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ISyPeM IIGiulia CappiÉcole polytechnique fédérale de Lausanne
Annual Plenary Meeting of the Nano-Tera programMay 4th, 2015
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Therapeutic drug monitoring (TDM)
time after dose (hrs)
Imati
nib
expo
sure
(ng/
ml)
Courtesy of N. Widmer
Modern anticancer agents
Immunosuppressants
Antiretrovirals
Antibiotics
2
Candidates for therapeutic drug monitoring
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Therapeutic Drug Monitoring Today
Abbott "Architect ci8200"
Blood sample taken at the doctor’s office or
at the hospital
Transfer to and measurement at
central clinical labsInterpretation of the
result by a specialized doctor
Today this procedure severely restricts the applicability of TDM
Treatment duration (days)
Drug
Con
cent
ratio
n
Patient
Target
?
http://e-sante.futura-sciences.com
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Drug quantification in body fluids
LC-MS/MS
Agilent
Used in Clinical facilities
Abbott "Architect ci8200”
FPIA (fluorescence polarization immunoassay) CMIA (chemiluminescent microparticle immunoassay) MIA (magnetic immunoassay)
Point-of-need
Syva RapidTest®
EMIT (Enzyme multiplied immunoassay technique)
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Point-of-need therapeutic drug monitoring
iSTAT, Abbott
ClonitStMicroelectronicsCobas product
lines Roche Diagnostics
Accu-Chek product lines. Roche Diagnostics
ISyPeM approach toTDM
Top bench
Hand-held
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ISyPeM. A solution to enable drug-based therapeutic drug monitoring
• Low-sample volumes
• Miniaturized reader
• Cartridge that can be stored with stable reagents
• Monitoring and prediction interface for clinicians and practitioners
• Local or remote
• Extension of eHealth medical standards
• Data integrity
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ISyPeM: A solution to enable drug-based therapeutic drug monitoring
• Cartridge that can be stored with stable reagents
In vitro selection of capture molecules for drug targets
HOMOGENEOUS AND HETEROGENEOUS ASSAYS
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In vitro selection of monoclonal antibodies for Tacrolimus
Heinis LabAntibody phage display
Sangram Kale
NO
OH
O
NO
O
H2NO COOH
NaOAc, rtdry MeOH, 16 h
HN NH
S
O
HN
OO
OO
HN
HN NH
S
O
HNOO
OOH2N
HATU, HOBt, DMF, DIEA, 5 h
Target:tacrolimus
Phage-antibodies were successfully selected for Tacrolimus and expressed
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In vitro selection of DNA-aptamers for Tobramycin
Spiga F. M. et al. ACS Comb. Sci. 2015, featured on the cover of May Issue
Guiducci Lab
F. M. SpigaNow with Creoptix Sensors
No drug derivatization High affinitySpecificFunctional in serum
DNA beacon aptamers Capture Selex
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Low-volume sample preparation on chip
FPIA of Tobramycin performed in glass microcapillaries
Squared glass capillary
Plasma extraction from whole blood
Yield outperforms any passive on-chip method reported to date http://dbs-system.ch
300 µm
0 2 4 6 8 10 120.140
0.160
0.180
0.200
0.220
0.240
0.260
Concentration [µg/ml]
Aniso
trop
y
Diana BurgheleaJ-M Segura
reference systemcapillary
David Forchelet
Renaud Lab
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Measurement system: miniaturization of an established assay technique
25 cm
20 cm
Fluorescence Polarization ImmunoassayCompetitive assayHighly-sensitiveCompatible with small volumes
Requires derivatization of drug molecules
Martial Geiser team
*
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Label-free quantification of drug from serum samples
200 nm
Layers of functionalized gold nanoislands
Heterogeneous assay based on binding kineticsNo drug derivatizationNo additional reagents
Giulia Cappi
Transmission Surface Plasmon ResonanceChange of optical properties of the interface
Cappi G. et al. Sensors and Actuators 2013
Guiducci Lab
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Hue readout of surface plasmonics
Hue readout of surface plasmonics
CMOS image sensorGuiducci Lab
Compact read-out system
Cappi G. et al. Analyt. Chem. 2015
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Measurement of binding kinetics of DNA aptamer-Tobramycin
KD = 200 nM, same as extracted from commercial SPR
Transmission SPR signal
Cappi G. et al. Analyt. Chem. 2015
Guiducci Lab
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Tobramycin quantification from serum
Jang, H. R., et al. Anal. Chem. 86, 814–819 (2013)
Chang, A. L. et al., Anal. Chem. 2014, 86, 3273−3278.
