WHOLE CELL INTEGRATED BIO-CHIP - Waseda University · Whole-cell biochips for early detection of water ... toxicity detection biochip Putting the components together for a functional

1

WHOLE CELL INTEGRATED WHOLE CELL INTEGRATED BIOBIO--CHIPCHIP

Yosi Shacham-Diamand

Tel-Aviv University, May 11, 2004

Whole-cell biochips for early detection of water

poisoning

Whole-cell biochips for early detection of water

poisoning


Tel-Aviv University Research InstituteFor Nano-Science and Nano-Technologies


Tel-Aviv University Research InstituteFor Nano-Science and Nano-Technologies

4

THE THREAT: Intentional poisoning of a drinking water source

THE NEED: A rapid broad-spectrum early warning device

Statement by Homeland Security Secretary Tom Ridge on Raising the Threat Level

For Immediate ReleaseOffice of the Press SecretaryMay 20, 2003

The Department of Homeland Security, in consultation with the Homeland Security Council, has made the decision to raise the national threat level from an Elevated to High risk of terrorist attack or Level Orange.

No such device exists today!No such device exists today!

How do we detect the presence of toxic

compounds in a water sample?

The obvious answer:

Chemical analysis

* Highly accurate and sensitive* Requires complex analytical

equipment* May be lengthy and costly

An alternative approach:

Toxicity bioassays

* Not “what does the sample contain”?, but“how toxic is the sample”?

TOXICITY BIOASSAYS

Standard toxicity bioassays, mostly designed for environmental purposes, are unsuitable for our needs.

They cannot answer the question:

“is the water safe to drink?”

Our solution: cell-based biosensorsOur solution: cell-based biosensors

Rather than use whole animals, we can genetically engineer live cells to emit a signal in the presence of toxicants

8

Human cell systems, assay screening:Ministry of Health, National Public Health LaboratoriesIsrael Laboratory Accreditation AuthorityDr. Efrat RormanDr. Orna Dreazen

æºØ

fił¢ª

ø¢łÆØ œ ØłÆ œØ ł

ªÆ

PUBLIC HEALTH LABO

RATO

RIES

Microbiology, yeast systems:Hebrew University of JerusalemProf. Shimshon BelkinDr. David Engelberg

Micro-System-Technology (MST) and integration :Tel-Aviv UniversityProf. Yosi Shacham-Diamand Prof. Yoram Shapira

Risk assessment and decision support models development:Israel Defense Forces, Medical CorpLieutenant Colonel Patrick BettaneDr. Lt. Colonel Yoav YehezkeliDr. Colonel Boaz Tadmor Dr. John S. Young, HUJ, Consultant

9

Cell-based toxicity sensor chipsCell-based toxicity sensor chips

Live cells, genetically engineered to emit a signal in the presence of toxicantsIncorporated into a disposable biochip that provides:

Live cell maintenanceMicrofluidics for sample introduction

The biochip is inserted into a Toxicity Analyzer that contains:

Electronic control and operation circuitsDetection opticsTemperature controlLogic circuits and decision algorithmsCommunication capacities

10

The final construct emits a dose-dependent signal in response to the presence of the target chemicals

The fusion of two genetic elements:The fusion of two genetic elements:

Light

gene promoter

Sensing element

luxCDABE

Reporting element

Engineering live cells for the detection of toxicants

Sensing element: A promoter of a gene involved in the response to the desired target

Reporting element: Fluorescence or bioluminescence genes

11

Liquid fireflies

12

Bacterial systems (E. coli)* Facilitated genetic manipulation* Proven concept validity* Limited relevance to human health

Human cell systems (liver, neuronal)* Maximum hum an exposure relevance* A much more complex technical challenge

Yeast cells (S. cerevisiae)* Eukaryotic structure and function* Relative facility in genetic manipulation

The original grand plan - three types of sensor cellsThe original grand plan - three types of sensor cells

14

Human cell constructs

Feasibility of engineering human and yeast cell systems has beendemonstrated, but performance is not yet in the required range

Clone # 43

Yeast constructs

No Induction 0.03ppm Cd++ Heat Shock

Clone # 31

No Induction 0.03PPM Cd++ 20PPM DDVP Heat Shock

15

YEGFP reporter is induced and quantitatively measurable in yeast

Bright Field

Fluorescence

0

5000

10000

15000

20000

25000

30000

35000

40000

Rel

ativ

e in

tens

ity

X 1

000

Uninduced(Dextrose)

