for detection of magnetic microbead...

Oak Ridge AACC 4-20-06 MagSensors

Microchip-based Sensors for Detection of

Magnetic Microbead Labels Mark Tondra

Diagnostic Biosensors, LLC; 1712 Brook Ave. SE; Minneapolis, MN 55414

[email protected] 331-3584

www.diagnosticbiosensors.com

mailto:[email protected]


Outline

• Using magnetic beads as assay labels• Magnetic sensor chips for tiny and disposable

integrated assay systems• Micromagnetic detection platforms

– Flowing magnetic labels / towards cell counting– Immobilized labels on the sensor chip– Immobilized labels on a separate chip + scan

• Conclusions


Acknowledgements

•Funded by NSF, DARPA

CAMDCAMDNVE GMR

sensor fab:Dexin WangZhenghong QianAnthony PoppleDave BrownellBob SchneiderKevin JonesLoren HudsonJohn TaylorAlbrecht Jander

LSU – CAMD microfluidics:

Jost GoettertChanggeng LiuZhengchung PengKun LianJosef HormezChalla Kumar

NRL assay development:

Lloyd WhitmanJack RifeCy TamanahaMike MillerShaun Mulvaney

ISU Dept. of Chemistry:

Dr. Marc PorterJohn NordlingRachel MillenNikola PekasToshi Kawaguchi


Making Biology Magnetic

• Attach magnetic particles to bio-species– Sizes range from 10 nm to 5 µm diameter– Commonly ~10% magnetic content

• Trade-offs– More magnetism may cause aggregation– Larger beads give bigger signal, but dominate the

dynamic properties of the analytes in solution• Unique chemical binding for each species

– Special surface coatings and treatments


Benefits of using Magnetic Labels for Biosensors

• Very low magnetic background• Single label detection• Magnetic forces can enhance hybridization

rates• Magnetic forces can reduce non-specific

binding• Wide range of sizes and magnetic properties

available


Immobilized analytes: “two-probe” DNA assay (NRL)

M M

Spintronic Detector Spintronic Detector

MM M

Spintronic Detector Spintronic Detector

M

A) Prepare array chip by spotting DNA “capture probes” onto surface

E) Read magnetic detector array by applying a magnetic field, measuring the resistance change

B) Introduce fluid sample carrying potential molecular match, allow to hybridize

C) Introduce magnetic labels with unique “label probes”

D) Magnetic labels bind only where they match


Stray Fields from Bound Magnetic Nanolabel

M

Spintronic Detector

Happlied

Hstray

Detector sees HTotal = Happlied + Hstray along a designed sense-axis


Motivation for Recent R&D EffortsMilitary / Homeland Defense wants bioassays that are:•Rugged•Lightweight / handheld•Cheap•Rapid •Highly sensitive and specific, multi-functional, fool-proof•Readers, sensors, and fluidics must be mass-manufacturable.


Laboratory-on-a-Chip •Shrink clinical or diagnostic laboratory setup onto a Si-type chip•Point-of-Care, Point-of-Use applications•Uses MEMS and microfluidics•Make technology available to: soldiers in field, security workers, eventually consumers•Not yet a commercial reality


Giant MagnetoResistive (GMR) design: Microfluidic Channel over Detector

RS1

RS2 RR2

RR1

E1

E2

Isrc

GND

Each detector has two reference and two sensing GMRs configured as a Wheatstone bridge

Channel passes over sensing GMRs

Top View

Two detectors are shown here. One is downstream from the other by 100 microns.


-20 -10 0 10 20

Field (Oe)

Vol

tage

/ Re

sista

nce

Pinned Layer

Sense Layerθ

Resistance ∝ R0 + sinθ

Idealized Linear GMR Detector Resistance vs. Magnetic Field

A GMR thin film resistor is typically a micron wide, 0.01 micron thick, and

arbitrarily long.


