varner 09-116 apm 271 final journal.

19 Pages·2009·1 MB·English

CRITICAL REVIEW www.rsc.org/jem | JournalofEnvironmental Monitoring
Sensors as tools for quantitation, nanotoxicity and nanomonitoring
assessment of engineered nanomaterials
O. A. Sadik,*a A. L. Zhou,a S. Kikandi,a N. Du,a Q. Wanga and K. Varnerb
Received30thJune2009,Accepted25thAugust2009
FirstpublishedasanAdvanceArticleontheweb14thSeptember2009
DOI:10.1039/b912860c
Thediscoveryoffullerenesin1985hasusheredinanexplosivegrowthintheapplicationsofengineered
nanomaterialsandconsumerproducts.Nanotechnologyandengineerednanomaterials(ENMs)are
beingincorporatedintoarangeofcommercialproductssuchasconsumerelectronics,cosmetics,
imagingandsensors.Nanomaterialsoffernewpossibilitiesforthedevelopmentofnovelsensing
andmonitoringtechnologies.Nanosensorscanbeclassifiedundertwomaincategories:(i)
Nanotechnology-enabledsensorsorsensorsthatarethemselvesnanoscaleorhavenanoscalematerials
orcomponents,and(ii)Nanoproperty-quantifiablesensorsorsensorsthatareusedtomeasure
nanoscaleproperties.Thefirstcategorycaneventuallyresultinlowermaterialcost,reducedweightand
energyconsumption.Thesecondcategorycanenhanceourunderstandingofthepotentialtoxiceffects
ofemergingpollutantsfromnanomaterialsincludingfullerenes,dendrimers,andcarbonnanotubes.
DespitetheenormousliteraturesandreviewsonCategoryIsensors,therearefewsensorstomeasure
nanoscalepropertiesorsensorsbelongingtoCategoryII.Thisclassofnanosensorsisanareaof
criticalinteresttonanotoxicology,detectionandriskassessment,aswellasformonitoringof
environmentaland/orbiologicalexposure.Thisarticlediscussesemergingfieldsofnanotoxicologyand
nanomonitoringincludingthechallengesofcharacterizingengineerednanomaterialsandthepotentials
ofcombiningexistinganalyticaltechniqueswithconventionalcytotoxicitymethods.Twocasestudies
areprovidedfordevelopmentofCategoryIInanosensorsforfullerenenanoparticlesandquantum
dots.Onehighlightstheuniquenessofaportable,dissolvedoxygenelectrochemicalsensorarrays
capableofdetectingtheENMsaswellasproviderapidnanotoxicologicalinformation.Thisreview
hasshownthataddressingthecomplexandcriticalissuessurroundingtheenvironmental
transformationandtoxicityofENMsmustbeaccompaniedbythecreationofnewapproachesor
furtherdevelopmentsofexistinginstrumentation.
1. Introduction
aCenter for Advanced Sensors & Environmental Monitoring (CASE), Nanotechnology—thescienceofassemblingorcontrollingmatter
Department of Chemistry, State University of New York-Binghamton, atom-by-atom at the nanoscale level and its potential applica-
P.O.Box6000,Binghamton,NY,13902
tions—presentsbothopportunitiesandchallenges.Opportunity
bUS-Environmental Protection Agency, National Exposure Research
existstocreatenovelmaterialsbasedontheenhancedcatalytic,
Laboratory, Characterization Research Division, P.O. Box 93478-3478,
LasVegas,NV,89193-3478 optical,magneticandelectricalpropertiesofnanomaterials.Some
Wunmi Sadik is a Professor of Chemistry at State University of New York at Binghamton (SUNY-
Binghamton)andthedirectoroftheCenterforAdvancedSensorsandEnvironmentalSystems.Sadik
receivedherPh.D.inChemistryfromtheUniversityofWollongonginAustraliaanddidherpostdoctoral
researchattheUSEnvironmentalProtectionAgency(US-EPA)inLasVegas,Nevada.DrSadikhas
held appointments at Harvard University, Cornell University and Naval Research Laboratories in
Washington,DC.Sadik’sresearchcurrentlycentersontheinterfacialmolecularrecognitionprocesses,
sensorsandbiomaterials,andimmunochemistrywithtandeminstrumentaltechniques.Herworkutilizes
electrochemical and spectroscopic techniques to study risk exposure assessment, endocrine disrupters,
andtoxicityofengineerednanomaterialsandnaturallyoccurringchemicalcompounds.Sadikhasover
370 scientific publications in the areas of biosensors, environmental and materials chemistry. These
include referred articles (110),patents/patent applications (10), invited/keynote presentations (125),
book chapters, andconference abstracts (135). Sadikis the recipient of HarvardUniversity’s Distin-
guished Radcliffe Fellowship, National Science Foundation’s Discovery Corps Senior Fellowship,
OmowunmiðWunmiÞA:Sadik SUNY Chancellor Award for Research, Australian Merit Award, Chancellor Award for Outstanding
Inventor,andNationalResearchCouncilCOBASEfellowship.