Induced(Galactose)

16

Transient TransfectionNeuroblastoma & Inducible

GFP

Induced

Bright Field

Fluorescent light

Non-induced

Induced Stable HepatomaClones

Promoter: HSP70BReporter: EGFP

Clone No. 8

Clone No. 12

17

Hepatopa cells, clone 8Response to a mixture of

Metals(including 0.056ppm Cd)

14 h exposure

Hepatoma Cells on a Chip Model

Confluent Monolayer Cells at the Edge

18

Obtaining a successful bacterial GFP induction in small volumes

Absolute protection against dryingPhysiological condition of cellsAppropriate cell density

0

2 104

4 104

6 104

8 104

0 100 200 300 400 500 600 700

FLU

OR

ES

CE

NC

E (R

FU)

TIME (min)

2 µl cell suspension

2x105 cells, recA::EGFP

Using bacterial systems we demonstrated dose-dependent responses to all target compounds so far tested, at or close

to the required detection thresholds

Using bacterial systems we demonstrated dose-dependent responses to all target compounds so far tested, at or close

to the required detection thresholds

190

10

20

30

40

50

60

70

80

Res

pons

e R

atio

lpxA

rfaDFCL

arcAlacAYZ

yigH

yhaN

nhoA

Choice of promoters

20

For maximizing the detection range and enhancing analysis potential: a panel of

sensor strainsor

aA

nhoA

mip

A

lacA

YZ

grpE

MethomylDDVP

N-MustardKCNParathionParaquat

0

10

20

30

40

50

60

Res

pons

e R

atio

oraA

nhoA

mip

A

lacA

YZ

grpE

MethomylDDVP

N-MustardKCNParathionParaquat

0

10

20

30

40

50

60

Res

pons

e R

atio

21

Panel response patternPanel response pattern

30 min 60 min 120 min

grpE

nhoA

oraA

lacZ

mipA

nitrogenmustard ethyl

paration paraquatpotassiumcyanide DDVP

sterile LB

0

50,000

100,000

150,000

200,000

250,000

grpE

nhoA

oraA

lacZ

mipA


parathion paraquatpotassiumcyanide DDVP

sterile LB

0

10,000

20,000

30,000

40,000

50,000

60,000

grpE

nhoA

oraA

lacZ

mipA



sterile LB

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

ABC

grpE

nhoA

oraA

lacZ

mipA


paration paraquatpotassiumcyanide DDVP

sterile LB

0

50,000

100,000

150,000

200,000

250,000

grpE

nhoA

oraA

lacZ

mipA



sterile LB

0

10,000

20,000

30,000

40,000

50,000

60,000

grpE

nhoA

oraA

lacZ

mipA



sterile LB

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

ABC

22

Chemical TDC(mg/l)# Concentration detected (mg/L)*

Paraquat 5,250 0.43

Potassium Cyanide 230 0.2

E-Parathion 70 13.0

DDVP 1,960 32.5

N-Mustard 350 6.1

2,4,5-Trichlorophenol 2,960 0.8

2,6-Dichlorophenol 6,550 1.2

2-Chlorophenol 12,110 2.8

Colchicine 203 14.3

Phosdrin 170 38.3

Metham Sodium 28,700 18

#) Chemical concentration needed to be present so that a 70 Kg person, consuming 2 liters of water, will be exposed to the published LD50 value (oral, Rat). The reporter’s detection threshold will need to be much lower then this number, as is indeed the case in most chemicals tested.

*) Concentration generating a 2:1 signal to noise ratio.

Sample detection thresholdsSample detection thresholds

23

We believe we can “tailor” our sensors to

respond to any group of toxicants, thus ensuring an unprecedented broad spectrum of bio-detection

24

Effect of Botulinum Toxin

0

5000

10000

15000

20000

25000

0 100 200 300Tim e (m in)

RLU

0.5m g/L0.250.1250.060.030.0150.0750

Effect of Botulinum Toxin Concentration

0.00

5000.00

10000.00

15000.00

20000.00

25000.00

0 0.05 0.1 0.15

Botulinum (m g/L)

