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EUROPEAN UNION
EUROPEAN REGIONAL DEVELOPMENT F UND
Microsystem solutions for biochemical and bioanalytical applications
Department of MicrobioanalyticsWarsaw University of Technology, POLAND
Michał Chudy
EUROPEAN UNION
EUROPEAN REGIONAL DEVELOPMENT F UND
Outline
• Aim of the project• Silicon-based electrochemical transducers
and miniaturized sensors• Microsystems for bioanalytical applications• Summary
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Aim of the project
This subproject aimed at the development of:
• novel silicon-based electrochemical (bio)sensors
for biochemical analysis
• hybrid microsystems for bioanalytical applications
EUROPEAN UNION
EUROPEAN REGIONAL DEVELOPMENT F UND
Electrochemical transducers and miniaturized sensors
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K. Wyglądacz, E. Malinowska, J. Jaźwiński, Z. Brzózka, Sensor. Actuat. B, 83 (2002) 109
Silicon-based gold transducers (Au electrode) with back-side contact
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Development of DNA sensorsMethylene blue(redox probe)
1. The apparent output signal change due to goldelectrode surface modification with ssDNA orafter hybridization
2. The redox potential shifts as the change inmethylene blue interactions with appropriatemonolayer
-0.245 V
Before surface modification After ssDNA
immobilization After hybridization
I II III
I
II
III
Au Au Au
-0.232 V -0.273 V
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Planar potentiometric 10-electrode sensor array on silicon support
electrodes
contact pads
Electrode type Ionophore [mg] Lipophilic salt [mg]
Plasticizer [mg]
Polymer [mg]
Na+– selective 1.7 sodium ionophore X
0.15 KTPClPB
65.3 DOS
32.6 PVC
K+ – selective 2.0 valinomycin
0.8 KTPClPB
64.6 DOS
32.3 PVC
Ca2+ – selective 2.0 ETH_129
0.85 KTPClPB
64.6 DOS
32.3 PVC
Cationselective - 1.0 KTFPB
66.0 DOS
33.0 PVC
Anionselective - 3.5 TDMAC
64.3 oNPOE
32.3 PVC
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Planar potentiometric 10-electrode sensor array on silicon support
MILK
WATER
ORANGE JUICE
Electrode type
Sca
led
sign
al
WATER
ORANGE JUICE
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Three-electrode transducerwith front-side contacts
1 mm
25,5 mm
7 mm
1 mm5 mm6 mm
5 mm
Au
Ag/AgCl
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Transducer Geometric area [mm2] Electrochemical area [mm2]
BVT 0.785 2.05Gold disc electrode 0.785 2.71
ITE transducer 0.694 0.71
Quality of various gold electrodeschronoamperometry measurements
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II
I
III
IV II
I
III
IV
Determination of dopamine in the presence of ascorbic acid
Bare gold electrode
N-acetyl-L-cystein monolayer
I
III
IV
II
I
III
IV
II
Non-modified transducer
N-acetyl-L-cysteinmodified transducer
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Bioanalytical microsystems
10 mm
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Technologies used for microsystems fabrication
* wet etching of microscopic glass slides for cell seeding microchambers
* capillary film-based replica moulding technique for PDMS plates with microchannel network
* bonding-less technology for PDMS microdevice for cell lysis based Gaucher disease diagnostics
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Photodynamic therapy procedures in the
microfluidic system
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mask
stamp
5- ALA LA MAL
Jedrych E., Pawlicka Z., Chudy M., Dybko A., Brzozka Z. Evaluation of photodynamic therapy (PDT)procedures using microfluidic system, Anal. Chim. Acta (2010)
Microdevice geometry and fabrication
precursor of photosensitizer
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Syringe pumpwith cells suspension
Microskope coupledwith CGG camera
Microsystem
computer
Scheme of lab procedure
cells cultured for 48h
precursor of photosensitizer (5-ALA)introduced through CGG
irradiation using a high power LED (λ=625nm)
4h incubation of cells with ALA
Set-up
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Results
• after 4 hours of incubation with different concentrations ALA proper growth and proliferation of the cells was observed
• tested concentrations of ALA did not have toxic effects on the cells• 24 hours after PDT procedures, viability test confirmed that the toxic effect of the
cell was depended on the ALA concentration
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Diagnostics of Gaucher disease
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Gaucher disease diagnosticsin microscale
GD is a genetic disease in which lipids accumulatesin cells and certain organs,Caused by the low activity of an enzyme -lysosomal β-glucosidase
Type I (or non-neuropathic type 1 in 50,000) - most common form of the disease,
Type II (or acute infantile neuropathic Gaucher's disease 1 in 100,000) typically begins within 6 months of birth. Affected children usually die by age 2.
