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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

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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

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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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EUROPEAN REGIONAL DEVELOPMENT F UND Spherical multicellular aggregates with interesting properties :

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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