RLU

The search for additional promoters

Preliminary responses to Botulinum toxin

25

PRINCIPLE OF OPERATION

Tran

sduc

er

Act

ivat

ion

resu

lts

biochipSample

portReaction Chamber

Water with ChemicalsSample

port

Suct

ion

Act

ivat

ion

Input: electrical, optical, acoustic, thermal, etc

Output: digital

Immobilized, dehydrated whole

cell biosensors

Rehydratedwhole cell biosensors

TransducerChemicals Analysis

Algorithm

Keyboard

Screen

Integrated System Control

Digitized SignalBio-signal

ecember 2003

26

Data Presentation &

Processing

Integrated System Prototype

Experimental System

Biochip Inserter

Biochip

Control Communication Link

Fast Communication Link

Blue light source

Emission filter

Video-

ControlModule

Temp.control

MEMS

Blue light source

Excitation filter

Video-cameraImage Sensor

Bio-SensorsWater channel uPumps activation

temperature control

intensity control

Sample portuPump

Emission optics Control Comm. Link

Fast Comm. Link

December 2003

27

28

Putting the components together for a functional toxicity detection biochip

Putting the components together for a functional toxicity detection biochip

Background

Actual chip

Chip design

Toxic sampleNeutral sample

Quantification

29

We can engineer live cells to generate optical signals in the presence of a practically unlimited spectrum of toxicants

In summaryIn summary

We can interface such cells with modern electronic circuitry and solid state platformsUnequivocal feasibility demonstrated in the course of a three-year, US $ 3.1 M DARPA-funded project

A functional prototype by the end of 2004

Two development options: a hand-held spot-check device or a continuous on-line toxicity monitor

Relevant patents filed

30

Acknowledgements

• Thanks to Dr. Colonel Boaz Tadmor(Medical Corps, IDF) whose

contribution was invaluable in getting DARPA involved.

• Thanks to all the students and researchers who are involved in this project.

31

Part II

• The electronics technology of bio-sensing

32

Table of Contents:IntroductionWhole cell biochips:

The signal source: Whole cell Biosensors on a microfluidicschip

Light emitting sources: Photoluminescent, BioluminescentElectrical sources: Electrochemical

The detectors: solid state photo-detectorsArrays – Diode arrays, CMOS, CCDSingle detectors: photodiodes, photo-multipliers

Example – Whole cell integrated systemSource - E. Coli integrated on chipDetector – CMOS array

Summary and conclusions

33

Motivation: cells can be integrated on a chipMotivation: cells can be integrated on a chipCells dimensions:

• Mammalian cells ~10-20 µm (typical)• Bacteria cell: ~ 0.1 – 10 µm

Biochip dimensions:

• Components ~1 – 1000 µm

• Chips ~1 mm – few cm

34

Motivation: cells can be integrated on a chipMotivation: cells can be integrated on a chip

New technologies became available: Micro-fluidics

- fluid systems on a chip using Micro-machining

Microelectronics - compatible with the micro-fluidics

Low power detectors - can be integrated with cells on chip

35

Why integrating live cells ?

Multi-cells:

Functional response

Emulating “real” life behavior

Emulating complex systems characteristics

Study cell behavior

Single cells:

All the above + cell sorting

36

Photograph from the "Welsh Coal Mines" Collection from the National Museum of Wales

The The ““Canary in Canary in a cagea cage”” conceptconcept

37

: Schematic outline of the integrated cell-based bio-chip.

Disposable Unit Nondis posable Unit

Sense & Data Analysis UnitWater inlet Toxicity InformationµFluidic

Sys tem

Cells chambers

38

System architectures

• Hybrid– Micro-fluidics biochip and – Sensor and signal processing ASIC chip

• Fully integrated –– Whole cell and the micro-fluidics

integrated with the detector and the electronics “front end”.

39

System architectures

Limited designComplex process

Simple processLow cost

Fully integrated

PackagingHigher cost

Optimized chipsHigher yield

Hybrid

problemsHighlights

40

MST scale: Micro-fluidics systems

Micri-pump made by MST (also known as

MEMS)

Micro-pump cross section

41

Micro-fluidics systems

No electronics on chip

Very low costSimple 3D integration

Plastic

No electronics on chip

AvailableDifficult

Glass

ExpensiveLong lead time

AvailablePrecise, availableElectronics on chip

SiliconProblemsProblemsHighlightsHighlightsSubstratesSubstrates

42

Silicon wet etch (KOH)

43

Silicon wet etch (KOH)

44

Silicon dry etch (ICP)

45

Optical signal

a. Low signala. Simpleb. Fast

Bioluminescence

a. Requires;1. Illuminator2. Transparent substrateb. λexitation~ λemission

c. Slow

Large signal at will

PhotoluminescenceProblemsHighlights

46

Light signal: Photo luminescent

Biosensor Cell

GFP

Modified DNAPromoter

toxicant

GFP

GFP

GFP

GFP

Plasmid

Modified DNA

Plasmid

47

Electrical signal: electrochemical

48

(a) Chip masks formed using standard CAD tools. (b) Two electrochemical cells, each containing three electrodes and

pads. (c) SEM picture of the electrochemical cell.

