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Quantum Dots as Reporters in Multiplexed
Immunoassays for Biomarkers of Exposure to
Agrochemicals
To cite this Article:Nichkova, M., Dosev, D., Davies, A. E., Gee, S. J., Kennedy, I.
M. and Hammock, B. D. , 'Quantum Dots as Reporters in Multiplexed Immunoassays
for Biomarkers of Exposure to Agrochemicals', Analytical Letters, 40:7, 1423 - 1433
To link to this article: DOI:10.1080/00032710701327088
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© Taylor and Francis 2007
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nia] IMMUNOLOGY
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Quantum Dots as Reporters in Multiplexed
of
y Immunoassays for Biomarkers of Exposure
sit
er to Agrochemicals
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y: [
B
d M.Nichkova, D.Dosev, A.E. Davies,S. J. Gee,
e I. M.Kennedy, and B. D. Hammock
d
a
o UniversityofCalifornia,Davis,California
nl
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o
D
Abstract: The application of quantum dots (QDs) as labels in immunoassay
microarrays for the multiplex detection of 3-phenoxybenzoic acid (PBA) and
atrazine-mercapturate(AM)hasbeendemonstrated.PBAandAMarebiomarkersof
exposure to the pyrethroid insecticides and to the herbicide atrazine, respectively.
Microarrays were fabricated by microcontact printing of thecoating antigens in line
patterns onto glass substrates. Competitive immunoassays were successfully
performed using QDs (QD560 and QD620) as reporters. The multiplexed immuno-
assays were characterized by fluorescence microscopy and SEM. The application of
QD fluorophores facilitates multiplex assays and therefore can contribute to
enhancedthroughputinbiomonitoring.
Keywords: Quantum dot, microarrays, immunoassay, microconatct printing, pyre-
throid,atrazine
Received20January2007;accepted27February2007
TheauthorswishtoacknowledgethesupportoftheNationalScienceFoundation,
grantDBI-0102662,theSuperfundBasicResearchProgramwithGrant5P42ES04699
fromtheNationalInstituteofEnvironmentalHealthSciences,NIHandtheCoopera-
tiveStateResearch,Education,andExtensionService,USDepartmentofAgriculture,
under Award No. 05-35603-16280. We gratefully acknowledge the leadership and
friendshipofDr.BertoldHockintheenvironmentalbiosensorfield.
Address correspondence to B. D. Hammock, Department of Entomology,
UniversityofCalifornia,Davis,95616California.E-mail:[email protected]
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1 INTRODUCTION
4
2
00: The microarray format has become an important tool for parallel (or multi-
At: plexed) monitoring of biomolecular interactions. Protein microarray techno-
a] logy plays a fundamental role in the miniaturization of biosensors, proteins
rni analysis,drugscreening,clinicalimmunologicalassays,andbiomarkeridenti-
o
alif fication(KusnezowandHoheisel2002;Espinaetal.2003).Althoughmicro-
C arrays are usually visualized and analyzed using organic fluorescent dyes
y of (Wiese 2003; Espina et al. 2004), their poor photostability, the decreasing
sit of the quantum yields upon conjugation to biomolecules and the self-
r
e quenchingathighlabelingratioslimittheireffectivenessinsuchapplications.
v
Uni New fluorescent probes for microarray-based bioanalysis such as europium
y: [ chelates (Scorilas et al. 2000), fluorophore loaded latex beads (Orth, Clark,
B and Craighead 2003), dye-doped silica nanoparticles (Zhou and Zhou
d
e 2004; Yao et al. 2006), lanthanide oxide nanoparticles (Dosev et al. 2005;
d
oa Nichkova et al. 2006) and inorganic nanocrystals (quantum dots, QDs)
wnl (Gerionetal.2003;Gehoetal.2005)havebeenexplored.QDsareveryattrac-
Do tiveforbiolabelingduetotheirpropertiessuchasexcellentbrightness,narrow
andprecisetunableemission,negligiblephotobleaching,fairlyhighquantum
yields, and photostability (Bruchez et al. 1998; Chan and Nie 1998). The
potential of QDs to address “multiplexing” has already been demonstrated
for biological imaging (Chan et al. 2002) and toxin analysis in a single well
of a microtiter plate (Goldman et al. 2004). Their broad excitation spectra
allowsimultaneousexcitationofdifferentparticlesizesatasinglewavelength
with emission at multiple wavelengths, each specific for an individual QD
fluorophore.QDfluorophoresenablemultiplexassaysrequiringasingleexci-
tationsourcefarfromtheQDemissionpeaks.AlthoughQDshaveprovento
be suitable labels in bioanalysis, their application in quantitative immunoas-
says for small molecules is still very limited (Goldman et al. 2002; Ding
et al. 2006).
