single cell biosensor

Single Cell Biosensor

Allan FierroDavid Sehrt

Doug TrujilloEvan Vlcek

Michael Bretz

Introduction

Allan FierroOptical

Detection Circuit

Flow Control

Cell Trapping

Fabrication

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 uses scatter of light

• Flow more expensive to run

• Flow samples take longer to prep

• Can’t use cells after

• Optofluidics uses refraction of light

• No fluorescent dye - cheaper and less prep time

• Optofluidics can keep cells after

Flow Cytometry Optofluidics

Microscope

Infrared LED

Cell

Light

Fabrication

David SehrtOptical

Detection Circuit

Flow Control

Cell Trapping

Fabrication

DEP Chip

Images courtesy of Weina Wang

DEP Chip Processing

1. 30 nm Chrome Deposition2. 120 nm Gold Deposition3. Spin Photoresist4. Exposure5. Development6. Gold Etch7. Chrome Etch8. 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 PDMS Channel

DEP Chip

Flow Control

Evan VlcekOptical

Detection Circuit

Flow Control

Cell Trapping

Fabrication

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

nanoport

channel

chip

200 µm

flow

*picture courtesy of L. Shao, Ph.D.from Ph.D. Defense Presentation

Nanoport Assembly

chipdrilled hole

adhesive o ring

nanoport

nanotube

TOP VIEW SIDE VIEWnanotube nanoport

from pump “waste”

adhesive o ring

Block Diagram

Pump? Off Cycle Stop?On Cycle Stop PumpSet

Parameters

Yes Yes

No

Labviewprogram

OrielController

DB9 RS-232

Actuator Syringe Nanotube

Chip

Actual Setup• Pump set to move actuator

0.5 µm/s

• On for 0.825 s, off for 7 s (≈10.5% duty cycle)

• 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!

Oriel Controller

nanotube

syringe

actuator

LabviewVI

RS 2320.5 µm/s

Actual Setupactuator

syringe

Labview program

Orielcontroller

Optical Detection Circuit

Doug TrujilloOptical

Detection Circuit

Flow Control

Cell Trapping

Fabrication

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

Amplifier Buffer

Signal to DAQ

Optical Detection of Cells

-Light Source-The light source is set up as shown in the figure. The LED is a high-intensity Infrared LED.

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.

50/125 multimode fiber

micro-fluidic sample

beam-splitter

infrared source LEDs

D1

PH

OTO

DIO

DE

1 2CircuitCircuit

Labview

DAQ

Traps

DAQ

Labview Traps

Optical Detection of Cells-Circuit Design-

0

0

0

+5 VDC

-5 VDC

-

+

U1A

LF412

3

21

84

R1250k

R2

3.2k

C19n

-

+

U1B

LF412

5

67

84

A1

C5

100n

0

C6

100n

R4100

0

+5 VDC

0

C7

100n

R5

124k

D1PHOTODIODE

12

C8

1n

0

+5 VDC

C9

100n

-5 VDC

0

C10

100n

~2mV Output from Diode

A1 signal to USB DAQ

Optical Detection

Figure of Time Response of Optical Detection Circuit

Rise Time: 128 µs

Peak Voltage:2.7 V

Cell Trapping

Michael BretzOptical

Detection Circuit

Flow Control

Cell Trapping

Fabrication

Automated Trapping

DetectionCircuit

Data Acquisition

Unit

Triggering Circuit

Analog Output Digital Output

AC SignalThe digital signal from the Data Acquisition Unit is input into an ADG-452 (basically a digital switch) chip. This allows an AC signal to be applied to the DEP traps 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

Ground

+AC Voltage

fluid flow

GroundAC voltage

The force caused by the electric field will push the spheres into the center of the trap.

Picture Courtesy of Weina Wang et al. powerpoint presentation “Lab-on-a-Chip Single Particle Dielectrophoretic (DEP) Traps” 3-6-2006.

Requirements1. Sphere must be traveling at 40

micron/second or less2. 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