1782 | J.Environ. Monit.,2009, 11, 1782–1800 Thisjournal isªTheRoyalSociety ofChemistry2009
engineered nanomaterials (ENMs) have already been incorpo- duetotheirlowcost,sensitivity,portabilityandsimplicity.The
rated into a range of commercial products, including pharma- present review focuses on the development of nanosensors for
ceuticals,automobileadditives,personalcare,suchassunscreens assessing the toxicity of engineered nanomaterials and case
and cosmetics, clothing, sporting equipment, tires, detergents, studies are described for the development of nanosensors for
and stain-repellants.1 ENMs are also being used as probes for fullerenes and quantum dots. This article also provides a short
ultrasensitive molecular sensing and diagnostic imaging, agents summaryofthedifferenttechniqueswhicharecurrentlyusedfor
for photodynamic therapy (PDT) and actuators for drug thecharacterizationand/orabsolutequantificationofengineered
delivery,triggersforphotothermaltreatment,andprecursorsfor nanomaterialsandnanotoxicity.
building solar cells, electronics and light emitting diodes.2
Despitetheincreasingapplicationsofengineerednanomaterials, 2. Toxicity of engineered nanomaterials
a complete understanding of the size, shape, composition and
aggregation-dependant interactions of nanostructures with bio- Currently, the number of engineered nanomaterials on the
logicalsystemsislimited.Also,thereareanumberofimportant, market is large and is expected to increase with advances in
unresolved questions concerning the safety of ENMS. The synthetic and technological developments. As shown in Fig. 1,
potentialexposurescenarios,andtheirinteractionwiththebio- a large number of engineered nanomaterials have been synthe-
logicalandenvironmentalsystemsarelargelyunknown.Hence, sizedtodate.Thisfigurealsoshowsthatsincethefirsttwopapers
subdisciplines of nanotechnology such as nanotoxicity, nano- on this topic appeared in 1991, the number of publications did
monitoring and/or nanomeasurements are emerging. Nano- not increase significantly until 2004, when the total number of
toxicologyfocusesonthecharacterizationandcategorizationof publicationsroseto754.From2005toAugust2009,therewere
thehealtheffectscausedbyengineerednanomaterialsinorderto 3,454articlespublishedthusbringingthetotalnumberofpapers
correlate the nanoparticles structure/function with toxicity.3 publishedtodateto4,208.
Nanomonitoring or nano-measurement refers to the science of While nanomaterials have numerous applied uses and the
isolating, detecting, characterizing, and quantifying ENMs in benefitsofnanotechnologyarewidelypublicized,thediscussion
complexenvironmental,biologicalorecologicalsamples. oftheirpotentialeffectsonhumanhealthandtheenvironmentis
Recentstudieshaveshownthatsomeofthespecialproperties justbeginning.Incontrasttothecomparativelylargenumberof
that make nanomaterials useful may also cause them to pose articlesonnanomaterialsynthesis(4208),asimilarsearchonthe
hazards to humans and the environment.4 For example, silver Web of Science with the search word: ‘‘nanotoxicology’’
nanoparticlesareharmfultotheenvironmentbecausetheymay producedonly171hitstodate.Theseinclude29reviewpapers,
destroyenvironmentallybenignbacteriathatareusedforwaste 103 articles, 16 meetings, 13 editorials and 10 news articles.