Microcontact printing (mCP) is a relatively new method for chemically
and molecularly patterning surfaces on a submicrometer scale (Kane et al.
1999). It has been successfully applied to the direct patterning of proteins
on a micrometer scale without loss of their biological activity on a variety
of surfaces (Bernard et al. 2000; Tan, Tien, nd Chen 2002; Graber et al.
2003). The low cost of fabrication and the simplicity of transferring
proteins to substrates, when compared to other techniques such as ink-jet
printers, lithography, etc., makes mCP-fabricated arrays very attractive (Lin
etal.2001).UsingdirectmCPofproteins,wewereabletodevelopmicroarray
immunoassaysforantibodies(Nichkovaetal.2006)andbiomarkersofpyre-
throid exposure (Nichkova et al. 2005) based on Eu:Gd O nanoparticles as
2 3
fluorescent reporters.
Immunoassays are used as screening tools for the assessment of human
exposure to a variety of agrochemicals (Jaeger, Jones, and Hammock 1998;
Shan et al. 2004). Because of the prevalent use of pesticide mixtures, people
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1 have simultaneous exposure to multiple pesticides that may cause enhanced
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2 toxicityviasynergisticeffects.Multiplexedassaysareneededforoccupational
00: toxicology, risk assessment, and epidemiological studies. In this work, we
At: focus on the detection of biomarkers of exposure to two major classes of
a] compounds: pyrethroid insecticides and triazine herbicides. Pyrethroids are
rni widelyusedinvectorcontrol,forestry,households,agriculture,andhorticulture.
o
alif As major pyrethroids contain the phenoxybenzyl group and phenoxybenzoic
C acid(PBA)isacommonmetaboliteformorintermediate,PBAcanbeusedas
y of agenericbiomarkerofhumanexposuretomostpyrethroids(seeFig.1)(Shan
sit etal.2004).AtrazineisoneofthemostwidelyusedherbicidesintheUS,and
r
e despite its low acute toxicitytohumans,it may pose a healthrisktohumans,
v
Uni especially to agricultural workers though occupational exposure. The main
y: [ urinary metabolite, atrazine-mercapturate (N-acetyl cysteine derivative of
B atrazine) (Lucas et al. 1993) has been suggested as relevant biomarker for
d
e atrazineexposureinhumans(seeFig.1)(Buchholzetal.1999).
d
oa TheaimofthisworkistoapplyQDsasfluorescentlabelsinmultiplexed
wnl micro-immunoassaysfordetectionofPBAandAMasbiomarkersofexposure
Do topyrethroidsandatrazine.MicroarraysarepreparedwithmCPandconfocal
microscopy is used for fluorescencemeasurements.
EXPERIMENTAL
Chemicalsand Materials
Atrazine mercapturic acid (AM) [N-acetyl-S-f4-(ethylamino)-6-
[(1-methylethylamino]-1,3,5-triazin-2-ylg-L-cysteine] was prepared in our
Figure1. Chemicalstructuresoftargetedagrochemicals(pyrethroidsandatrazine)
andtheirbiomarkersofexposure(PBAandAM).
