Introduction: GFAP as a promising biomarker for brain-related...
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1. Introduction: GFAP as a promising biomarker for brain-related pathologies 3. Conclusion: strategy comparison and perspectives 2. Experimental: biomarker detection with three strategies GLIAL FIBRILLARY ACIDIC PROTEIN (GFAP) DETECTION IN SERUM: A COMPARISON BETWEEN THREE FUNCTIONALIZATION STRATEGIES M. Agostini*, G. Greco, F. Amato, M. L. Vieri, M. Cecchini Laboratorio NEST, Scuola Normale Superiore and Istituto di Nanoscienze - CNR, Pisa (Italy) * [email protected] [1] J. Tichy et al., J. Neuroncol., 126 (2016) [2] J. Song et al., Adv. Func. Mater., 27 (2017) [3] M. Agostini et al., Sensor. Actuat. B-Chem, 254 (2018) References: web.nano.cnr.it/neurosens The glial fibrillary acidic protein (GFAP) is a promising biomarker for severe brain-related pathologies in humans. The interest on GFAP as a biomarker is related to two main aspects. First, the impor- tance of the pathologies that can be addressed: severe or mild traumatic brain injuries (TBIs), glioblastoma multiforme (GBM) cancer with or without blood-brain-barrier disruption. Second, the possi- bility of detecting this biomarker in circulating blood [1,2]. In the presented work, we compare three different functionalization strategies and antibodies for GFAP detection both in clean buffers (phosphate saline buffer, PBS) and in fetal bovine serum (FBS). F1 exploits a streptavidin-polyethyleneglycol(2kDa)-thiol (pegSTREP) and a biotinylated antibody (antiGFAP) as a probing layer. F2 is based on the capability of the protein-G (prG) to bind the heavy-chain of a non-conjugated antiGFAP. In F3 the antiGFAP is split in two halves (sp-antiGFAP) with a reducing agent (TCEP) and the surface directly functionalized. The resulting limits of detection (LoDs) are extrapolated from the Langmuir isotherm fit. The experiments are performed with gold-plated quartz-crystal-microbalance (QCM) sensors. The picture that emerges from the experiments highlights the superior capability of the prG-antiGFAP (F2) and sp-antiGFAP (F3) layers to detect GFAP dissolved in PBS. The resulting limits of detec- tion are of the order of tens of nanomolar. However, this result is reversed in the case of serum as buffer. The high molecular concentration of FBS (e.g., albumin ~40 mg/mL) shields the GFAP detec- tion for F2 and F3. On the other hand, the pegSTREP-antiGFAP layer (F1) confirms the capability of detecting GFAP concentrations higher or equal to 34 nM, as found for PBS, probably owing to the PEG antifouling properties. It is worth noting that the investigated functionalizations are designed for a gold surface, which is one of the most common type of sensing interface owing to its optical properties, material stability, and ease of fabrication, patterning, and functionalization [3]. This work is supported by the “FAS14 GLIOMICS” project of “Regione Toscana”: proteomics/genomics/metabolomics for Glioblastoma biomarkers discovery and their detection with an ultrasensitive platform. S Au streptavidin antiGFAP GFAP biotin S S S PEG 2k prG antiGFAP GFAP S Au S S S sp-antiGFAP S S S S S S S S Au GFAP F1 F2 F3 1p 10p 100p 1n 10n 100n 1μ -60 -50 -40 -30 -20 -10 0 LoD = 34 nM Langmuir isotherm fit QCM frequency shift, Hz GFAP concentration in PBS, M 1p 10p 100p 1n 10n 100n 1μ -60 -50 -40 -30 -20 -10 0 LoD = 12 nM Langmuir isotherm fit QCM frequency shift, Hz GFAP concentration in PBS, M 1p 10p 100p 1n 10n 100n 1μ -60 -50 -40 -30 -20 -10 0 LoD = 14 nM Langmuir isotherm fit QCM frequency shift, Hz GFAP concentration in PBS, M 0 20 40 60 80 100 120 140 160 180 -70 -60 -50 -40 -30 -20 -10 0 rinsing QCM frequency shift , Hz Time, min antiGFAP pegSTREP rinsing 0 20 40 60 80 100 120 140 160 -180 -150 -120 -90 -60 -30 0 QCM frequency shift , Hz Time, min rinsing antiGFAP prG rinsing 0 10 20 30 40 50 60 70 80 90 -90 -75 -60 -45 -30 -15 0 QCM frequency shift, Hz Time, min rinsing sp-antiGFAP <2 20p 2 .2n 22n 220 n 2 .2 u -20 -10 0 10 20 GFAP concentration in FBS, M QCM frequency shift, Hz <220p 2.2n 22n 22 0n 2.2 u -4 -3 -2 -1 0 1 ** GFAP concentration in FBS, M QCM frequency shift, Hz <220p 2.2n 22n 22 0n 2.2 u -15 -10 -5 0 5 ** **** GFAP concentration in FBS, M QCM frequency shift, Hz
Transcript of Introduction: GFAP as a promising biomarker for brain-related...