-20 -10 0 10 20

Field (Oe)

Vol

tage

/ Re

sista

nce

Pinned Layer

Sense Layerθ

Resistance ∝ R0 + sinθ

Resistance changes when magnetic labels are present

Resistance with labels

Signal is resistance difference


Array of 20 GMR biosensors under serpentine microfluidic channel

~200 µm diameter

sensor spot (matches pin spotter size)

~150 µm wide fluidic

channel


Micromagnetic Detection Platforms

1. Detect magnetized objects in a microfluidic flowstream (e.g. cytometer)

2. Detect magnetic beads bound to the sensor surface (may include microfluidics)

3. Detect magnetic beads bound to a different surface (e.g. glass slide)


Detection of Flowing Magnetics

• Towards a cell counter or “cytometer”


Flow Cytometer working definition

•Counts cells in a continuously flowing system•Usually needs to discriminate between various cell types (e.g. all healthy cells vs. cancerous cells)


Magnetic Flow Cytometer Phases

1. ISOLATE cells of interest magnetic labels

2. IDENTIFY them magnetic labels

3. DIRECT them to detector magnetic / fluidic forces

4. COUNT the cells in flow Giant Magnetoresistive (GMR) detector


1. ISOLATE Cells of interest






Prototypical Example: Cancer Cell Isolation

•Want to grab only cancerous cells, count them, and store them for further analysis•Could be only 1 : 109

•May not be distinguishable by size or color


Magnetic isolation is a common approach

•Add special magnetic particles to container


Biochemically specific attachment•Add special magnetic particles to container•Allow specific binding of labels to cell


Apply magnetic force•Add special magnetic particles to container•Allow specific binding of labels to cell•Use magnet to attract cells to corner


Remove “chaff” from system•Add special magnetic particles to container•Allow specific binding of labels to cell•Use magnet to attract cells to corner•Dump out waste


2. Give cells of interest a unique IDENTITY






Cell is IDENTIFIED with same labels

•Add special magnetic particles to container•Allow specific binding of labels to cell•Use magnet to attract cells to corner•Dump out waste•Add water•Repeat as needed


3. DIRECT Cells to a Detector 1. ISOLATE cells of interest magnetic labels




Single-Wire Magnetic Director

Hext

Flow

Flow

Electrical current

Top view X-sections

Channel: 400 µm long

50 µm wide channel

Simple situation: Qualitative force calculation

Current into plane 1 µm x 1 µm wire under channel center

50 µm wide channel1 µm diameterParamagneticSmall wire x-sectionHexternal across channelHexternal parallel Hwire

x

Hx = Hexternal = 100 Oe

35 µ

m d

eep

X-section

Simple situation: Qualitative force calculation

Current into plane 1 µm x 1 µm wire under channel center

Particles are attracted(Flip sign of current, particles are repulsed)

1 µm diam. ParticlesparamagneticSmall wire x-sectionHexternal across channelHexternal parallel Hwire

x


50 µm wide channel

35 µ

m d

eep


Magneto-fluidic Dynamics

magadtdm Fvv +−= π η3

−=

− tma

mag ea

Ftv

π η

π η

3

13

)(

a: particle diameter = 1 micronn: viscosity (water)m: particle mass Fmag: Force in channel cross-section due to Hwire and Hexternal

v: velocityt: time

Equation of motion

Integrate to get velocity

For a given Fmag, one can calculate: “terminal velocity” “characteristic time”

Viscous drag

Magnetic force

Initial motion of particle far from wire

Current = 10 mA

Initial Fmag ~ 9 picoNInitial Vterminal ~ 1100 µm/secCharacteristic time = 87 nsecMax travel time = 0.03 sec.

Because the characteristic time is so much smaller than the total travel time of the particle [overdamped], one can basically say that the particle trajectory follows the magnetic lines of forcexFlow into page


50 µm wide channel

35 µ

m d

eep


Two-way Diverter Design and Results

• A uniform external field magnetizes particles

• Current lines induce field gradients of 102-103 T/m

• Resulting force diverts particles to a desired channel

Top view

X-section


Magnetic Flow Sorting Demonstration

Bangs Labs, 28% magnetite, 1 µm

Flow rate: 6 nL/min

85% of the beads in desired channel

Top Views


4. COUNT the Cells in Flow 1. ISOLATE cells of interest magnetic labels





Detection of magnetic objects in flow

•Giant Magnetoresistive (GMR) detector•Microfluidic flow channel passes directly over GMR detector


Proof of principle: GMR Sensing of Magnetic Picodroplets

• Wheatstone bridge configuration

GMR sensitivity 0.07%/Oe

Picoliter-sized droplets of ferrofluid formed at a fluidic junction

Pekas et al., Appl. Phys. Lett., 85, (2004)

