Transcript Single Cell Biosensor - Colorado State University

Single Cell Biosensor
Allan Fierro
David Sehrt
Doug Trujillo
Evan Vlcek
Michael Bretz
Introduction
Fabrication
Allan Fierro
Flow Control
Optical
Detection
Circuit
Cell Trapping
What is Flow Cytometry?
• Technique for
counting cells
• Examining cells
• Sorting cells
Example of Flow Data
Analysis of a marine sample of photosynthetic picoplankton by flow cytometry
showing three different populations (Prochlorococcus, Synechococcus and
picoeukaryotes)
What is Optofluidics?
• Optofluidics combines microfluidics and optics
• Able to see the difference in light refraction
wavelength
wavelength
*diagrams courtesy of L. Shao, Ph.D.
from Ph.D. Defense Presentation
Flow Cytometry vs. Optofluidics
Flow Cytometry
• Flow uses scatter of
light
• Flow more expensive to
run
• Flow samples take
longer to prep
• Can’t use cells after
Optofluidics
• Optofluidics uses
refraction of light
• No fluorescent dye cheaper and less prep
time
• Optofluidics can keep
cells after
Microscope
Light
Cell
Infrared LED
Fabrication
Fabrication
David Sehrt
Flow Control
Optical
Detection
Circuit
Cell Trapping
DEP Chip
Images courtesy of Weina Wang
DEP Chip Processing
1. 30 nm Chrome Deposition
2. 120 nm Gold Deposition
3. Spin Photoresist
4. Exposure
5. Development
6. Gold Etch
7. Chrome Etch
8. Resist Removal
PDMS Channel
•
•
•
•
PDMS- Polydimethylsiloxane
Suitable optical properties
Adhesive to glass
Channel mold made of a
silicon substrate and SU-8
Photoresist
• PDMS poured into mold and
baked to form elastic solid
DEP PDMS Bonding
• Oxygen-plasma treated
• Bonding is performed
with a mask aligner
• Minute Pressure is
applied for bonding to
transpire
DEP Chip
PDMS Channel
Flow Control
Fabrication
Evan Vlcek
Flow Control
Optical
Detection
Circuit
Cell Trapping
Chip Design for Fluid
• Two drilled holes at each
end
• Nanoports for each hole
• 200 µm wide, 25 µm
deep, channel
• Need flow rate of
approximately 40 µm/s
drilled hole
chip
channel
200 µm
flow
nanoport
*picture courtesy of L. Shao, Ph.D.
from Ph.D. Defense Presentation
Nanoport Assembly
TOP VIEW
drilled hole
SIDE VIEW
chip
nanotube
nanoport
adhesive
o ring
adhesive
o ring
from pump
nanoport
nanotube
“waste”
Block Diagram
Labview
program
DB9  RS-232
Oriel
Controller
Actuator
Syringe
Nanotube
Chip
Set
Parameters
Yes
Pump?
Yes
On Cycle
Off Cycle
Stop?
No
Stop Pump
Actual Setup
• Pump set to move actuator
0.5 µm/s
actuator
Oriel
Controller
• On for 0.825 s, off for 7 s (≈10.5%
duty cycle)
0.5 µm/s
• π cm2 syringe area * 0.5 * 10-4
cm/s * 0.105
 1.66 * 10-5 cm3/s
through channel
• This is 1.43 liters being pumped
through the channel every 24
hours!
RS 232
syringe
Labview
VI
nanotube
Actual Setup
actuator
Labview program
syringe
Oriel
controller
Optical Detection Circuit
Fabrication
Doug Trujillo
Flow Control
Optical
Detection
Circuit
Cell Trapping
Optical Detection of Cells
-Purpose• Detect the presence of a cell in proximity of a trap
• Interface with the pump flow controller
• Provide triggering for the RF traps
-Implementation• Photo diode coupled via fiber optic cable from microscope
to detect light modulation
• Monitor the change of the reverse bias current from the
diode through the use of a USB Data Acquisition unit
• Digital switch to trigger RF traps
Optical Detection of Cells
-Requirements•
•
•
Able to detect light modulation of cells traveling 40 µm/sec
Output a voltage in the range of 2V - 3.8V. Any higher voltage output may damage the
DAQ
Photodiode must be responsive to light source of 860-910 nm
Circuit Design Flow Chart
Microscope
output
Photo Diode
Low pass filter
Signal to
DAQ
Amplifier
Buffer
Optical Detection of Cells
-Light SourceThe light source is set up as shown in the figure. The LED is a high-intensity Infrared
LED.
50/125
multimode
fiber
Circuit
Labview
Traps
1
2
D1
PHOTODIODE
DAQ
beamsplitter
Lear, Kevin L., Hua Shao, Weina Wang, and Susan E. Lana. "Optofluidic
Intracavity Spectroscopy of Canine Lymphoma and Lymphocytes." IEEE
Explore (2007). 4 Dec. 2007.
microfluidic
sample
infrared
source
LEDs
Optical Detection of Cells
-Circuit Design+5 VDC
C5
0
+5 VDC
100n
2
D1
PHOTO DIODE
C9
0
0
100n
1
3.2k
8
8
R2
100n
U1A
3
+
2
-
1
5
6
+
U1B
7
-
4
R1
250k
C1
9n
LF412
C6
4
~2mV
Output
from Diode
+5 VDC
C7
LF412
0
C10
100n
0
- 5 VDC
0
0
100n
- 5 VDC
R5
124k
C8
1n
R4
100
0
A1
A1 signal
to USB
DAQ
Optical Detection
Rise Time:
128 µs
Peak Voltage:
2.7 V
Figure of Time Response of Optical Detection Circuit
Cell Trapping
Fabrication
Michael Bretz
Flow Control
Optical
Detection
Circuit
Cell Trapping
Automated Trapping
Analog Output
Detection
Circuit
Digital Output
Data
Acquisition
Unit
The digital signal from the Data
Acquisition Unit is input into an ADG452 (basically a digital switch) chip.
This allows an AC signal to be applied
to the DEP traps
Triggering
Circuit
AC Signal
DEP Traps
Dielectrophoretic(DEP) Trapping
• Uses theory of
electromagnetics to trap cells
• A non-uniform electric field
causes polarization within
individual molecules of
spheres/cells.
•This polarization along with
electric field apply a force
that will move spheres to a
desired location.
Electric field
Electromagnetic modeling
AC voltage
Ground
fluid flow
+AC Voltage
Picture Courtesy of Weina Wang et al. powerpoint presentation “Lab-on-a-Chip Single Particle
Dielectrophoretic (DEP) Traps” 3-6-2006.
The force caused by the electric
field will push the spheres into the
center of the trap.
Ground
Requirements
1. Sphere must be traveling at 40
micron/second or less
2. Signal used to generate the electric
field must be a time varying signal in
order to produce a non-uniform
electric field.
3. Signal used to generate the electric
field must have an amplitude greater
than 5 Volts peak to peak
Both of these requirements are necessary
in order for the for the force caused by
the electric field to overcome the force
caused by the flow of water. i.e. an
object in motion stays in motion unless
acted upon by a net external force.
Pictures of DEP traps
Video of DEP Trapping
Budget
• ADG 452 Digital Switch -- $15
• Various circuit elements -- $10
• Hytek iUSBDAQ U120816 -- $105
• TOTAL EXPENSES = $130
Future Work
• Improve glass chip fabrication process
• Fine-tune optics for better optical detection
• Incorporate spectrometer and automate data
acquisition
• Research best trap design to allow for faster
flow rate
• Explore methods to allow for cells to be
analyzed faster