Micro-Scale Engineering –III Lab-on-a-Chip and Cancer ... · Micro-Scale Engineering –III...

Micro-Scale Engineering –IIILab-on-a-Chip and Cancer Detection

Y. C. LeeDepartment of Mechanical Engineering

University of ColoradoBoulder, CO 80309-0427

[email protected]

September 16, 20081

2

Three Lectures

• Tuesday, September 2- MEMS Introduction

• Tuesday, September 9- BioMEMS, Lab-on-a-Chip

• Tuesday, September 16 - BioMEMS, Cancer cell analysis, implantable MEMS

Contents• Biochip and bioMEMS• Cancer cell detection

- molecular markers- electrophoresis, electro-osmosis and dielectrophoresis- fluid flow fractionation- cancer detection

• Injectable MEMS- microneedles

• Implantable MEMS- drug delivery devices

3

4

Microprocessor in 2007

An Intel microprocessor announced in November 2007: 214mm2 (~12mmX18mm) and 820 million transistors 5

Moore’s Law

6

An Integrated Nanoliter DNA Analysis Device

Mark Burns et al., Science, 1998

47mm X 5mm X 1mm

7

Application-Specific Integrated Circuits

8





9

Molecular Fingerprints of Cancer Cells

10

Presenter

Presentation Notes

The p16INK4a protein is comprised of 156 amino acids and contains four ankyrin repeats that are required for protein-protein interaction.30 p16INK4a acts as a tumor suppressor by blocking cdk4 and cdk6-mediated pRb phosphorylation, resulting in inhibition of E2F dependent transcription and inhibition of cell cycle progression at the G1 to S checkpoint.31 The repression of p16INK4a gene expression by hypermethylation or mutation is a common occurrence in a wide range of cancer cell lines and primary human tumors. However, in most cervical carcinomas, the functional inactivation of pRb by HPV E7 results in the reciprocal overexpression of p16INK4a, due to a negative feedback loop between pRb and p16INK4a. 32-36 Thus, p16INK4a over-expression in cervical neoplasia is a surrogate marker of HPV E7-mediated pRb catabolism and may reflect disruption of the mechanisms that control cell proliferation.37,38

Antibody

11

Antigen Binding to Antibody

12

BB CA

Antibody + Fluorescence Label

Intrinsic

Fluorescent ProteinQuantum

Dots

13

Localization of p16INK4a in squamous mucosa. Panel A, benign squamous mucosa; Panel B, CIN 1; Panel C, CIN 3; Panel D, SCC.

14

Biopsy and Cancer Detection

• Precancerous molecular or chromosomal abnormalities are undetectable.• Geographic misses frequently occur leading to false negative biopsies.• Cancers are often multifocal. A positive biopsy at one site may not give a full picture of the

extent of disease if the cancer is multifocal or has extended to “skip areas”.• There is no standard technique for determining genetically abnormal tissue at resection

margins in the operating room. Such abnormalities are clinically undetectable and may lead to recurrence despite “complete resection” as determined by frozen section technique.

Punch Microtome Detection system

15

A micro-thin tissuetaken from the abnormalarea using micropunchwithout anesthetic

Lab-on-a-ChipCarcinoma detection by measuring light absorption and fluorescence of tissue

InstantBiopsy

Light-Guided Micropunch-Based Instant Biopsy

16

Epoxy with glass beads as spacers

Micro-thin Tissue

Silicon

Window etched

Sharp blades made bydouble-side polishing

Micropunch

17

Lab-on-a-Chip for Cancer DetectionA

F

G

D C B

J

H

E

18





19

Electrophoresis (CoventorWare)

Vep= μep E

Velocity of Charged Species = Eletrophoretic mobility of the ion in the carrier species X Electrical Field

Strength

Note: in most cases, the carrier does not move under electrophoresis.20

Electrophoresis

21

Electrophoresis and Electroosmosis• Electrophoresis :

The movement of a charged surface plus attached material relative to stationary liquid by an applied electric field. Mobile particles, Stationary liquid.

• Electroosmosis :The movement of liquid relative to a stationary charged surface by an applied electric field.Mobile liquid, Stationary cell wall.