T. Thorsen, R. W. Roberts, F. H. Arnold, and S. R. Quake, Phys. Rev. Lett., 86, 4163 (2001)

H. Song, J. D. Tice, and R. F. Ismagilov, Angew. Chem. Int. Ed. 42 (7), 768 (2003)

Plug dimensions: 13 µm wide 18 µm deep 85 µm long

FerroTec 307 10nm ferrite particles ~1% by volume


flow

Excitation field 15 Oe; Flow rate 250 nL/min; 1.2% magnetite v/v

Direct Flow Velocity Monitoring

Velocity determined by cross-correlating the signals from two bridges

Detection of single ~5 micron beads in flow

Top View

Model data from cell covered by “shell” of magnetic labels

Hx component, 2 µm below the 200 nm shell

Hx component, 2 µm below the 200 nm shell

FEMLAB packageProtozoan cell 8x6x6 µm48 1-µm spheres, χ=0.3 (Dynal MyOneTM)Homogeneous 1-µm shell, χ=0.18Homogeneous 200-nm shell, χ=0.2 (Micromod nanomag-D, χ=2)

Hypothetical cell covered with magnetic labels

Detection of labels and cells in small channels is magnetically easy

But, channel gets plugged

GMR detector

2 µm x 2 µm detector area 3 µm x 15 µm

detector area

12 µm x 15 µm channel x-section

old designnew design50 µm wide channel

35 µ

m d

eep

Design of Cell – Label splitter

Hext

Flow

Gather

Discriminate

Split

Redirect

Electrical current

Top View


Cell sorter, director, and detector


Fluid dynamics provide additional tools for microfluidic control

This talk has largely ignored the fluid dynamics. However, they are very important!

Mostly, a detailed account would show that there are additional tools that can be designed in to aid in sorting and detecting.


New low profile fabrication process design

a) Motivation: 1. Need wider channels to avoid plugging2. Lower surface topography for better flow and sealing3. Want thin cover for closest microscope working distance4. Improved manufacturability

b) Features1. Buried interconnects formed using damascene process2. Allows arbitrary channel width and alignment3. Much lower surface step height (<100 nm vs. 2000 nm)4. Thin passivation is viable (<100 nm)5. Facilitates electrodes for electrochemistry and applying electric forces6. Fluidic through-holes for better optical access and fluidics options


Detecting Magnetic Labels bound to the Biosensor Surface

1. Multi-sensor array is convenient2. Dynamic range of better than 3 logs

(example: one detector can quantify from 5 to 5,000 labels on a 200 micron diam. Spot)

3. Low cost microchip is disposable4. Requires microfluidics integration


Array of GMR sensors under serpentine microfluidic channel

~200 µm diameter

sensor spot

~150 µm wide fluidic

channel


NRL “cBASS” system


Courtesy L. Whitman, Naval Research Lab.1) Single label detection is possible2) >3 decades of dynamic range 3) Better than 1 fMolar with fluidics4) Magnetism enhances specificity

2.8 micron DynalBeads

NRL “cBASS” system, Array of GMR sensors


NRL “cBASS” system

cBASS™ Prototype compact Bead Array Sensing System


Detecting Magnetic Labels bound toa separate surface

1. Multi-sensor array is convenient2. “Scan” the assay slide by the reader chip3. Dynamic range of better than 3 logs

Detector is “permanent” in reader4. Much simpler sample handling

technology5. Increased mechanical engineering

challenges


Sample Strip Reader

• Developed a GMR test station capable of detecting samples which are scanned across a sensor (e.g., magnetic card reader)

• Developed a method for normalizing the GMR signal from samples at varying separations

• Investigating analytical figures of merit

• Potential utilization of streptavidin magnetic particles as a universal label


GMR Test Station

Breakout Box

Coil

Coil

Digital VoltMeter

Current Source

Sample

GMRElectromagnetic Power Supply

Breakout Box

Breakout Box

Coil

Coil

Digital VoltMeter

Current Source

Sample

GMRElectromagnetic Power Supply

John NoldingToshikazu Kawaguchi

John NoldingToshikazu Kawaguchi

µm400

RS1 & RS2

Single GMRs

GMR and PC connector

Test Station: power supply, voltmeter,

and current source

All controlled by software written in house

RR1 & RR2Sample Above GMR

SAMPLE MOVEMENT


Sample Stick Experiment

Move sample across GMR sensing area

200 x 200 μm

External field at 150 Oe Sample is held near the

GMR (~50 µm) and moved across sensing area

SAMPLE MOVEMENTPermalloy

Gold

500 μm


Effect of Sample Leveling

Time (s)