✔ Small molecules✔ Complex Matrix
✖ Small molecules✔ Complex Matrix
✔ Small molecules✖ Complex Matrix
Cappi G. et al. Analyt. Chem. 2015
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Supporting clinical interpretation
Population-based percentiles
Individual concentration vs time profile
Dosing schedule suggestion
Div. Clin. Pharmac.
Aline Fuchs
Yann Thoma
EzeCHiel interface
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Data management
MOLIS, CHUVEzeCHielRSDB
“translate” from one representation to another
Data Exchange between EzeCHiel and DB of the
Medical Institution
Health data
POC
Medical DB
Dubovitskaya, A. Privacy Preserving Interoperability for Personalized Medicine, in: Swiss Medical Informatics, Swiss Society for Medical Informatics, Switzerland, 2014
Alevtina DubovitskayaData securityDynamic Health Data Aggregation
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Population pharmacokinetics
Div. Clin. Pharmac.
Br J Clin Pharmacol. 2014 Nov;78(5):1090-101
Meta-analysis. Implementation and validation
Pharmacokinetic models
Aziz ChaouchAline Fuchs
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BACK UP
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Filtration and FPIA on paper
FPIA for tacrolimus with antibody selected by EPFL (tacrolimus fluorescent derivate needed)
Aptamer for imatinib
EzeCHiel start up and beta testers
Interoperability with MOLIS database
Population pharmacokinetic studies on tacrolimus and tobramycin
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Personalized Medicine and TDM Therapeutic drug monitoring (TDM) is foundational to the concept of
personalized medicine. The concept has later evolved to include pharmacogenomic and
other biomarker-driven strategies for patient segmentation
In the clinics: TDM transformed drug therapy by affording the ability to characterize sources of variability in drug disposition and response to individualize drug dosing. Initially, TDM formed the key conceptual basis for personalized medicine
Interest from Pharma:Personalized medicine takes into account the fact that 30% of drugs investigated in clinical trials fail because of lack of efficacy*, and its premise is that stratifying patients and diseases into molecular subtypes and treating with subtype-specific drugs will improve drug efficacy.
JD Momper and JA Wagner, Clinical pharmacology & therapeutics, VOLUME 95 NUMBER 2 2014 Li and Jones Genome Medicine 2012, 4:27 T. Buclin, Who is in charge of assessing therapeutic drug monitoring? The case of imatinib. Lancet Oncology 2012Kola I, Landis J: Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug Discov 2004, 3:711-715
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Research level drug quantification approaches
MEDICUCSB
Griss R, Schena A, Reymond L, Patiny L, Werner D, Tinberg CE, Baker D, Johnsson K. | . Nature Chemical Biology
The sensor molecule works by binding the drug circulating in the patient’s bloodstream and changing color accordingly. The molecule itself is made up of four components. One component is a receptor protein, which can bind the molecules of the target drug. The second component is a small molecule similar to the target drug, which can bind the drug receptor. The third component is a light-producing enzyme called luciferase, and the fourth is a fluorophore molecule that can modify the color of the luciferase’s light when it comes close to it.
LUCIDEPFL
We conjugated its 3 end to a methylene blue (MB) reporter and its 5 ′ ′end to an alkane thiol for attachment to gold working electrodes within the MEDIC chip micro- channel . Target binding induces a conformational change in the aptamer that modulates electron transfer between MB and the electrode . This modulation is expected to produce a readily measurable change in current at the MB reduction peak when the sensor is interrogated using square-wave voltammetry (SWV).
Real-Time, Aptamer-Based Tracking of Circulating Therapeutic Agents in Living Animals, Brian Scott Ferguson et al.Sci Transl Med 5, 213ra165 (2013)
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Where we stand. Why we are unique.