water treatment5,6 although they are widely used in different Clearly, there is an increasing interest in the toxicity of engi-
productsinthemarketsuchasdetergents,wounddressingsand neerednanomaterials.
disinfectants. The effects of engineered carbon nanotubes and In general, nanoscale particles, whether termed ultrafine,
metal oxide nanoparticles on diverse microbial communities,7 engineered, intentional, or incidental, pose challenges for phys-
algae,plants,andfungiaswellasonaquaticinvertebrateshave ical, chemical and biological characterization. Meantime, these
also been reported.8 The toxicity mechanisms of such nano- newmaterialscouldhaveanumberofpotentialcausesoftoxicity
materials has been attributed to the production of reaction orconcerns:(1)nanostructureshavebeendemonstratedtohave
oxygen species and accidental release of metal ions.8 Smaller electronic, optical, and magnetic properties that are related to
particles in ambient air have been demonstrated in inhalation their physical dimensions, and the breakdown of these nano
studiestoexhibitadversehealtheffectsduetodeeperpenetration structurescouldleadtouniquetoxiceffectsthataredifficultto
intolungsandlargesurfaceareas.9Theseparticlesarecapableof predict. (2) Nanostructured surfaces are utilized in many
bypassing the blood-brain barrier through the olfactory bulb.
Somemetaloxidenanoparticleshavebeenreportedtoaffectthe
inflammatoryprocessesofthecentralnervoussystem.10Further,
a set of predictive measures of nanomaterial toxicity have also
been identified11–14 relying on the detection of the generated
reactive oxygen species (ROS),15,16 mitochondrial perturbation,
inflammation response pathways, lipid peroxidation, protein
denaturation and degradation, and DNA damage. Nel et al.16
havestressedtheimportanceofpragmaticandmechanism-based
approach in testing the potential harmful effects of engineered
nanomaterials. Oberdo€rster et al. have outlined three key
elementsofnanoparticletoxicityscreeningstrategies:17(i)phys-
icochemicalcharacterization(size,surfacearea,shape,solubility,
aggregation), elucidation of biological effects involving (ii) in
vitro and (iii) in vivo studies. In that respect, a broad array of
analytical tools and methods are needed to perform such char-
Fig. 1 Published papers on nanomaterials synthesis from 1990–2009.
acterizations. These statistics were generated from the ISI Web of Science using
Chemicalandbiosensorsarewellsuitedtocomplementstan- a combination of search terms that represent ‘‘nanomaterials and
dard analytical methods for detection of environmental toxins synthesis’’.
ThisjournalisªThe RoyalSociety ofChemistry 2009 J.Environ. Monit., 2009,11,1782–1800 | 1783
catalyticandoxidativereactions.Ifthesereactionsinducecyto- translate to in vivo interaction. These and many similar con-
toxicity, the toxicity can be greater than what is observed for trasting reports make it difficult for researchers and policy
asimilarbulkmaterialsbecauseoftheenhancedsurfacearea-to- makers to determine which nanomaterials to study and howto
volume ratio for nanoscale materials, (3) some nanostructured regulatetheindustry.
materialscontainmetalsorcompoundswithknowntoxicityand
thus the breakdown of these materials can elicit similar toxic 3 Environmental detection and characterization of
responses to the components themselves.2 For example, the
ENMs-EPA perspectives
removaloftetrahydrofuran(THF),thesolventusedinpreparing
(cid:2)30 nm–100 nm particles of C resulted in a loss of toxicity: The mission of the United States Environmental Protection
60
Biomaterials27(29):5049–58,2006.(4)Synergisticand/antago- Agency(EPA)istoprotecthumanhealthandtheenvironment.