1. Introduction: GFAP as a promising biomarker for brain-related pathologies
3. Conclusion: strategy comparison and perspectives
2. Experimental: biomarker detection with three strategies
GLIAL FIBRILLARY ACIDIC PROTEIN (GFAP) DETECTION IN SERUM: A COMPARISON
BETWEEN THREE FUNCTIONALIZATION STRATEGIES
M. Agostini*, G. Greco, F. Amato, M. L. Vieri, M. Cecchini
Laboratorio NEST, Scuola Normale Superiore and Istituto di Nanoscienze - CNR, Pisa (Italy)* [email protected]
[1] J. Tichy et al., J. Neuroncol., 126 (2016)[2] J. Song et al., Adv. Func. Mater., 27 (2017)[3] M. Agostini et al., Sensor. Actuat. B-Chem, 254 (2018)
References:web.nano.cnr.it/neurosens
The glial fibrillary acidic protein (GFAP) is a promising biomarker for severe brain-related pathologies in humans. The interest on GFAP as a biomarker is related to two main aspects. First, the impor-tance of the pathologies that can be addressed: severe or mild traumatic brain injuries (TBIs), glioblastoma multiforme (GBM) cancer with or without blood-brain-barrier disruption. Second, the possi-bility of detecting this biomarker in circulating blood [1,2].In the presented work, we compare three different functionalization strategies and antibodies for GFAP detection both in clean buffers (phosphate saline buffer, PBS) and in fetal bovine serum (FBS). F1 exploits a streptavidin-polyethyleneglycol(2kDa)-thiol (pegSTREP) and a biotinylated antibody (antiGFAP) as a probing layer. F2 is based on the capability of the protein-G (prG) to bind the heavy-chain of a non-conjugated antiGFAP. In F3 the antiGFAP is split in two halves (sp-antiGFAP) with a reducing agent (TCEP) and the surface directly functionalized. The resulting limits of detection (LoDs) are extrapolated from the Langmuir isotherm fit. The experiments are performed with gold-plated quartz-crystal-microbalance (QCM) sensors.
The picture that emerges from the experiments highlights the superior capability of the prG-antiGFAP (F2) and sp-antiGFAP (F3) layers to detect GFAP dissolved in PBS. The resulting limits of detec-tion are of the order of tens of nanomolar. However, this result is reversed in the case of serum as buffer. The high molecular concentration of FBS (e.g., albumin ~40 mg/mL) shields the GFAP detec-tion for F2 and F3. On the other hand, the pegSTREP-antiGFAP layer (F1) confirms the capability of detecting GFAP concentrations higher or equal to 34 nM, as found for PBS, probably owing to the PEG antifouling properties. It is worth noting that the investigated functionalizations are designed for a gold surface, which is one of the most common type of sensing interface owing to its optical properties, material stability, and ease of fabrication, patterning, and functionalization [3].This work is supported by the “FAS14 GLIOMICS” project of “Regione Toscana”: proteomics/genomics/metabolomics for Glioblastoma biomarkers discovery and their detection with an ultrasensitive platform.
S
Au
streptavidin
antiGFAPGFAP
biotin
S SS
PEG2k
prG
antiGFAP
GFAP
S
Au
S SS
sp-antiGFAP
SS SSSS SS
Au
GFAP
F1 F2 F3
1p 10p 100p 1n 10n 100n 1µ
-60
-50
-40
-30
-20
-10
0
LoD = 34 nM
Langmuir isotherm fit
QC
M fr
eque
ncy
shift
, Hz
GFAP concentration in PBS, M1p 10p 100p 1n 10n 100n 1µ
-60
-50
-40
-30
-20
-10
0
LoD = 12 nM
Langmuir isotherm fit
QC
M fr
eque
ncy
shift
, Hz
GFAP concentration in PBS, M1p 10p 100p 1n 10n 100n 1µ
-60
-50
-40
-30
-20
-10
0
LoD = 14 nM
Langmuir isotherm fit
QC
M fr
eque
ncy
shift
, Hz
GFAP concentration in PBS, M
0 20 40 60 80 100 120 140 160 180
-70
-60
-50
-40
-30
-20
-10
0
rinsing
QC
M fr
eque
ncy
shift,
Hz
Time, min
antiGFAPpegSTREP
rinsing
0 20 40 60 80 100 120 140 160-180
-150
-120
-90
-60
-30
0
QC
M fr
eque
ncy
shift,
Hz
Time, min
rinsing
antiGFAPprG
rinsing
0 10 20 30 40 50 60 70 80 90
-90
-75
-60
-45
-30
-15
0
QC
M fr
eque
ncy
shift
, Hz
Time, min
rinsing
sp-antiGFAP
<220p 2.2
n22
n22
0n 2.2u
-20
-10
0
10
20
GFAP concentration in FBS, M
QC
M fr
eque
ncy
shift
, Hz
<220p 2.2
n22
n22
0n 2.2u
-4
-3
-2
-1
0
1
**
GFAP concentration in FBS, M
QC
M fr
eque
ncy
shift
, Hz
<220p 2.2
n22
n22
0n 2.2u
-15
-10
-5
0
5
**
****
GFAP concentration in FBS, M
QC
M fr
eque
ncy
shift
, Hz