Well-defined application objective (in the short-medium term) candidate diseases clearly identified point-of-care use for therapeutic drug monitoring in clinical or private
practice settings support to MDs. Development of comprehensive, flexible, but user
friendly code
Unique analytical approach, combining development of novel in vitro selection protocols of drug capture
molecules (small drugs, even with no receptor or antibody available) assay development (new molecules employed in traditional or
alternative detection techniques)
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Where we stand. Why we are unique. (cont.)
Committed to the feasibility of our solutions Data management: compliancy and interoperability Assay development: integration, sample size, sensor characteristics
responding to realistic requirements for development
Leveraging nearby companies’ potential interest Pharma: Novartis, Roche, DebiopharmAssays/Systems: STMicroelectronics, Abionics, Mycartis, DBS systems, …
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PROJECT STRUCTURE AND TIMELINE
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Research areas – Internal structureNovel capture probes and quantification in complex matricesC. Guiducci, CHUV, STMicroelectronics and C. Heinis
Miniaturization of sample preparation and FPIAJ.M Segura, C. Heinis, Ph. Renaud, CHUV
Interpretation and dose adjustmentDatabase exchange and interoperabilityY. Thoma, CHUV, M. Schumacher
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Internal organization in teams and team leaders
System integrationM. Pfeifer
Assays for drug quantification/sem
i-quantificationJ-M Segura
Data interpretation and data
managementY. Thoma
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Milestones expected by May 2015WORKPACKAGE 1 - Sample preparationLMSI- EPFL, LDI-HES-SO Valais (Prof. Marc Pfeifer), CHUVM1.1: demonstration of volumetric sampling of blood plasma, concept validation of FP measurements in paper. Concept of the whole system architecture.
WORKPACKAGE 2 - Highly selective capturing molecules for the target drugsLPTT-EPFL, CLSE-EPFL and CHUVM2.1: Phage selection and characterization of antibody specific for imatinib (will be provided to Segura for FP assay development); expression of tacrolimus-specific Ab and characterization (will be provided to Segura).M2.4: report on SELEX-driven convergence of the DNA library and choice of the 10 (aptamer) candidates
WORKPACKAGE 3 - Drug detection by miniaturized systemsCLSE-EPFL, LDI-HES-SO Valais, STMicroelectronics, CHUVM3.1: fluorescent and biotin derivatesM3.2: validated FPIA protocols for tacrolimus and tobramycinM3.5: choice of an opto-electronic device (including light source and detection) for the FP method and functioning table-top system.
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Milestones expected by May 2015 (cont.d)
WORKPACKAGE 4 - Data Analysis, Interoperability and Intelligent DatabasesHEIG-VD, AISLab HES-SO Valais, CHUV
M4.1: Local connected version of the database integrated with EzeCHiel
WORKPACKAGE 5 - DemonstratorsLDI-HES-SO Valais, STMicroelectronics, CHUV
WORKPACKAGE 6 - Consolidation of pharmacokinetic/pharmacodynamic reference data, clinical exploitation of results and validation of TDM at point of careCHUV, ASILab HES-SO Valais, HEIG-VDM6.1: theoretical elaboration of a PKPD meta-analysis concept
M6.4: validation report about the interpretation algorithms as implemented in the EzeCHieL tool
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FORTHCOMING MILESTONES
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Milestones expected by Oct 2015WORKPACKAGE 1 - Sample preparationLMSI- EPFL, LDI-HES-SO Valais (Prof. Marc Pfeifer), CHUVM1.2: concept validation of fluid transfer between two paper layers by contact and sample concentration by evaporation
WORKPACKAGE 2 - Highly selective capturing molecules for the target drugsLPTT-EPFL, CLSE-EPFL and CHUVM2.5: report on the chosen aptamer binding characteristics towards the target drug M2.2: Affinity and/or stability maturation of imatinib-specific antibody according to the needs of Segura; CDR grafting of tacrolimus-specific Ab into stable IgG framework (will be provided to Segura).
WORKPACKAGE 3 - Drug detection by miniaturized systemsCLSE-EPFL, LDI-HES-SO Valais, STMicroelectronics, CHUVM3.3: comparison of assays in Microsystems M3.7: full description of the requirement specification for the demonstrators to be deployed.