nisticreactionsofENMswithotherchemicalsmaybedifficultto EPAmusthaveasoundscientificbasistocarryoutthismission.
isolate and (5) ENMs could be used as potential chemical/bio- EPA conducts and supports programs that address human
logicalweapons. health and the environmental effects of substances; assesses
Thefirststepstoidentifyanumberofcriticalriskassessment potential risk management approaches; and finds innovative,
issues regarding manufactured nanomaterials9 began at a 2004 cost-effective ways of reducing risks. According to the EPA
workshopcosponsoredbytheNationalScienceFoundationand Nanotechnology White Paper, ‘‘a challenge for environmental
theNationalInstituteofEnvironmentalHealthServices.Critical protection is to help to fully realize the societal benefits
issuesidentifiedincludeexposureassessment,toxicology,ability of nanotechnology while identifying and minimizing any
to extrapolate, environmental and biological fate, transport adverse impacts to humans or ecosystems from exposure to
mechanism, persistence, transformation, and overall sustain- nanomaterials’’.
ability. This workshop was instrumental in bringing about the EmergingareasofconcernfortheEPAincludeunderstanding
NationalScienceandTechnologyCouncil(NSTC).TheNSTCis health and environmental effects of ENMs and being able to
theprincipalmeansbywhichtheExecutiveBranchcoordinates communicatetheexposure(healthandsafety)riskstothepublic.
science and technology policy across the diverse entities that Extensive research is needed which provides for accuracy,
make up the Federal research and development enterprises. A precision and sensitivity of analytical techniques for under-
subcommittee of the NSTC is the Nanoscale Science, Engi- standingENMs.Researchareasfocusontheengineeringfactors
neeringandTechnology(NSET),whichisresponsibleforcoor- involved in the transport, transformation and longevity of
dination of the National Nanotechnology Initiative (NNI). ENMs (Draft Nanomaterial Research Strategy (NRS), EPA/
Recently, as part of a nationaleffort to stimulate new research 600/S-08/002). EPA’s National Exposure Research Laboratory
and knowledge in this area, the National Institute for Occupa- (NERL) Environmental Sciences Division (ESD) is responsible
tional Safety and Health (NIOSH) collaborated with NSET to forconductingstudiesfordetecting,characterizing,quantifying,
co-sponsortheworkshoponHumanandEnvironmentalExpo- and monitoring of nanotechnology and emerging contaminates
sure Assessment of Nanomaterials from Feb. 24–25, 2009 in in the environment. Areas of research include source, fate, and
Bethesda,MD. transport, exposure, preventing and mitigating risks of nano-
The cytotoxic potential, detection and characterization of materialsthroughidentifyingtechnologiesthatcanbeappliedto
nanomaterials are dependent on factors such as functionaliza- measure and minimize exposure to ENMs. ESD concern is
tion, geometry, and material. One study evaluated a variety of greatly vested with the transport of nanomaterials in the envi-
carbon nanomaterials and found single walled nanotubes ronmentwhichrequiresunderstandingthemechanismsofenvi-
(SWNT) to be much more cytotoxic than fullerenes or multi- ronmentalmedia,thevariousenvironmentalparameters,andthe
wallednanotubes(MWNT).14Conversely,anotherstudyevalu- various nanomaterial parameters. Nanomaterial parameters
ating MWNT, carbon nanofibers and carbon black found that include particle size and charge, chemical or elemental compo-
thesmallerthesizeofthenanomaterial,thegreaterthetoxicity.18 sition,andsurfacemodifications.Environmentalmediaparam-
AkeynanotoxicitystudyworthyofnoteisthatreportedbyA. etersincludepH,ionicstrength,flowrate,composition,andthe
Tagaki et al. (2008).152 These authors demonstrated that intra- presence of naturally occurring (such as dissolved organic
peritoneal administration of MWNT led to the induction of carbon) and anthropogenic contaminants.20 EPA is seeking to
mesothelioma in p53 heterozygous mice after these model discoveranddefinepreviouslyunknownandpoorlyunderstood
animals were injected with micrometer sized MWNTs, with vulnerabilities and provide quantitative data to exposure risk
lengthsreachingtensofmicrometersthatcorrespondtothesize assessors which would make it possible to predict estimates of
and shape of asbestos. The result of this study points to the nanomaterials that most likely would be of concern (US EPA
possibility that carbon-made fibrous or rod-shaped micrometer NanotechnologyWhitePaper,EPA100/B-07/001).
