light scattering particle immunoassays in lab-on- a-chip

UA-NVRQS International Collaboration

Light Scattering Particle Immunoassays in LabLight Scattering Particle Immunoassays in Lab--onon--aa--Chip for Infectious Agents in Livestock BarnsChip for Infectious Agents in Livestock Barns

Jae-Young Song, D.V.M., Ph.D.Virology Division, National Veterinary & Quarantine Service, Korea

Brian C. Heinze, Jin-Hee HanDepartment of Agricultural & Biosystems Engineering, University of Arizona, USA

Young Hee Oh, D.V.M., Ph.D.Research Institute of Public Health & Environment, Seoul Metropolitan Government, Korea

Jeong-Yeol Yoon, Ph.D.Department of Agricultural & Biosystems Engineering, University of Arizona, USA


•• National Veterinary Science & Quarantine Service, Republic of KNational Veterinary Science & Quarantine Service, Republic of Koreaorea

Mission statementMission statement

Quarantine and inspectionQuarantine and inspection

Sanitary control for livestock productsSanitary control for livestock products

Animal disease controlAnimal disease control

Research and development (in veterinary science)Research and development (in veterinary science)

Animal medicineAnimal medicine

Animal protectionAnimal protection

Veterinary diagnosticsVeterinary diagnostics


Veterinary diagnostic tool that is:Veterinary diagnostic tool that is:

EarlyEarly

PointPoint--ofof--carecare

UltraUltra--low detection limitlow detection limit

AutomatedAutomated

To detect infectious diseases in livestock and poultry:To detect infectious diseases in livestock and poultry:

Bovine Viral Diarrhea (BVD) - cattle

Porcine Reproductive and Respiratory Syndrome (PRRS) - swine

Salmonella – poultry

Avian influenza (AI) - poultry

•• UAUA--NVRQS International CollaborationNVRQS International Collaboration


•• BVDV & PRRSVBVDV & PRRSV

BVDV: one of the most significant viral infections in cattleBVDV: one of the most significant viral infections in cattle

Affects both dairy and beef cattleAffects both dairy and beef cattle

Infected cattle are more susceptible to many respiratory and Infected cattle are more susceptible to many respiratory and

intestinal pathogensintestinal pathogens

PRRSV: positivePRRSV: positive--strand RNA virusstrand RNA virus

50-65 nm (enveloped virus); 25-35 nm (nucleocapsid core)

Abortions, stillbirths, weak piglets

Respiratory diseases

Secondary infections in adult pigs


•• Salmonella Salmonella typhimuriumtyphimurium

Salmonella Salmonella typhimuriumtyphimurium: Gram: Gram--negative bacterianegative bacteria

Cylindrical rodCylindrical rod

22--3 3 µµm (length); 0.6m (length); 0.6--0.9 0.9 µµm (diameter)m (diameter)

Food poisoning (gastroenteritis)Food poisoning (gastroenteritis)


•• Detection limits of current methodsDetection limits of current methods

ELISA: 50 pgELISA: 50 pg PCR: 10PCR: 10--100 TCID100 TCID5050/ml/ml


•• LabLab--onon--aa--chip (LOC)chip (LOC)

A semiconductor chip used to analyze the composition of a A semiconductor chip used to analyze the composition of a

personperson’’s body fluids (blood, saliva, urine) for medical purposes. It s body fluids (blood, saliva, urine) for medical purposes. It

combines combines microfluidicsmicrofluidics and semiconductor processing on the and semiconductor processing on the

same chip.same chip.

Eliminate human effort such as Eliminate human effort such as pipettingpipetting..

Many biological reactions are diffusionMany biological reactions are diffusion--limited; Miniaturization limited; Miniaturization

can speed up the reaction.can speed up the reaction.

Hundreds of assays can be performed simultaneously, saving Hundreds of assays can be performed simultaneously, saving

considerable time and effort.considerable time and effort.

We will use LOC for veterinary diagnosticsWe will use LOC for veterinary diagnostics


LIA has NOT been demonstrated in LOC (diffusional mixing issue)

First demonstrated by UA Biosensors Lab in 2006Han, Kim & Yoon, Anal. Chim. Acta 584: 252 (2007)

•• Latex Latex immunoagglutinationimmunoagglutination assay (LIA)assay (LIA)


Antibody-conjugatedstructured particles

Target solution

Light scatteringdetection w/ optical fibers

Duplicate experimentsw/ two-well slide(75 µl, 800 µm depth)

•• ProcedureProcedure390-, 510- or 920-nm (mostly 510-nm)Highly carboxylated polystyrene particles

45 degree forwardlight scattering detection

380-nmLED

miniaturespectrometer

positioningstages

syringepump


•• Bovine Viral Diarrhea Virus (BVDV): 2Bovine Viral Diarrhea Virus (BVDV): 2--wellwell

Heinze, Song & Yoon, Proc. SPIE, 6886: 688605 (2008)

