Microchip-based Sensors for Detection of Magnetic Microbead Labels

Transcription

Microchip-based Sensors
for Detection of
Magnetic Microbead Labels
Mark Tondra
Diagnostic Biosensors, LLC;
1712 Brook Ave. SE; Minneapolis, MN 55414
Mark@diagnosticbiosensors.com
612 331-3584
www.diagnosticbiosensors.com
Oak Ridge AACC 4-20-06 MagSensors
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
CAMD
ISU Dept. of
Chemistry:
Dr. Marc Porter
John Nordling
Rachel Millen
Nikola Pekas
Toshi Kawaguchi
NVE GMR
sensor fab:
LSU – CAMD
Dexin Wang
microfluidics:
Zhenghong Qian
Jost Goettert
Anthony Popple
Changgeng Liu
Dave Brownell
Zhengchung Peng
Bob Schneider
Kun Lian
Kevin Jones
Josef Hormez
Loren Hudson
Challa Kumar
John Taylor
Albrecht
Jander
NRL assay
development:
Lloyd Whitman
Jack Rife
Cy Tamanaha
Mike Miller
Shaun Mulvaney
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)
C) Introduce magnetic
labels with unique
“label probes”
D) Magnetic labels
bind only where they
match
B) Introduce fluid
sample carrying
potential molecular
match, allow to
hybridize
M
M
M
E) Read magnetic
detector array by
applying a
magnetic field,
measuring the
resistance change
Spintronic Detector
Spintronic Detector
A) Prepare array chip by spotting DNA
“capture probes” onto surface
Stray Fields from Bound Magnetic
Nanolabel
Detector sees HTotal = Happlied + Hstray along a designed sense-axis
Happlied
M
Hstray
Spintronic Detector
Motivation for Recent R&D Efforts
Military / Homeland Defense wants bioassays that are:
•Rugged
•Lightweight / handheld
•Cheap
•Rapid
•Highly sensitive and specific, multi-functional, foolproof
•Readers, sensors, and fluidics must be massmanufacturable.
Laboratory-on-a-Chip
•Shrink clinical or diagnostic laboratory setup onto a Sitype 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
Two detectors are shown
here. One is downstream
from the other by 100
microns.
Each detector has two reference
and two sensing GMRs configured
as a Wheatstone bridge
E1
RR1
GND
Isrc
RS1
RS2
RR2
Top View
E2
Channel passes over sensing GMRs
Voltage / Resistance
Idealized Linear GMR Detector
Resistance vs. Magnetic Field
Resistance ∝ R0 + sinθ
θ
Sense Layer
Pinned Layer
-20
-10
0
10
20
Field (Oe)
A GMR thin film resistor is typically a
micron wide, 0.01 micron thick, and
arbitrarily long.
Voltage / Resistance
Resistance changes when magnetic
labels are present
Resistance ∝ R0 + sinθ
Signal is resistance difference
θ
Pinned Layer
Resistance
with labels
-20
-10
0
10
Field (Oe)
Sense Layer
20
Array of 20 GMR biosensors under
serpentine microfluidic channel
~150 µm
wide fluidic
channel
~200 µm
diameter
sensor spot
(matches pin
spotter size)
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
2. IDENTIFY them
magnetic labels
(GMR) detector
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
•Allow specific binding of
labels to cell
Apply magnetic force
labels to cell
•Use magnet to attract cells to
corner
Remove “chaff” from system
labels to cell
•Use magnet to attract cells
to corner
•Dump out waste
2. Give cells of interest a unique
IDENTITY
2. IDENTIFY them
magnetic labels
(GMR) detector
Cell is IDENTIFIED with same
labels
labels to cell
•Use magnet to attract cells
to corner
•Dump out waste
•Add water
•Repeat as needed
3. DIRECT Cells to a Detector
2. IDENTIFY them
magnetic labels
(GMR) detector
Single-Wire Magnetic Director
Top view
X-sections
Channel: 400 µm
long
Hext
Electrical current
Flow
50 µm wide channel
Simple situation:
Qualitative force calculation
Hx = Hexternal = 100 Oe
35 µm deep
50 µm wide channel
x
X-section
1 µm diameter
Paramagnetic
Small wire x-section
Hexternal across channel
Hexternal parallel Hwire
Current into plane
1 µm x 1 µm wire
under channel center
Simple situation:
Qualitative force calculation
50 µm wide channel
Particles are attracted
(Flip sign of current,
particles are repulsed)
35 µm deep
1 µm diam. Particles
paramagnetic
Small wire x-section
Hexternal across channel
Hexternal parallel Hwire
x
Current into plane
1 µm x 1 µm wire
under channel center
Magneto-fluidic Dynamics
For a given Fmag, one can calculate:
Equation of motion
dv
m
= − 3π η av + Fmag
dt
Viscous
drag
Magnetic
force
Integrate to get velocity
3π η a
−
t 
Fmag 
 1− e m 
v(t ) =