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Milestones expected by Oct 2015 (cont.d)
WORKPACKAGE 4 - Data Analysis, Interoperability and Intelligent DatabasesHEIG-VD, AISLab HES-SO Valais, CHUV M4.2: Interoperable server prototype with eHealth standards
WORKPACKAGE 5 - DemonstratorsLDI-HES-SO Valais, STMicroelectronics, CHUV M5.1:Advanced Breadboard (MAY 2016)
WORKPACKAGE 6 - Consolidation of pharmacokinetic/pharmacodynamic reference data, clinical exploitation of results and validation of TDM at point of careCHUV, ASILab HES-SO Valais, HEIG-VDM6.2: publication of a computer too implementing PKPD meta-analysis M6.5: validation report about the measurement results produced by the analytical tool
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Timeline for demonstrators1ST YEAR
Nov 2013 – Oct 20142ND YEAR
Nov 2014 – Oct 2015
FUNCTIONAL MODULES PRE-PROTOTYPES
FIRST ASSEMBLED PROTOTYPE FIRST
TESTING PHASE
SYSTEM PROTOTYPE
SECONDTESTING PHASE
MODELING SOFTWARE
INTELLIGENT DATABASES
3RD YEARNov 2015 – Oct 2016
4th YEARNov 2016 – Oct 2017
ADVANCED PROTOTYPE
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PROJECT CONTEXT AND AIMS
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DEADLINES AND DATES
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Deadlines
28th of March send out my presentation for the REVIEW MEETING (6th of May in Bern)
13th of April updated scientific report which will cover (Nov. 1st, 2013 – March 2015). Please, send me your updated contribution, according to the usual template:https://www.dropbox.com/sh/5asho7dmnqmcowv/AADjc7ULkxvbZV9Ds2wBYyDva?dl=0
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Mark your calendars
REVIEW MEETING 6th of May (afternoon) in Bern. Co-PIs
ANNUAL N-T MEETING 4-5th of May
in Bern. All ISyPeM participants. We have to choose a PhD student to present existing achievements of our work!
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CLSE and ISyPeM
DNA capture molecules
Analysis of matrix
effects
Label-free measurement techniques
Nano-patterned surfaces
CHUV• Identification of drug candidates• Comparison with standard measurement systems• Samples
J-M/HES-SO• Use of DNA aptamers
for FPIA assays
Philippe Renaud• Integration of sample
preparationSTMicroelectronics
• Optical readout
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aptamers
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Challenges in small molecules assays
Lack of suitable capture molecules ANTIBODIES
150 kDaAPTAMERS10 kDa - 30 kDa
Sensing in matrices:• Non specific
adsorption • Interference
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Selection strategy for DNA aptamer
Ligand-binding oligos amplified by PCRLonger randomized region
Tobramycin.Selected best binders (CLSE) better or comparable to existing aptamers
Fabio M. Spiga, Paolo Maietta and Carlotta Guiducci, “More DNA−aptamers for small drugs: a capture−SELEX coupled with Surface Plasmon Resonance and High Throughput Sequencing”, ACS Combinatorial Science, accepted for publication
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Minimizing the number of cycles
SPR monitoring Specificity arises for sublibrary at cycle 8
High throughput sequencingThe most enriched sequences are already visible after only two capture-SELEX cycles
Tobramycin
Fabio M. Spiga, Paolo Maietta and Carlotta Guiducci, “More DNA−aptamers for small drugs: a capture−SELEX coupled with Surface Plasmon Resonance and High Throughput Sequencing”, ACS Combinatorial Science, accepted for publication
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Selectivity towards Kanamycin
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Surface preparation45
Controlaptam
er
Passivation
Analysis
50μm
500μm
~2m
m
Specific
aptamer
Detection is label-free Does not require drug derivatization
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46Binding kinetics of Tobramycin on SPR in serum Tobramycin concentration range of initial serum samples: [5µM
- 100µM] Derived sample characteristics:
Serum 10%, Tween 20 0.01%, Tobr [0.5µM - 10µM]
Detection of sample concentration is preceded by calibration
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R2=0.993