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1 laboratory previously (Lucas et al. 1993). 3-Phenoxybenzoic acid (PBA) and
4
2 bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St. Louis,
00: Missouri). The preparation of the coating antigen BSA-PBA, and the anti-
At: PBA specific polyclonal rabbit antibody (As 294) (Shan et al. 2004), and the
a] coating antigen BSA-AM (7-BSA) and the polyclonal sheep anti-AM (As
rni 266)(Jaeger,Jones,and Hammock 1998) havebeenreported previously. The
o
alif IgG fractions fromAs294 and As226 were obtainedby protein A purification
C using Affi-gel protein A MAPSw II kit (Bio-Rad Laboratories, California).
y of IgG226 was biotinylated using sulfo-NHS-biotin (Pierce, Illinois). Goat anti-
sit rabbit IgG F(ab’)2 conjugated to Birch Yellow EviTag (anti-rabbit QD580,
r
e 5mg/ml protein concentration) and streptavidin conjugated to Maple Red
v
Uni EviTag(SA-QD620,5mg/mlproteinconcentration)wereobtainedformAnti-
y: [ bodiesInc.(Davis,California).ClayAdamscoverglassfromFisherScientific
B (Pittsburgh,Pennsylvania)wasusedasasubstrateformicrocontactprintingof
d
e proteinsandfluorescencecharacterizationandan-dopedSiwaferwasusedfor
d
oa SEMcharacterization.Thepolydimethylsiloxane(PDMS)stampwasprepared
wnl accordingtotheliteraturemethod(KumarandWhitesides1993)usingSylgard
Do 184 silicon elastomer (Dow Corning, Midland, Michigan). The stamp used in
themicrocontactprintinghadapatternedmatrixof10(cid:1)10mmstrips.
Instrumentation
Fluorescent images were acquired with a Leica TCS-SP Laser Scanning
Confocal Microscope equipped with UV, Argon, Krypton, and Helium-
Neon lasers for excitation and photomultiplier tubes (PMTs) for detection.
Leica Confocal Software LCS Lite 2.0 was used for image acquisition. A
UV laser beam was used for the excitation of QD580 and 620. For SEM
characterizationaFEIXL30-SFEGScanningElectronMicroscopewasused.
Multiplexed Fluorescence Microimmunoassay for PBA and AM
Substrate Preparation
Cover glass was used as a solid substrate for microcontact printing of the
coating antigen BSA-PBA. Prior to use, the glass substrate was cleaned by
immersion in a solution of H SO :H O (70:30v/v) for 2h and then
2 4 2 2
washed extensively in running deionized water. Then the glass substrate
was thoroughly washed in an ultrasonic bath in ethanol and deionized water
for 10min each. Finally, it was dried under a blowing nitrogen stream. The
PDMS stamp was washed by sonication in ethanol (3(cid:1)10min), dried
under nitrogen and exposed to the solution of the inking protein mixture
(BSA-PBA and BSA in PBS in different ratios with 50mgml21 total
protein concentration) for 40min. Excess solution was removed, and the
stamp was dried under nitrogen. After inking, the stamp was brought into
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1 contactwiththeglasssubstrateandaverysmallamountofforcewasapplied
4
2 tomakeagoodcontactbetweenbothsurfaces.TheBSA-PBAprintingstageis
00: schematically presented in Fig. 2A. Printed slides were incubated with the
At: 2mgml21BSAforblockinginthecaseofsingleanalyteassays.Ifsubstrates
a] were prepared for multiplex analysis, they were incubated with the second
rni coating antigen (BSA-AM and BSA in PBS in different ratios with
o
alif 2mgml21totalproteinconcentration)for1handrinsedwithwater(Fig.2B).
C
of
sity Competitive Micro-Immunoassay
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ni Thesubstrateswereincubatedfor1hwiththespecificantibodies(50mgml21
U
y: [ IgG294 and 20mgml21 biotinylated IgG226) in 0.5% BSA/PBS, allowing
B the specific interaction between antibody and antigen to occur (Fig. 2C). In
d the competitive assays, PBA and AM at 100mgl21 concentration were
e
d
a added to the antibody solution. After washing with buffer, the substrate was
o
nl incubated for 1h with anti-rabbit QD580 (1/50 dilution in 0.5%BSA/PBS)
w
o andSA-QD620(1/30dilutionin0.5%BSA/PBS)(Fig.2D).Aftertheinter-
D
action took place, the substrate was rinsed with buffer, water, dried under
nitrogen, and scanned for fluorescence evaluation.