particlesmaysharethecarcinogeneticmechanismspostulatedof
asbestos. Some researchers have also shown that functionaliza-
4. Conventional methods for assessing nanotoxicity
tioncanchangethecytotoxicityofnanomaterials.Forexample,
the cytotoxicity of fullerenes can be decreased after fullerenes Asdescribedearlier,theincreasedprevalenceofnanomaterialsin
were hydroxylated with 24 hydroxyl groups,19 while the cyto- consumergoodshaslaidthefoundationfortheemergingfieldof
toxicity of carbon nanomaterial may be enhanced if it was nanotoxicologyandnanomonitoring.Thishasbroughtattention
functionalized with carboxylic acid moieties which promote to the research needs regarding the toxicological assessment of
aqueous phase dispersion.7 These studies, as others, have ENMs.Atremendousamountofactivityhasbeenwitnessedin
primarilybeentestedinvitroandtheresultsmaynotaccurately thisfieldasreviewedbyMarquisetal.3However,itisimportant
1784 | J.Environ. Monit.,2009, 11, 1782–1800 Thisjournal isªTheRoyalSociety ofChemistry2009
to recognize that particle or agglomerate size distribution can, nanoparticles3,21,22–25 before and after exposure to cells. High
and often does, change as a material is prepared and used in resolution transmission electron microscopy (HRTEM) can
toxicological studies. The size and shape of the material inter- especiallybethebestchoicetoidentifythecrystallinestructures
actingwithanorganismmaydifferdramaticallyfromitsoriginal of particles, as reported by Petri-Fink et al.26 for super-para-
form. Thus, the size distribution ‘‘as doses’’ might be quite magnetic ironoxidenanoparticlesandbyWarheit etal. quartz
differentfromthatof‘‘as-generated’’or‘‘as-received’’material.3 particles.22 However, HRTEM is subject to artifacts caused by
Conversely, hardly any single assay is readily available on samplepreparationorspecialanalysisconditions.Forexample,
quantification, measurement and monitoring technologies for itrequireshighvacuumandthinsamplesectionsorparticlesof
nanomaterials. Techniques are best used in concert with each limiteddiametertoallowtheelectronbeamtopenetratethrough
othertoprovideamorecompleteunderstandingofuptake. the sample. Tissue sample preservation, fixation, and staining
Inthissection,wesummarizedtheexistingmethodologiesfor require skills to preserve the sample details and to avoid intro-
detection of nanotoxicity including microscopy, spectrometry, ductionofartifacts.27TheSEMcanbeusedtoimagethesample
classical cell culture techniques, sensors as well as computer- surface by scanning it with a high-energy beam of electrons in
assisted molecular modeling techniques. As shown in Fig. 2, ascanningpattern.Linetal.hasexaminedthephototoxicityof
thereis anintersection betweenthe characterization techniques ZnO nanoparticles by Lolium perenne (ryegrass) using SEM.23
for ENMs and the detection of its nanotoxicity. Relevant The results indicated that ZnO nanoparticles greatly adhered
propertiesthatcouldbemeasuredincludedose,purity,particle ontotherootsurface.Scanningprotonmicroscopy(SPrM)isan
size & distribution, shape, surface chemistry, surface charge, analogueofelectronmicroscopyandenablestheuniquefeature
surfaceareaandsurfaceactivityaswellastheirabilitytopene- of elemental mapping down to the parts-per-million level with
tratethebiologicalsystems. highaccuracy.However,thetechnologyforfocusingprotonsis
technologicallymorecomplexthanforelectronsduetothemuch
higher momentum of protons. Tong et al.28 has probed the
4.1 Microscopytechniques
cytotoxicityofnanoparticles(ZnO,Al O andTiO )andorganic
2 3 2
Microscopy is one of the most powerful techniques to provide compoundsusingSPrMonaTlymphoblasticleukaemiaJurkat
valuableinformationregardingthesize,shape,andmorphology celllines.
