry, (Ed. R.A. Meyers), John Wiley: Chichester., 2010. DOI: 9780470027318.

• M. Franko, “Bioanalytical Applications of TLS”, in: Thermal wave physics and related photothermal techniques: Basic principles and recent developments, ISBN 978-81-7895-401-1 (Ed. E. Marin), Research Signpost Press, 2009

• M. Franko, Thermal Lens Spectrometric Detection in Flow Injection Analysis and Separation Techniques, Appl. Spectrosc. Rev. 43, 2008, 358-388.

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• Kitamori, T., Tokeshi, M., Hibara, A. and Sato, K., Thermal lens microscopy and microchip chemistry. Anal. Chem., 76, 2004, 52A-60A.

• M. Franko, C.D. Tran, Analytical Thermal Lens Instrumentation, Rev. Sci. Instrum. 67, 1996, 1-18.

• Liu M., Franko M.: Progress in thermal lens spectrometry and its application in microscale analytical devices, Crit. Rev. Anal. Chem. 44, 2014, 328-353.

39

Basic literature on TLS

Acknowledgements• Mingqiang Liu• Tatjana Radovanović• Ambra Delneri• Ana Čevdek• Lea Pogačnik• Jasmina Kožar-Logar• Franja Prosenc• Dr. Mitja Martelanc• Prof. Dorota Korte• Prof. Chieu D. Tran• Prof. Igor Plazl et al.• Funding: ARRS,

– Trans2Care, MIZŠ,– AdFutura