60 80 100 120 140 160 180 200 220

Sign

al (m

V)

218

219

220

221

222

223

224

225

Signal Before Sample Leveling

1 mm

A N

Time (s)

80 100 120 140 160 180Si

gnal

(mV

)218

219

220

221

222

223

224

225

Signal After Sample Leveling

A N A N

1.2 ± 0.1 mVDifference:

N

A

Peak ID

0.073.72

0.074.91

Std. Deviation

Signal (mV)

0.05 ± 0.06 mVDifference:

N

A

Peak ID

0.054.97

0.035.02

Std. Deviation

Signal (mV)

n = 8

Internally referenced sample: 20-nm thick permalloy squares


Sample Volume (µ m3)

200 400 600 800Nor

mal

ized

Sig

nal (

sign

alx/

sign

al80

0 µm

3)

0.0

0.2

0.4

0.6

0.8

1.0

1.20 µ m50 µ m100 µ m150 µ m200 µ m

Multiple Sample Signal Normalization

Sample Volume (µ m3)

200 400 600 800

Sign

al (m

V)

0.0

0.5

1.0

1.5

2.0

2.5

3.0"0" µ m+50 µ m+100 µ m+150 µ m+200 µ m

Raw Signal at varied Sample/GMR Separation

Normalized Signal at varied Sample/GMR Separation

996.00192.1034.1

2

3

=

+×= −

rxy

1 mm


Permalloy Limit of Detection Measurements

1 mm Sample • Arrays of 24 x 24 µm squares• 20 nm thick permalloy

Parameters

Sample volume (µ m3)

0 50 100 150 200 250 300

Raw

Sig

nal (

mV

)

0.0

0.2

0.4

0.6

0.8

972.0014.1097.1

2

3

=+×= −

Rxy

Predicted Limit of Detection (LOD):

• 12.2 µm3 permalloy which translates to ~31 magnetic particles (40% permalloy) on a 200 x 200 µm sensor

• 0.1 1µm MP/µm2 sensor detectable

Ways to Improve:• Smaller GMR sensor

• Decrease GMR/Sample Separation

• Faster signal acquisition (signal averaging)

Raw signal for permalloy sample


MP Binding to Sample StickGold Square Permalloy Square

Results:Binding to biotinylated gold square is preferredNon-specific binding to permalloy and pyrex is an issue

*10 μL of MP on the patterned sample overnight


Signal w/ Referenced Sample Stick

Magnetic field: 150 OeGMR-sample stick separation: ≤ 50 μm

B C D E F G H I J K L MA

Permalloy

Gold

13260.268M86610.352L6010.115K

28400.156J00.049I00.013H00.038G00.007F00.040E50.008D60.032C

6080.061B17660.141A

# of MP

Signal (mV)Sample


GMR Signal vs. MP Concentration

Limit of detection (3x SNB) is 0.005 MP/μm2, or 215 MP per gold square.


Magnetic Microchip FabricationCross Section Diagram

SU-8 Lid

ChannelLayer

Silicon wafer

GoldBonding

Pad

EncapsulatedChannelGMR Sensor

Fluid Port

CopperInterconnects and

Strip Lines

M

M

M


Magnetic Excitation and Data Collection Module

•8 On-board signal

preamps

•Jumpers for sensor

channels

•Jumpers for coil driver


Research and Development on Magnetic Biosensors around the globe

• Nat. Labs and Universities– EU, Korea, China, Several US Labs

• Commercial Efforts– Siemens, Seahawk Biosystems,

MagneBiosensors, Magnesensors


Other magnetic detection technologies

• Hall effect• Anisotropic magnetoresistance (AMR) • SQUID• Coils


Chip-based detection and manipulation advantages

• Sensors and manipulators are on same length scale as labels, cells

• Opens up many opportunities for very small systems:– Implanted diagnostic sensors– Catheters


Conclusions

• Magnetoresistive sensors are a versatile tool for biochemical research and development

• Some sensors operate “wet,” some “dry”• Microfluidics and / or mechanics are next• Packaging and testing issues are current

barriers to low-cost commercial disposable biosensor products

• Laboratory standards are lacking


Company Goal

• Develop technology leading to commercially available diagnostic biosensors.

for detection of magnetic microbead...

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