of nanomaterials. Examples include electron and proton Asaresultofcontinuousdevelopmentsinsamplepreparation,
microscopy, atomic force microscopy (AFM), scanning probe imaging techniques, and instrumentation, AFM is regarded as
microscopyandscanningtunnelingmicroscopy(STM),andeach a companion of both X-ray crystallography and electron
has specialized skills. Table 1 provides a summary of the microscopy.AFMhasalsoevolvedintoanimagingmethodthat
microscopic techniques for characterizing nanomaterials. The yields structural details of biological samples such as proteins,
advantages and drawbacks of each technique are also high- nucleicacids,membranes,andcellsintheirnativeenvironment.
lighted. Specifically, TEM has provided the most detailed Ithasbeenusedinvariousbio-medicalapplicationsincludingthe
information regarding in vitro nanoparticle uptake and locali- testing of cellular toxicity of nanoparticles and carbon-nano-
zation by allowing both visualization of nanoparticle location tubes.29Inaddition,AFMisusedtomeasurethesurfaceareaof
within a cell or tissue and, in conjunction with spectroscopic nanoparticles,whichisthoughttoplayanimportantroleinthe
methods,characterizationofthecompositionoftheinternalized toxicity of nanoparticles.54–56,153 Chen et al. used AFM to
Fig.2 Majortechniquesemployedforthecharacterizationofengineerednanomaterialsandforassessingnanotoxicity.
ThisjournalisªThe RoyalSociety ofChemistry 2009 J.Environ. Monit., 2009,11,1782–1800 | 1785
Table1 MicroscopicTechniquesforCharacterizingNanomaterialsa
Method/References Applications Nanomaterials Advantagesanddrawbacks
TEM3,21,23–25 Invitrouptake/localization Organic,inorganic (cid:3)VisualizationofENMs
&hybridnanomaterials (cid:3)TEMimagesprovidepoorresolutionofdiffuse
electronmaterials
HRTEM22,26,27 Crystallizationofparticles Ironoxide,quartzparticles (cid:3)Individualparticlesandagglomeratescanbe
resolved
(cid:3)Providesvaluableinformationonsize,chargeand
morphology
(cid:3)Highresolutionmicroscopyissubjecttoartifacts
causedbysamplepreparationconditions
(cid:3)Requiresthinsamplesectionsorparticlesoflimited
diametertoenabletheelectronbeamtopenetrate
throughthesample
SEM23 Invitro/invivo PhototoxicityofZnO, (cid:3)Requireshighvacuumconditions
surfacemorphology AlO,TiO (cid:3)Aspecimenisnormallyrequiredtobecompletely
2 3 2
dry
SprM28 Tlymphoblasticleukaemia Organic,inorganic (cid:3)Nuclearmicroscopycanquantitativelymapall
&hybridnanomaterials elementsintheperiodictable
Celllines (cid:3)Providessimultaneousstructuralimaging
(cid:3)Providesuniquefeaturesofelementalmapping
(cid:3)Suitableforparts-permillionlevelsensitivity
(cid:3)Highmomentumofprotonscreatesamorecomplex
focusingproblemsforprotonsthanforelectrons
AFM29 BiomedicalImaging Carbonnanotubes (cid:3)AllowsthedeterminationofsurfaceareaofENMs
&cellulartoxicity (cid:3)Providessub-nanometerresolutionatareasonable
ofnanoparticles signal-to-noiseratiounderphysiological
conditions
(cid:3)AFMisregardedasacompanionofbothX-ray
crystallographyandelectronmicroscopyandhas
evolvedintoanimagingmethodthatyields
structuraldetailsofbiologicalsamplessuchas
proteins,nucleicacids,membranes,andcellsin
theirnativeenvironment
AFFM30 Biomedicalimaging (cid:3)Providesbiochemicalidentification
SNUL Imagingnanoparticles (cid:3)Applicableforimaging
Fluorescence Cellularcytotoxicity ImagingofSWCNTs (cid:3)Qualitativelydeterminesthebindinganduptakeof
microscopy31,32 nanomaterials
Nanoparticleuptake DendrimerENMs (cid:3)Visualinspectionofcellswithbrightfield
andlocalization microscopyforchangesincellularornuclear
morphology
(cid:3)Quantitativeassessmentcanbeachievedin
amannersimilartoICP-AESthroughuseofbulk
fluorescenceoronacell-to-cellbasisusing
confocalfluorescence
(cid:3)Cellularuptakeisanecessaryprerequisite
aAbbreviationkey:TEM:transmissionelectronmicroscopy,SEM:scanningelectronmicroscopy,SPrM:Scanningprotonmicroscopy,AFM:atomic
forcemicroscopy, AFFM:atomicforcefluorescence microscope,SNULscanningnear-fieldultrasonicholography, SWCNTs:singlewalledcarbon
nanotubes.