Determination of Tobramycin concentration in serum
47
Tobramycin concentration range of initial serum samples: [5µM - 100µM]
Derived sample characteristics:Serum 10%, Tween 20 0.01%, Tobr [0.5µM -
10µM]• LoD 0.15 µM
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Conclusive remarks48
Tobramycin detection [0.5µM - 100µM] in serum samples Linearity up to 10 µM Label-free, direct detection Volume of serum needed: 20µl
Sample preparation time: 45 min per sample (from serum) Analysis time: half an hour per sample
Tobramycin-specific DNA aptamers successfully selected with KD <1 µM
No drug derivatization Relatively simple and fast aptamer selection protocol
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Direct detection of drug molecules in patient sera
6% error24% error(Tolerance in this range: 20%)
Standard addition methodDirect detection by SPR based on immobilized DNA aptamers specific for Tobramycin
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Classic Capture
• Evolution-like iterative selection system• Long(er) sequences, immobilised oligo• More specificity - more possibilities for binding detection• And they are selected on a surface
New SELEX concept50
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Design of the assay
Sensitivity in the physiological range Specificity in presence of concomitant
drugs Simple and fast sample preparation Transferability of the analytical
approach to other drugs Avoid drug derivatization Straightforward selection of new probes
Sample size efficiency
51
Specifications for a companion diagnostic device in TDM
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Serum sample preparation for drug concentration analysis
Precipitation upon protein denaturation
Filtering with low cut-off (eg: < 3 kDa)
Anti-fouling solutions to prevent non specific adsorption on the surface
52
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Determination of Tobramycin concentration53
Tobramycin concentration range of initial serum samples: [5µM - 100µM]
Derived sample characteristics:Serum 10%, Tween 20 0.01%, Tobr [0.5µM -
10µM]Some numbers: Volume of patient serum needed: 20µl
Sensing region covered by aptamers: 1.05 mm2
Moles of aptamers required: 150pmol Manipulation time: ~10min per sample Run time: ~12min per cycle (2 cycles per
sample)
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Variability in treatment response
54
T. Buclin, et al.“Who is in charge of assessing therapeutic drug monitoring? The case of imatinib”. Lancet Oncol. 2011;12(1):9-11. 0 1 2 3 4 5 6 7 8 9 10 11 12
Treatment duration (days)
Drug
Con
cent
ratio
n (μ
g/m
L)
0.0
0.4
0.8
1.2
1.6
2.0
2.4
Patient
Target
Risk of adverse effects
Risk of inefficacy
Imatinib 400 mg qd
800 mg qd
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Therapeutic drug monitoring at the point-of-care Compatibility/
Suitability Acceptable and suitable
setting for in-field drug measurement, considering disease, frequency of monitoring, treatment toxicity, costs
Need for stable and specific molecular assay for drug measurement, compatible with on field biosensors
55
Information and Interpretation
Missing statistical population dataNeed for formal and accessible models of interpretationData exchange
DA RISITEMARE
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tSPR
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Transmission SPR. Direct detection in undiluted serum.
0 5 10 15 200
2
4
6
8
10
12
14
16
x 10-4
Tobramycin concentration (µM)
hue
Aptamer (average)Langmuir interpolationControl
KD= 0.26 µM
G. Cappi, E. Accastelli, V. Cantale, M. A. Rampi, L. Benini, C. Guiducci ., Sensors and Actuators B: Chemical, 2013G. Cappi, Spiga, F.M., Moncada, Y., Ferretti, A., Beyeler, M., Bianchessi, M., T. Buclin, L. Decosterd, Guiducci, C. Analytical Chemistry, accepted for publications
Undiluted serum
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Portable system for array measurements
Power supply
Plasmonic
sensor
Computer
White LED
CMOS image sensor
Diffuser
Lens
58
• Power supply via USB• Real-time display of images
registered
Regions of interest
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Do Hue and Peak have the same plasmonic information?