3π η a 

“terminal velocity”
“characteristic time”
a: particle diameter = 1 micron
n: viscosity (water)
m: particle mass
Fmag: Force in channel cross-section
due to Hwire and Hexternal
v: velocity
t: time
Initial motion of particle far from
wire
35 µm deep
50 µm wide channel
Flow into page x
Initial Fmag ~ 9 picoN
Initial Vterminal ~ 1100 µm/sec
Characteristic time = 87 nsec
Max 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
Current = 10 mAmagnetic lines of force
Two-way Diverter
Design and Results
Top view
X-section
• A uniform external
field magnetizes
particles
• Current lines induce
field gradients of
102-103 T/m
• Resulting force
diverts particles to a
desired channel
Magnetic Flow Sorting
Demonstration
Top Views
Bangs Labs, 28%
magnetite, 1
µm
Flow rate: 6
nL/min
85% of the beads
in desired
channel
4. COUNT the Cells in Flow
2. IDENTIFY them
magnetic labels
(GMR) detector
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

Picoliter-sized droplets of
ferrofluid formed at a fluidic
junction
Plug dimensions:
13 µm wide 18 µm deep 85 µm long
FerroTec 307 10nm ferrite particles
~1% by volume
 GMR sensitivity 0.07%/Oe
Pekas et al., Appl. Phys. Lett., 85, (2004)
• Wheatstone bridge
configuration
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)
Direct Flow Velocity Monitoring
flow
Excitation field 15 Oe; Flow rate 250 nL/min; 1.2% magnetite v/v
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
Hypothetical cell covered
with magnetic labels
Hx component, 2 µm below the
200 nm shell
FEMLAB package
Protozoan cell 8x6x6 µm
48 1-µm spheres, χ=0.3 (Dynal MyOneTM)
Homogeneous 1-µm shell, χ=0.18
Homogeneous 200-nm shell, χ=0.2
(Micromod nanomag-D, χ=2)
Detection of labels and cells in
small channels is magnetically easy
But, channel gets plugged
new design
35 µm deep
50 µm wide channel
old design
12 µm x 15 µm
channel x-section
2 µm x 2 µm
detector area
GMR detector
3 µm x 15 µm
detector area
Design of Cell – Label splitter
Top View
Hext
Redirect
Split
Discriminate
Electrical current
Gather
Flow
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.
2.
3.
4.
Need wider channels to avoid plugging
Lower surface topography for better flow and sealing
Want thin cover for closest microscope working distance
Improved manufacturability
b) Features
1.
2.
3.
4.
5.
6.
Buried interconnects formed using damascene process
Allows arbitrary channel width and alignment
Much lower surface step height (<100 nm vs. 2000 nm)
Thin passivation is viable (<100 nm)
Facilitates electrodes for electrochemistry and applying electric forces
Fluidic through-holes for better optical access and fluidics options
Detecting Magnetic Labels bound to
the Biosensor Surface
1. Multi-sensor array is convenient
2. 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 disposable
4. Requires microfluidics integration
Array of GMR sensors under
serpentine microfluidic channel
~150 µm
wide fluidic
channel
~200 µm
diameter
sensor spot
NRL “cBASS” system
NRL “cBASS” system,
Array of GMR sensors
2.8 micron DynalBeads
1)
2)
3)
4)
Courtesy L. Whitman, Naval Research Lab.
Single label detection is possible
>3 decades of dynamic range
Better than 1 fMolar with fluidics
Magnetism enhances specificity
NRL “cBASS” system
cBASS™ Prototype compact Bead Array Sensing System
Detecting Magnetic Labels bound to