22

Electrochemical double layer and Electroosmosis

-------

-------

+ -- ++ -- ++ -- +

++++++

++++++

1

2

1

Electrolytepulled byelectricalfield23

Electroosmosis

Arrows showvelocity vectors

24


F

G

D C B

J

H

E

25

Dielectrophoresis

1)

2)

3) reverse the field?- time to build up charges- AC frequency effect

26

Presenter

Presentation Notes

First, it will be observed that if the direction of the field in the above figure is reversed then so too will be the sign of the induced polarization on the particle and the dielectrophoretic force will end up being in the same direction as before! Therefore, if we use an alternating electrical field, dielectrophoretic collection will still occur because the polarization will reverse each time the field does. Of course, we are assuming in this case that the induced polarization has time to build up after each reversal of the alternating field. In reality, the rate with which electrical polarization can occur are governed by the properties of the particle, and, if the polarization does not have time to build up fully in response to changes in the field then the dielectrophoretic force will be less. By studying the way in which the dielectrophoretic collection force varies with the frequency of the applied alternating field, the dielectric properties of the particle can be deduced. This is significant: it means that the dielectric properties of particles can be measured without making any electrical connections to them “simply” by observing their motion in response to an inhomogeneous, alternating electrical field. It also means that particles having different dielectric properties will have different responses.��Second, the figure illustrates an electrically-polarizable particle within a poorly polarizable medium (e.g. a gas). But what if the particle itself were non-polarizable and it were suspended in a polarizable medium (e.g. a gas bubble suspended in water)? In this case, more charge will build up in the polarizable medium close to the non-polarizable particle than in the particle itself. The polarity of this polarization will be opposite to the case of the highly polarizable particle. Result: the non-polarizable particle will experience a force that acts in the opposite direction to before - away from the high field region. It follows that if one suspends poorly polarizable particles in a polarizable medium then dielectrophoresis can be used to push them away from high field regions. This is extremely useful because it is possible to design electrode configurations with small, well defined low field regions. Dielectrophoresis can be used to focus particles towards these low field regions away from the electrodes. We usually refer to the repulsive dielectrophoretic force as negative DEP and to its attractive counterpart as positive DEP.��

1)

2)

-

What if the particle itself were non-polarizable and it were suspended in a polarizable medium (e.g. a gas bubble suspended in water)?

Negative Dielectrophoresis

27

Presenter

Presentation Notes

First, it will be observed that if the direction of the field in the above figure is reversed then so too will be the sign of the induced polarization on the particle and the dielectrophoretic force will end up being in the same direction as before! Therefore, if we use an alternating electrical field, dielectrophoretic collection will still occur because the polarization will reverse each time the field does. Of course, we are assuming in this case that the induced polarization has time to build up after each reversal of the alternating field. In reality, the rate with which electrical polarization can occur are governed by the properties of the particle, and, if the polarization does not have time to build up fully in response to changes in the field then the dielectrophoretic force will be less. By studying the way in which the dielectrophoretic collection force varies with the frequency of the applied alternating field, the dielectric properties of the particle can be deduced. This is significant: it means that the dielectric properties of particles can be measured without making any electrical connections to them “simply” by observing their motion in response to an inhomogeneous, alternating electrical field. It also means that particles having different dielectric properties will have different responses.��Second, the figure illustrates an electrically-polarizable particle within a poorly polarizable medium (e.g. a gas). But what if the particle itself were non-polarizable and it were suspended in a polarizable medium (e.g. a gas bubble suspended in water)? In this case, more charge will build up in the polarizable medium close to the non-polarizable particle than in the particle itself. The polarity of this polarization will be opposite to the case of the highly polarizable particle. Result: the non-polarizable particle will experience a force that acts in the opposite direction to before - away from the high field region. It follows that if one suspends poorly polarizable particles in a polarizable medium then dielectrophoresis can be used to push them away from high field regions. This is extremely useful because it is possible to design electrode configurations with small, well defined low field regions. Dielectrophoresis can be used to focus particles towards these low field regions away from the electrodes. We usually refer to the repulsive dielectrophoretic force as negative DEP and to its attractive counterpart as positive DEP.��

Dielectrophoresis

Two different particles in a non-uniform electric field. The particle on the left is more polarisable than the surrounding medium and is attracted towards the strong field at the pin electrode, whilst the particle of low polarisability on the right is directed away from the strong field region.

28

Negative Dielectrophoresis

29

Positive Dielectrophoresis

30

Molecules

Inert particles

Entities with a singlemembrane (virus, prokaryotesand eukaryotes with small nucleii)

Complex cells (plant cellsor mammalia cells with large nucleii)

Simulation

Interface between particle and medium

fCM is the Clausium-Mossotti factor which describes the frequency-dependent dielectri characteristics of the particle and its surroundings.

Applied AC frequency31

Dielectric Constant

21

21),( 2

0

02 VzACVzxU rεε−=−=

metal

metaldielectric

dzo

32

Electrochemical double layer and Electroosmosis

-------

-------

+ -- ++ -- ++ -- +

++++++

++++++

1

2

1

Electrolytepulled byelectricalfield33

Dielectric constant

Electrical conductivity

Dielectrophoretic properties

point

Solid particle

A compartmentwith an envelop

Two compartmentswith an envelop

Applied AC frequency

Clausius-Mossotti factor

34

DEP Trapping to Collect Tumor Cells (repelling by negative DEP)

Electrode

Slow fluid flow

Breast tumor cells

Peripheral blood mononuclear cells (PBMNC)

One tumor cell out of 2 x 106 PBMNCs. 35

DEP Trapping (positive DEP)

Electrode

Slow fluid flow

To collect all viable cells but reagents, dead cells or debris.