Scanning andEvaluation
AUVlaserbeamwasusedfortheexcitationofthetwoQDsusedinthiswork.
The detection wavelength ranges of the two PMTs were independently
adjusted to 625–635nm for QD 620 and 565–575nm for QD 580. The
gain of each PMT was individually adjusted to obtain similar fluorescence
intensities from the QD 620 labeled strips and the QD 580 labeled strips.
The average of five strips was used for evaluation of signal intensity. Each
experiment was performedin duplicate.
RESULTS AND DISCUSSION
With the aim to develop multiplexed immunoassays for biomarkers for
exposure we have chosen two targets: PBA and AM (see Fig. 1). The
specificantibodiesagainstthoseanalytesarerabbitandsheeppolyclonalanti-
bodies, respectively. We expected low cross-reactivity in the multiplexed
system. Initially we tested anti-rabbit and anti-sheep secondary fragments
labeledwithQDots,buttheS/Nratioobtainedfortheanti-sheepsystemwas
very low. Therefore, we biotinylated the sheep antibody and used biotin-
streptavidinamplificationdetection.AsreporterswehaveselectedQDot580
andQDot620becauseunderUVexcitationtheyhavewelldefinedseparateflu-
orescenceemissionpeakscenteredat570and630nm,respectively(seeFig.3).
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Figure2. SchemeofthemultiplexedimmunoassayforPBAandAM.(A)microcon-
tact printing of the coating antigen (BSA-PBA); (B) immobilization of the coating
antigen (BSA-AM); (C) immunoreaction with anti-PBA antibody and biotinylated
anti-AMantibody;(D)incubationofthemicro-patternswiththesecondaryimmuno-
reagents(anti-rabbitQDot580andstreptavidin-Qdot620).
Single Analyte Microimmunoassay
Initially we tested the QD detection system in a single analyte format. Glass
substrates patterned with BSA-PBA (or AM) and blocked with BSA were
Figure 3. Fluorescent spectra of anti-rabbit QDot 580 and streptavidin-Qdot 620
(excitationat350nm)recordedinSpectramaxM2microplatereader.
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1 used and different concentrations ofsecondary reagents were tested. Typical
4
2 fluorescent image and the corresponding line intensity profile of the
00: obtained micropatterns for the PBA assays are shown in Fig. 4A and B. A
At: series of alternating bright (green) and dark strips can be observed. The
a] actual width of the strips (10mm) is defined by the PDMS stamp used. The
rni green strips correspond to the QDots 580 specifically bound to the printed
o
alif BSA-PBA and the dark strips correspond to the BSA-blocked spacings
C between them. The average signal-to-noise ratio (S/N), i.e. specific
y of intensity/backgroundintensityof(cid:1)10demonstratessuccessfulimmunoreac-
sit tionwithverylownonspecificbinding.Figure4Crepresentstheeffectofthe
r
e coatingantigenconcentration(BSA-PBA)usedintheprintingprocessonthe
v
Uni fluorescence signal of QD580. Saturation typical for this type of
y: [
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Figure 4. Single analyte micro-immunoassay for PBA (A) Fluorescence image of
QD580 specifically bound to anti-PBA rabbit IgG bound to microcontact printed
BSA-PBAantigen;(B)Intensityprofilecorrespondingtolineshownin(A);(C)Fluor-
escent intensity of QD580 as a function of the concentration of the microcontact-
printedBSA-PBAcoatingantigen.