49

Example:

E. coli producing β-galactosidase, entrapped in agar media coupled to the chip surface was monitored by amperometric technique (V=220mV). The oxidation current observed is generated by the enzyme activity.

It is based on the work of Prof. Yehudit Rishpon on large scale cell containers at Tel-Aviv University that was applied to a biochip with volumes in the range of 100-1000 nL.

50

Current from bacteria producing β-galactosidase in thin layer LB-agar (1.8%) medium placed on the chip surface following addition of PAPG substrate. .

0.00E+00

2.00E-08

4.00E-08

6.00E-08

8.00E-08

1.00E-07

1.20E-07

1.40E-07

1.60E-07

0 500 1000 1500 2000 2500 3000

Time, sec

I, A

mpe

r

Agar with bacteia Agar without bacteria

51

Electrical signal: electrical

52Joseph J. Pancrazio, Center for Bio/Molecular Science and Engineering Naval Research Laboratory Washington, DC, Proc. WTEC workshop 2000.

Single cell integration

53

The signal source: Whole cell Biosensors on a microfluidics chipLight emitting sources: Photoluminescent,

BioluminescentElectrical sources: Electrochemical

The detectors: solid state photo-detectorsSingle detectors: photodiodes, photo-multipliers

Arrays – Diode arrays, CMOS, CCD

Whole cell biosensors:

54

The signal problem from a biochip

1. Weak signals emitted to 4π, low signal to noise ratio

2. Spatial nonuniform signal

3. Complex optics – molecular sources in liquid in micro-containers with many reflecting surfaces.

4. Require miniaturized optics – micro-machined or micro-coupled.

5. Difficult temperature control– both of the sources (cells) and the detectors (Solid state)

55

Integrated Solid state detectors

• Single detectors – photodiodes, avalanche photodiodes, photomultipliers

• Detector arrays – CMOS imagers, CCDs

56

Single detectorsSingle detectors

1. Single container

2. Large volumes

3. Can be optimized – electronics area is not limited by the biochip

57

Photodiodes – convert photons flux to electrical current

diodeph Aqi ⋅Φ⋅⋅= η

areadiodeAfluxPhotonΦηq

i

diode

ph

efficiancy quantum chargeelectron

currentdiode

====

=

58

Response to PL biosensor

Bio chip

Cell’s container

Photodiode

Excitation chip (Blue)

Emission (green)

59

PL biosensor-Photodiode Response

diodethetodistance

4Ann 2

diodecellsph,diodeph,

−−

⋅⋅=

rfactorlGeometricaGF

GFrπ

Assume small detector:

60


diodediodephdiode AncmPhotons /]sec/[ ,2 =⋅Φ

GFr

n ⋅⋅= 2diode

cellsph,diodeph,4

Anπ

GFr

Annqi diodecellphotons

phcellsph ⋅⋅⋅⋅⋅= ⋅ 2sec 4][

πη

61


cellsph

cellphotons

ph

npowerExcitationi

powerExcitationn

⋅∝

⋅∝⋅

][ sec

Assumptions

• No absorption of PL signal

• Linear PL effect

62

Photodiodes – limits

diode29-

A Amp/cm 10 ×≈

TempRoomleakageiTypical

leakagediodeph iAqi >⋅Φ⋅⋅= η

secphotons/cm 10~ /q][Amp/cm 10 21029- ⋅>Φ η

The limit is on ncellsx Excitation power

63

Photodiodes – limits

Solutions:

Lower temperature

Lower leakage (better process)

64

Avalanche Photodiodes – converts photons flux to electrical current with internal gain

diodeph AqGi ⋅Φ⋅⋅⋅= η

areadiodeAfluxPhotonΦηq

gainernalG

i

diode

ph

efficiancy quantum chargeelectron

int

currentdiode

====

=

=

65

converts photons flux –Photomultipliers to electrical current with high internal gain

Highlights:

Very sensitive

Problems

Slow

Expensive

Very difficult to integrate

66

Michael L. Simpson et al., Sensors and actuators B, 2002, 179-185

Example: integrated photodiode with CMOS circuit

Micro-luminescent detection ASIC:

Chip area: 2mm X2mm

Photodiode area: 1.47 mm2

67

Michael L. Simpsona,b, Gary S. Saylerc, Greg Pattersonb, David E. Nivensc, Eric K. Boltonb, James M. Rochelleb, James C. Arnottb, “An integrated CMOS microluminometer for low-level luminescencesensing in the bioluminescent bioreporter integrated circuit”, Sensors and Actuators B 72 (2001) 134±140

A bioluminescent bioreporter integrated circuit is formed by placing genetically engineered bioluminescent cells on an optically sensitive integrated circuit (IC). The molecular specificity is provided by the cells, while the IC provides the advantages of a microelectronic format.