Detection limit

1~2 TCID50/ml

Monoclonal antibodyTissue: MDBKWashed by centrifuging


•• BVDV: BVDV: microfluidicmicrofluidic

Detection limit

<1 TCID50/ml

BVDV: microfluidic

-500

0

500

1000

1500

2000

2500

3000

blank -1 0 1 2 3

log TCID50 /ml

Detection limit: 1 TCID50/ml


•• BVDV: RTBVDV: RT--PCRPCR


•• BVDV: RTBVDV: RT--PCRPCR

Gel electrophoresisof BVDV PCR productsranging from 1-104 TCID50/mL

Bands observedas low as 103 TCID50/mL

Heinze, Song & Yoon, Proc. SPIE, 6886: 688605 (2008)


•• Porcine Reproductive & Respiratory Syndrome Virus (PRRSV): 2Porcine Reproductive & Respiratory Syndrome Virus (PRRSV): 2--wellwell

PRRSV: 2-well (MAb)

6000

6500

7000

7500

8000

8500

blank -3 -2 -1 0 1 2 3 4 5

log TCID50 /ml

PRRSV: 2-well (PAb)

11000

11500

12000

12500

13000

13500

blank -3 -2 -1 0 1 2 3 4 5

log TCID50 /ml

Live vaccine (attenuated virus)Similar to BVDV


PRRSV: microfluidic (PAb)

5000

7000

9000

11000

13000

15000

17000

19000

blank -3 -2 -1 0 1 2 3

log TCID50 /ml

•• PRRSV: PRRSV: microfluidicmicrofluidic

Detection limit: 10-3 TCID50/ml


•• PRRSV: timePRRSV: time--course detectionscourse detections

PRRSV: 2-well (PAb)

4100

4200

4300

4400

4500

4600

4700

4800

4900

5000

5100

0 1 2 3 4 5

time (min)

PRRSV: microfluidic (PAb)

4400

4500

4600

4700

4800

4900

5000

5100

0 1 2 3 4 5 6 7 8 9

time (min)

Maximum signal at 2 min.


5 4 3 2 1 0 -1 (log TCID50/ml)

•• PRRSV: RTPRRSV: RT--PCRPCR

Gel electrophoresis of PRRSV PCR productsranging from 10-1-105 TCID50/ml

Bands observed as low as 102 TCID50/ml


Salmonella: 2-well (MES, KPL antibody)

3000

3500

4000

4500

5000

5500

6000

6500

7000

blank 5 50 500 5E+3 5E+4 5E+5 5E+6

CFU/ml

Salmonella: 2-well (PBS, KPL antibody)

2500

3000

3500

4000

4500

5000

5500

blank 5 50 500 5E+3 5E+4 5E+5 5E+6 5E+7

CFU/ml

•• Salmonella Salmonella typhimuriumtyphimurium: 2: 2--wellwell

Cultured in media, washed by centrifugePolyclonal antibodies


•• Salmonella: 2Salmonella: 2--well & well & microfluidicmicrofluidic

Salmonella: 2-well (PBS, Biodesign antibody)

6000

6500

7000

7500

8000

8500

blank 0.5 5 50 500 5E+3 5E+4 5E+5 5E+6

CFU/ml

Salmonella: microfluidic

9000

10000

11000

12000

13000

14000

15000

blank 0.5 5 50 500 5E+3 5E+4 5E+5 5E+6 5E+7

CFU/ml

Detection limit: 0.5-50 CFU/ml


•• Salmonella: Salmonella: immunoagglutinationimmunoagglutination images with 910images with 910--nm beadsnm beads


•• Other targets under testingOther targets under testing

Brucella abortus

Influenza A (H3N2)


•• ConclusionConclusion

Detection limits:Detection limits: BVDV <1 TCIDBVDV <1 TCID5050/ml (/ml (MAbMAb))

PRRSV 10PRRSV 10--33 TCIDTCID5050/ml (/ml (PAbPAb))

Salmonella <5 CFU/ml (Salmonella <5 CFU/ml (PAbPAb))

Maximum signal achieved at 2 minMaximum signal achieved at 2 min

Total assay time <5 minTotal assay time <5 min

•• Future workFuture work

Integrate optical fibers (optical waveguide)Integrate optical fibers (optical waveguide)

Replace miniature spectrometer with Avalanche photodiode Replace miniature spectrometer with Avalanche photodiode

(APD) and computer/software with op(APD) and computer/software with op--amp circuit/voltmeteramp circuit/voltmeter

Test for field sampleTest for field sample


•• AcknowledgmentsAcknowledgments

Yoon Group at UA:Yoon Group at UA:JeongJeong--Yeol Yoon, Yeol Yoon, KeesungKeesung Kim,Kim,TremaineTremaine Powell, JinPowell, Jin--HeeHee Han,Han,Brian Brian HeinzeHeinze, , PhatPhat Tran,Tran,JennineJennine CheslerChesler, , AnbarAnbar NajamNajam

Song Group at NVRQS:Song Group at NVRQS:JaeJae--Young Song, ChangYoung Song, Chang--HeeHee Lee,Lee,EunEun--Jin Jin ChoiChoi

Funding was provided by:Funding was provided by:National Veterinary Research & Quarantine Service (NVRQS)National Veterinary Research & Quarantine Service (NVRQS)Award # CAward # C--AD14AD14--20062006--1111--0101

light scattering particle immunoassays in lab-on- a-chip

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