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12 immunoreactionsisobserved.Wehavechosenaconcentrationof4mgml21for
4
2 thecompetitiveassay.Similarly,wehavetestedtheexperimentalconditionsfor
00: theAMimmunoassayinasingleanalyteformat(datanotshown).Micropat-
At: ternswithredstripscorrespondingtoSA-QDot620boundtothebiotinylated
a] anti-AMantibodyrecognizingtheprintedBSA-AMwereobtained.
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C SEM Characterizationofthe Microarrays with QD
of
y
sit Themicroarrayswerecharacterizedbyscanningelectronmicroscopy(SEM)
r
ve for more detailed studies at single nanocrystal level. For this purpose, the
Uni micro-patterns were prepared on a silicon wafer instead of glass slide
y: [ following the procedure described above and keeping all other experimental
B conditions identical. The use of the silicon wafer assured a perfectly flat
d
e surface and conductivity of the substrate—conditions required for the SEM
d
a
o studies. The SEM image presented in Fig. 5 illustrates a segment of a strip
wnl formed by the specifically bound anti-rabbit QD620 to anti-PBA rabbit IgG
o
D boundtomicrocontactprintedBSA-PBAcoatingantigen.Thewaferwaspas-
sivated by BSA blocking. The QDots can be individually distinguished as
white spots on the dark background. It can be seen that the distribution of
theQDs isnotperfectlyuniformandthe QDs are notdensely packedwithin
the strip. A similar effect was observed by other authors when immobilizing
polystyrene beads through specific avidin-biotin binding (Andersson et al.
2002)andalsowhenusinglanthanideoxideparticlesaslabelsinsimilarcon-
figurations(Nichkovaetal.2006).Webelievethatthisismainlyduetosteric
hindranceandlimiteddiffusionoftheQDsnearthesubstrateduringtheincu-
bation.Inspiteofthat,theQDsarearrangedintoagoodmatchwiththeprinted
patterns.OnlyafewindividualQDscanbeobservedinthenon-printedareas
blockedwithBSA,thusshowingverylownon-specificbinding.
Figure 5. Scanning Electron Microscopy (SEM) image of specifically bound anti-
rabbitQD580toanti-PBArabbitIgGboundtomicro-contactprintedBSA-PBAcoating
antigen(10(cid:1)10mmlinepatterns).BSAisusedforblockingthenonprintedareas.
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B Figure 6. Multiplexed immunoassay for PBA and AM. (A). Confocal microscope
d fluorescentimagecorrespondingtoQD580(PBA)andQD620(AM);(B).Competi-
e
ad tivemicro-immunoassay(signalinhibitioninpresenceofbothanalytes).
o
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D Multiplexed Fluorescence Microimmunoassay for PBA and AM
The multiplexed micro-immunoassay was performed with glass substrates
containing strips formed by the two coating antigens (BSA-PBA and BSA-
AM).Fig.6Arepresentsthefluorescenceimageobtainedforthemultiplexed
assay at zero analyte concentration. Strips with alternating green and red
colorscorrespondingtoQD580andQD620canbeobserved.Thedifference
inthespectralemissionofthetwoQDs enables theirsimultaneousdetection
without cross-talk. In a mixture of the analytes (PBA and AM), the fluor-
escence signal was inhibited because fewer amounts of specific antibodies
bind to the substrate resulting in less binding of the secondary reagents
(QDs) (see Fig. 6B). Further optimization of the quantitative measurements
would require the incorporation of internal fluorescence calibration of the
detection system (Nichkova et al. 2005) and optimization of the sensitivity
of the multiplexed immunoassay. Using europium oxide nanoparticles as
labelsinsimilarconfiguration,wehaveobtainedsensitivityforPBAcompar-
able tothatof the ELISA (Nichkova et al. 2005).
CONCLUSIONS
Quantumdotscanbeusedassuitablelabelsforimagingofmicroarraysforthe
detectionofsmallmolecules(biomarkersofpesticideexposure).Theirphoto
stabilitypermitsprolongedexcitationforimageobservationandoptimization.
The spectral properties of quantum dots allow the multi-analyte detection of
the targets. The same approach could be successfully applied to the
detection ofproteinsand DNA with different bioanalytical applications.