light scattering particle immunoassays in lab-on- a-chip
TRANSCRIPT
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
UA-NVRQS International Collaboration
•• 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
UA-NVRQS International Collaboration
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
UA-NVRQS 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
UA-NVRQS International Collaboration
UA-NVRQS International Collaboration
UA-NVRQS International Collaboration
•• 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)
UA-NVRQS International Collaboration
•• Detection limits of current methodsDetection limits of current methods
ELISA: 50 pgELISA: 50 pg PCR: 10PCR: 10--100 TCID100 TCID5050/ml/ml
UA-NVRQS International Collaboration
•• 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
UA-NVRQS International Collaboration
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)
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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
UA-NVRQS International Collaboration
•• 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
UA-NVRQS International Collaboration
•• 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
UA-NVRQS International Collaboration
•• BVDV: RTBVDV: RT--PCRPCR
UA-NVRQS International Collaboration
•• 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)
UA-NVRQS International Collaboration
•• 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
UA-NVRQS International Collaboration
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
UA-NVRQS International Collaboration
•• 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.
UA-NVRQS International Collaboration
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
UA-NVRQS International Collaboration
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
UA-NVRQS International Collaboration
•• 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
UA-NVRQS International Collaboration
•• Salmonella: Salmonella: immunoagglutinationimmunoagglutination images with 910images with 910--nm beadsnm beads
UA-NVRQS International Collaboration
•• Other targets under testingOther targets under testing
Brucella abortus
Influenza A (H3N2)
UA-NVRQS International Collaboration
•• 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
UA-NVRQS International Collaboration
•• 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