a separate surface
1. Multi-sensor array is convenient
2. “Scan” the assay slide by the reader chip
3. Dynamic range of better than 3 logs
Detector is “permanent” in reader
4. Much simpler sample handling
technology
5. 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
Electromagnetic
Power Supply
GMR
John Nolding
Digital
VoltMeter
Current
Source
Sample
Break
out
Box
Toshikazu
Kawaguchi
Single GMRs
Coil
Coil
RR1 & RR2
Sample Above GMR
GMR and PC
connector
SAMPLE MOVEMENT
Test Station: power
supply, voltmeter,
and current source
RS1 & RS2
400 µm
All controlled by software written in house
Sample Stick Experiment
Move sample
across GMR
sensing area
200 x
200 μm
SAMPLE MOVEMENT
 External field at 150 Oe
 Sample is held near the
GMR (~50 µm) and moved
across sensing area
Permalloy
Gold
500 μm
Effect of Sample Leveling
A
N
1 mm
225
225
224
224
223
223
Signal (mV)
Signal (mV)
Internally referenced sample: 20-nm thick permalloy squares
Signal After Sample Leveling
Signal Before Sample Leveling
222
221
220
N
A
219
222
221
220
219
218
A
N
218
60
80
100
120
140
160
180
200
220
80
100
Time (s)
120
140
160
180
Time (s)
Peak ID
Signal (mV)
Std.
Deviation
Peak ID
Signal (mV)
Std.
Deviation
A
4.91
0.07
A
5.02
0.03
N
3.72
0.07
N
4.97
0.05
Difference:
1.2 ± 0.1 mV
n=8
Difference:
0.05 ± 0.06 mV
1 mm
Raw Signal at varied
Sample/GMR Separation
3.0
Signal (mV)
2.5
2.0
1.5
"0" µ m
+50 µ m
+100 µ m
+150 µ m
+200 µ m
1.0
0.5
0.0
200
400
600
800
Normalized Signal (signalx/signal800 µ m3)
Multiple Sample Signal
Normalization
Normalized Signal at varied
Sample/GMR Separation
1.2
0µm
50 µ m
100 µ m
150 µ m
200 µ m
1.0
0.8
0.6
y = 1.34 × 10 − 3 x + .0192
r 2 = 0.996
0.4
0.2
0.0
200
Sample Volume (µ m3)
400
600
Sample Volume (µ m3)
800
Permalloy Limit of Detection
Measurements
Sample
1 mm
Parameters
• Arrays of 24 x 24 µm squares
• 20 nm thick permalloy
• 12.2 µm3 permalloy which
translates to ~31 magnetic
particles (40% permalloy) on
a 200 x 200 µm sensor
Raw signal for permalloy sample
0.8
Raw Signal (mV)
y = 1.97 × 10 − 3 x + .014
0.6
Predicted Limit of
Detection (LOD):
R 2 = 0.972
• 0.1 1µm MP/µm2 sensor
detectable
0.4
Ways to Improve:
0.2
• Smaller GMR sensor
0.0
• Decrease GMR/Sample
Separation
0
50
100
150
200
250
300
•
3)
Sample volume
(µ
m
Faster signal acquisition
(signal averaging)
MP Binding to Sample Stick
Gold Square
Permalloy Square
*10 μL of MP on the patterned sample overnight
Results:
 Binding to biotinylated gold square is preferred
 Non-specific binding to permalloy and pyrex is an issue
Signal w/ Referenced Sample Stick
A
B
C
D
E
F
G
H
I
J K L M
Sample
Permalloy
Gold
Magnetic field: 150 Oe
GMR-sample stick separation: ≤ 50 μm
Signal
(mV)
# of
MP
A
0.141
1766
B
0.061
608
C
0.032
6
D
0.008
5
E
0.040
0
F
0.007
0
G
0.038
0
H
0.013
0
I
0.049
0
J
0.156
2840
K
0.115
601
L
0.352
8661
M
0.268
1326
GMR Signal vs. MP Concentration
 Limit of detection (3x SNB) is
0.005 MP/μm2, or 215 MP per
gold square.
Magnetic Microchip Fabrication
Cross Section Diagram
SU-8 Lid
Gold
Bonding
Pad
M
Channel
Layer
GMR Sensor
M
Encapsulated
Channel
M
Fluid Port
Silicon wafer
Copper
Interconnects and
Strip Lines
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.

Microchip-based Sensors for Detection of Magnetic Microbead Labels

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