• Evaluation of the response of the NIs to different RI• Test with glucose/sucrose solutions alternated with water• Elaboration from the same RGB raw data
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Validation of the hue for plasmonic evaluation
Bulk refractive index change
Surface binding events Small molecules in saline
buffer Small molecules in serum
matrix
60
• Tobramycin 10 μM in TE 1X buffer
• DNA aptamer spot
10 20 30 40 50 60 7000.198
0.1985
0.199
0.1995
0.2
0.2005
0.201
0.2015
0.202
Hue
580
580.5
581
581.5
582
582.5
583
583.5
584
Pea
k lo
catio
n (n
m)
Time (min)
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Hue-peak correlation to surface events
580 581 582 583 5840.2
0.201
0.202
0.203
0.204
0.205
Peak location (nm)
Hue
h33 correlation hue peak in TE
Surface RI change in TE buffer
580 580.1 580.2 580.3 580.40.2001
0.2002
0.2003
0.2004
Peak location (nm)
Hue
0.0715 nm
1E-4
35
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Small molecules detection in TE buffer
37
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 60.195
0.196
0.197
0.198
0.199
0.2
Time (hours)
Hue
AptamerControl
TE 1X
Serum (control)
NaCl 1M
TE 1X
TE + Tobramycin
0.5 µM
1 µM
2 µM
5 µM
10 µM
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DNA aptamer and control spots Array of aptamers/control HCR/blank NIs 2-channels microfluidics
Ref FTO
NIs
SH-aptamControl HCRBlank NIs
NaCl
NaCl
NaCl
TE TE TE TE TETobr
20µM
Tobr200µM
500 1000 1500 2000 2500 3000 3500 4000 4500 50000.184
0.186
0.188
0.19
0.192
0.194
0.196
0.198
Numero Immagine
Valo
re P
ixel
Tinta in FTO & NIs
HFTO
HCONTR
HDNA
HDNA
HNIS
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THERAPEUTIC DRUG MONITORING FOR PERSONALIZED MEDICINE
ISyPeM
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MULTI-SCALE APPROACH FOR POINT-OF-CARE TDM
POINT-OF-CARE TESTING Low volume
blood testing Quantitative Specificity
towards metabolites
Miniaturized
INTERPRETATION AND DOSE ADJUSTMENT Is the result
expected? Is the drug still
suitable? Prediction and dose
adjustment
DATA EXCHANGE AND INTEROPERABILITY Upload patient’s data Extension of eHealth
medical standards Data integrity
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INNOVATE IN VITRO ASSAYS
DNA aptamers
Monoclonal antibodies
In-Check - STMicroelectronics
Aptamer-based analysis of tobramycin in serum samples
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SUPPORT INTERPRETATION OF DRUG CONCENTRATION DATA
ezeCHiel
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EXPLOITATION PARTNERS
Division of Clinical Pharmacology Collect patient samples
Validation in field conditions
Elaborate a protocol for large-scale randomized clinical trials
Readers and prototyping
Contribution in the development of a demonstrator
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Candidate treatments for therapeutic drug monitoring
69
Modern anticancer agents
Antiretrovirals
Immunosuppressant
Antibiotics
IMATINIB 1000 ng/ml493.60 Da
TACROLIMUS 10 ng/ml804.02 Da
EFAVIRENZ 2000 ng/ml315.70 Da
TOBRAMYCIN 1000 ng/ml467.5 Da
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From surface confined to volume confined systems
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Clinical Pharmacology, UNIL CHUV (CH)Thierry Buclin, DirectorLaurent Decosterd
European Institute of Oncology, Milan (I)Marco Giorgio
Ludwig Institute for Cancer Research (CH)Immanuel Luescher
CEA LETI (F)Thomas Ernst
EPFL
Microelectronic Systems Laboratory, Yusuf Leblebici
CMi
LMIS4, Philippe Renaud
Nanophotonics and Metrology Laboratory Olivier Martin
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• Dielectric properties of single cell : Discrimination- Main leukocyte sub-populations (monocytes, lymphocytes, neutrophils) in human blood(Label-Free Differential Leukocyte Counts Using a Microfabricated, Single-Cell Impedance Spectrometer, D Holmes et al.)- Red blood Cells ghost and RBCs fixed in glutaraldehyde (Impedance spectroscopy flow cytometry: On-chip label-free cell differentiation, K. Cheung et al.) - Monocytes and dendritic cells(On-chip non-invasive and label-free cell discrimination by impedance spectroscopy, G.Schade-Kampmann et al.) viability - MFC-7 cell death (Label-free single cell analysis with a chip-based impedance flow cytometer, A.Pierzchalski et al)- Living and dead yeast cells (Multiple-frequency impedance measurements in continuous flow for automated evaluation of yeast cell lysis , G. Mernier et al) Infection - Babesia bovis infected erythrocytes (Label-free detection of Babesia bovis infected red blood cells using impedance spectroscopy on a microfabricated flow cytometer, C. Küttel )
• Dielectric properties of labels decorating the cell Polysterene particles (Single Cell Impedance Cytometry for Identification and Counting of CD4 T-Cells in
human Blood Using Impedance Labels, D. Holmes et al.)
