tl-spectrometry and applications in biomedical...
TRANSCRIPT
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
19
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)
34
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.
38
• 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