Type III (the chronic neuropathic form 1 in 100,000) can begin at any time. Slow progress. Patients often live into their early teen years and adulthood.
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Gaucher disease diagnostics
- sample - cells containing lysosomal β-glucosidase
- the enzyme activity - deficiency (<30% of normal levels) -can be determined only after lysis of cells
REACTION - catalyzed by lysosomal β-glucosidase (β-Glc)SUBSTRATE - 4-methylumbelliferyl-β-D-glucopyranoside (MUG)FLUORESCENT PRODUCT - 4-methylumbelliferone (4-MU)lex = 365 nm, lem 455 nm
enzyme
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f.o. from source f.o. to detector
Sheath flow zone
detection area
7.5 nl
cells inlet
Buffer + MUG inlet
Microsystem for GD diagnostics
Kwapiszewski R., Ziolkowska K., Jedrych E., Skolimowski M., Chudy M., Dybko A., Brzozka Z., A microfluidic device with fluorimetric detection for intracellular components analysis, Biomedical Microdevices (2011) in press
analytical system cell line
enzyme activity
[µU/105 cells]MACRO A549 34,6±7,5MACRO L929 110,2±19,6MICRO L929 95,0±15,0
analytical system cell line
enzyme activity
[µU/105 cells]MACRO A549 34,6±7,5MACRO L929 110,2±19,6MICRO L929 95,0±15,0
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Multicellular tumor spheroids....- a new model for tumor studies
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Intercellular interactions and connections (desmosomes)
Cytoskeleton structure like in vivo
Extracellular matrix
Concentration gradients od substances inside spheroids
Nectotic core
Alive cells
HT-29
HT-29
Centre of a spheroid
environment
Oxygen and nutrients’ gradient
Metabolitesgradient
Multicellular tumor spheroids (MTS)....
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Microsystem - design
Flow-through mictochambers - volume 0.2 µL
Microchannels 300 µm x 50 µm
3D structure
Photolithography
10 mm
inletoutlet
microchamber Spheroid
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HT 29 cells
24hagregacjaPłukanie medium
Spheroids’ formation
Microsystem - results
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Summary
• silicon-based electrochemical transducers with smooth electrode surface
• possibility of modification with self-assembled monolayers and various analytes determination
• microdevices for various bioanalytical applications
• microdevice suitable for PDT procedures evaluation
• microsystem for different Lysosomal storage diseases diagnostics (e.g. Gaucher disease)
• microsystem for a new tumor model studies (MTS)
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Dr Barbara Czartoryska from Department of Genetics, Institute of Psychiatryand Neurology, Warsaw, Poland is acknowledged for her scientific advicesconcerning lysosomal storage disorders diagnostics.
This work was realized with a frame of project MNS-DIAG, which is financed by the European Union through the European Regional Development Fund and the Polish state budget in the framework of the Operational Programme Innovative Economy 2007-2013, contract No. UDA-POIG.01.03.01-00-014/08-01
Thank You for Your attention!
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Cell seeding in MCCS • cells flushed with PBS buffer
• MCCS and medium prewarmed at 37oC
• introduction of cell suspension in the culture medium (1.2 mlmin for 50 min)
• cells observation and temperature control
• after proper cell adherence observation (medium was changed every day in MCCS)
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Bonding-Less (B-Less) fabrication of polymeric microsystems,Microfluidics and Nanofluidics, 2009, 7(5), 733-737
PDMS- bonding-less
bottom of the structure
3D microchannels network
top of the structure
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microchannels 20 - 500 mm
Capillary film - based replica moulding technique
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6-(ferrocenyl) hexanethiol
Self-assembled monolayer formation
CV
SWV
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PDT mechanism
Reactive oxygene species
Cells dead (necrosis, apoptosis)photosensitizer
intracellular oxygen
irradiation
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