• Change in ionic force of medium cell lysis detection- CD4+ T (Cell detection and counting through cell lysate impedance spectroscopy in microfluidic devices , X. Cheng)
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Back up
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Quantification of small molecules in serum
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EMIT(Enzyme Multiplied Immunoassay Technique )
requires derivatization of the drug molecules Drug of abuse
FPIA: (Fluorescence Polarization Immunoassay)
hard to integrate, based on antibodies requires derivatization of the drug molecules Therapeutic drug monitoring: employed only for
specific diseases (difficult to interpret and measure)
Syva RapidTest®
FPIA
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New approaches to small molecules quantification. DNA aptamer based
cANTIBODIES
150 kDaAPTAMERS
10 kDa - 30 kDa YFerguson, B. S. et al. (2013). "Real-Time, Aptamer-Based Tracking of Circulating Therapeutic Agents in Living Animals." Science Translational Medicine 5(213): 213ra165.
Chang, A. L. et al., Anal. Chem. 2014, 86, 3273−3278.
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Do our aptamerswork in complex matrices?
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Test on SPR label-free
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Point of care biosensor
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Localised Surface Plasmon Resonance
Cappi, G., Accastelli, E., Spiga, F.M., Cantale, V., Rampi, M.A., Benini, L., Guiducci, C. (2013) Mat Res Soc Symp Proc
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DNA Aptamer Selection against tobramycin
Tobramycin
Aminoglycoside antibiotic Adverse effects on kidney
and ears
Tested in buffer:• 0.23 M KD
• No affinity toward carbenicillin
• 5 time higher KD toward kanamycin
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Tobramycin on T-LSPR in undiluted serum
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5
-0.4
0
0.4
0.8
1.2
1.6
Time (hours)
Nor
mal
ized
sig
nal
Aptamer
10 µM 20 µM 40 µM 60 µM 80 µM0 µM 0 µM 0 µM 0 µM
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Point of care biosensor:can monitor small molecules?
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Tobramycin on T-LSPR
• T-LSPR setup: Kd= 0.26 µM
• Biacore SPR: Kd= 0.23 µM
0 5 10 15 200
2
4
6
8
10
12
14
16
x 10-4
Tobramycin concentration (µM)
hue
Aptamer (average)Langmuir interpolationControl
Cappi, G., Spiga, F.M., Moncada, Y., Ferretti, A., Beyeler, M., Bianchessi, M., Guiducci, C. (2014) ACS Nano (in prep.)
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Point of care biosensor:small molecules in complex matrices?
83
Tobramycin on T-LSPR in undiluted serum
Linear trend Theoretical minimum
resolvable concentration of tobramycin 3.4 µM.
Good linearity in almost all the analytical range (1-80µM)Cappi, G., Spiga, F.M., Moncada, Y., Ferretti, A., Beyeler, M., Bianchessi, M., Guiducci, C. (2014) ACS Nano (in prep.)
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Making aptamers - SELEX
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Classic Capture
In-vitro evolution-like process
No need to immobilize the target: good for small molecules!
Spiga, F.M., Maietta, P., Guiducci, C. (2014) JACS (in submission)
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Capture-SELEX with control
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In stream control of library affinity via Surface Plasmon Resonance
Spiga, F.M., Maietta, P., Guiducci, C. (2014) JACS (in submission)
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Tobramycin Carbenicillin