AC frequencies > 200 KHz 36

Field Flow Fractionation (FFF)

37


• Narrow channel with the maximum velocity along the centerline.

• Large particles Small diffusion coefficients Closer to the wall Leave the column late.

• Particles susceptible to the field applied Closer to the wall Leave the column late.

38


39

Particles in a Flow Stream

with Positive

DEPFive frequencies close tothe crossover frequency.

40

DEP Collection Spectra

Breast Cancel Cell MDA231

T-lymphocytes

Erythrocytes

Crossoverfrequencies

Repulsion

Trapping

41

Parameters Affecting Cell Properties

• Membrane thickness• Effective area• Dielectric constant• Electrical conductivity

0.8 μF/cm2 for smooth biological membrane15 μF/cm2 for highly convoluted hepatocyte membrane1.2 to 4 μF/cm2 are typical for mammalian cells

42

DEP Crossover Data (σs = 56 mS/m)

43

Particles in a Flow Stream with Negative DEP

44

Eletrosmear for normal

and cultured tumor cells

At their crossover frequencies, cells touch down on the slide surface and are captured by the binding agent. 45

Magnetaphoretic-dielectrophoretic FFF for Cells with Magnetically Labeled Surface Markers

46


F

G

D C B

J

H

E

47

Spiral Electrodes to Concentrate

Cells

48

Spiral Electrodes to Concentrate

Cells

Human erythrocytes infectedby the malarial agent Plasmodium falciparumwere discriminated fromuninfected cells and focusedto the center.

49

Alternative: Dielectrically Engineered Carrier Beads

2.5 to 10 um in diameter.50

Carrier Beads with Different Chain Lengths

51

Tagged Carrier Beads Going through Cell During Lysis Step

52

Molecular Recognition and Sensing

• 5-um beads• 100 tumor cells+250 beads (10 types)

focused on an area with 50-um in diameter

109 cells/ml held in contact with 2x 109 beads/ml carrying molecular probes.

53





54

Out-of-Plane Microneedles

200 um tall, 425 um base diameter tapering to 40 um lumen and 750 um pitch 55

Processing Steps

56

Sharp Microneedles

57

Painless Transdermal Delivery

58

Injectable MEMS

• Microneedles• Injectable Micromodules

- deliver electronic devices such as neuromuscular stimulators to the human body through large-gauge hypodermic needles.

59




• Implantable MEMS ( in vivo devices)- drug delivery devices

60

Biocompatibility• Cytotoxic?Irritants?

- single crystal silicon, polycrystalline silicon, silicon dioxide, single-crystal silicon carbide and titanium are O.K.

• Biofouling?- adsorption of biomolecules (peptides and proteins) followed by cells frequently leads to device fouling and failure.

61

Surface Modification

• Surface Immobilized Polymers- poly (ethylene glycol) (PEG) to inhibit protein adsorption.

• Self-assembled monolayer (SAM)- Oligo(ethylene glycol) terminated alkanethiol moiety to passivate surfaces against protein and cellular adsorption.

62

Nanoporous Gold Film for Therapeutic Retention and Elution

63

A Controlled Release Microchip

17mmX17mmX310um34 reservoirs

< 2mm possible

0.3 um gold

64

Removal of an anode membrane to initiate release from a reservoir

Gold was chosen as a model membrane material because it is easily deposited and patterned, has a low reactivity with other substances and resists spontaneous corrosion in many solutions over the entire pH range. However, the presence of a small amount of chloride ion creates an electric potential region which favours the formation ofsoluble gold chloride complexes.

65

Sodium Fluorescein released into phosphate-buffered saline

1.04 Volt w.r.t. saturated calomel reference electrodefor 30 seconds

66

Release Rates

67

Pulsatile release of multiple substances from a single microchip device

Release rate of 45Ca2+ ions (open triangles; vertical scale in units of 53 nCi min-1) and sodium Fluorescein (filled circles; in units of ng min-1) into 0.145M NaCl solution over several hours

68

Implantable MEMS

• Biosensors- for in vivo diagnostics- pH, analytes, blood pressure, tissue and body fluids.

• Stents• Immunoisolation Devices

- cell containing microcapsule to prevent immunorejection.

• Drug Delivery Systems

69

Summary• Biochip and bioMEMS• Cancer cell detection




70

Date post:	11-Apr-2018
Category:	Documents
Upload:	vudien
View:	215 times
Download:	2 times

Micro-Scale Engineering –III Lab-on-a-Chip and Cancer ... · Micro-Scale Engineering –III...

Documents