68Michael L. Simpson et al., Sensors

and actuators B, 2002, 179-185

Bio-luminescence from a growing culture of P. fluorescens 5RL. At t=0 1ppm salycilate was added to induce the lux genes.

Example: integrated photodiode with CMOS circuit

69

Detector arraysHighlights

• Many containers – battery on a chip

• Can be software controlled for the collection area

Problems:

• Electronics area is limited by the biochip

• Complex optical constraints

70

71

• SXGA resolution: 1280 x 1024 pixels

• High sensitivity 20 µV/electron

• High fill factor 60 %

• Quantum efficiency > 50% between 500 and 700 nm.

• 20 noise electrons = 50 noise photons

• Dynamic range: 66 dB (2000:1)

• 7 x 7 µm2 pixels

• Low fixed pattern noise (1 % Vsat p/p)

• Low dark current: 344 pA/cm2 (1055 electrons/s, 1 minute auto saturation)

Special low-noise CMOS imager (IBIS4)

72

Example

Integrated E.Coli whole cell photo-luminescent

detection of acute toxicity in water

73

:System Technology (IMST)-Integrated Micro

Specific technologies:

Integrated fluidic systems for miniature analytical instruments,

Embedded sensors and actuatorsfor activating bio-assays and sensing their response

CCD Imager and Mixed signal VLSI chipfor signal detection, processing, storage

and communication

74

The Bio-chip Technology• Miniature propelling mechanisms

– Internal - nano/micro-litters levels – External – micro-litters levels

• Capable of sequential operations using multi-reagents.

• Capable of parallel operations of different tests• Low-cost mass production disposable chip -

currently we use Si substrate, however, the technology can be applied on plastics.

75

Bio-material on chip

• Photoluminescent bio-sensors• Bio-material was deposited on the chip.• recA::gfp, E. coli• Nalidixic acid induction

76

300 350 400 450 500 550 600250

300

350

400

450

500

550

376.5 -- 400.0 352.9 -- 376.5 329.4 -- 352.9 305.9 -- 329.4 282.4 -- 305.9 258.8 -- 282.4 235.3 -- 258.8 211.8 -- 235.3 188.2 -- 211.8 164.7 -- 188.2 141.2 -- 164.7 117.6 -- 141.2 94.12 -- 117.6 70.59 -- 94.12 47.06 -- 70.59 23.53 -- 47.06 0 -- 23.53

Emission Wavelength, nm

Exci

tatio

n W

avel

engt

h, n

m

Dependence of Excitation Spectrum on Emission Spectrum

77

Light

W F

Data Processing

CMOSimager

Luminescence

Control Sync

Cells chamber

WaterSample

Filtering unit

Whole cell integrated system: Photoluminescence & CMOS imagers

78

wafer level

79

The MEMS Bio-chip• Contains inspection chambers, micro-pumps, water channels and inlet ports• A 12-well modular design

1 mm

80

Integrated level

81

Illuminator- GaN blue LED array ( ~ 470 nm)

Detector and signal processor- 1 Mpixel CMOS imager

- Signal detection and cell container signal acquisition

- Signal compression and storage

82

Background

Actual chip

Chip design

Toxic sampleNeutral sample

Quantification

83

Typical output data

I

ttrigger

High concentration

Mediumconcentration Low concentration

hours

84

Summary

• Whole cell biosensors require special micro fluidic and sensor design

• Micro-fluidic system requires fluid containers, micro-pipes, micro pumps inlet and outlet.

• We demonstrated open loop system, however closed loop system is possible

• Bio-material deposition is still a problem• Both optical (gfp, lux) and electrical sensing is

possible.,

Documents

WHOLE CELL INTEGRATED BIO-CHIP - Waseda University · Whole-cell biochips for early detection of water ... toxicity detection biochip Putting the components together for a functional