determinetheshapeandsizeofcoppernanoparticles,andthen holography can now image nanoparticles buried below the
calculatedtheaveragesurfaceareapergram.However,themost surfacesofcells,whichcouldproveusefulinnanotoxicology.31,32
common method to measure the surface area is Engelhard, the
multipointBrunaeur,EmmettandTeller(BET)method.Inthis
4.2 Classicalcellculturetechniques
case, the gas absorption to the surface of these particles is first
measured,andthenthesurfaceareacanbecalculatedoutusing Invitrocytotoxicityassaysarethemajoralternativemethodsto
BET method. Instruments for BET method are commercially animal testing for basal cytotoxicity assessment of chemicals,
available from Quantachrome Instruments, Beckman Coulter typically indicating the number of cellswhich are dead oralive
and Micromeritics. Besides, scanning mobility particle sizer afterexposuretotestchemicals.Conventionalinvitrocell-based
(SMPS),transmissionelectronmicroscopy(TEM),anddiffusion assaysarecommonlyusedtoscreencytotoxiceffectsinducedby
charging (DC) have also been reported for measurements of chemicals in a variety of cell systems. Examples include
some nanoparticles.154 Atomic force fluorescence microscope biochemical methods such as 3-(4,5-dimethylthiazol-2-yl)-2,5-
(AFFM)hasbeen developedthatcombinesthe high-resolution diphenyltetrazolium bromide test (MTT), neutral red uptake
topographicalimagingofAFMwiththereliable(bio)-chemical (NRU), ATP and lactate dehydrogenase (LDH) measurement,
identificationcapabilityofopticalmethods.30Arelatedscanning Sulforhodamine B (SRB) assay, WST assay. Others include
probemicroscopytechniquecalledscanningnear-fieldultrasonic growthassayssuchascolonyformingefficiency(CFE),cytokine
1786 | J.Environ. Monit.,2009, 11, 1782–1800 Thisjournal isªTheRoyalSociety ofChemistry2009
assay, phagocytosis assay, nitric oxide assay, and glutathione oxidative stress, mitochondrial perturbation, inflammation
assays. These techniques are equally applicable to measure the responsepathways,lipidperoxidation,proteindenaturationand
cytoxicity of nanoparticles. Several recent peer-reviewed publi- degradation, and DNA damage. Table 2 provides a list of the
cations have focused on a set of predictive measures of cyto- classical cell cultures and other techniques with utilities for
toxicity of nanomaterials11–14 through the detection of the assessingtherisksposedbyengineerednanomaterials.
generated reactive oxygen species (ROS),15,16 focusing on
4.2.1 Colorimetric assays. Colorimetric methods are the
major tools employed in cytotoxicity assessment throughout
Table2 ListofInstrumentalandClassicalCellCultureTechniquesfor publishednanomaterialsstudies.Thesecolorimetricmethodscan
CharacterizingNanomaterialsa befurthercategorizedintoteststhatmeasureplasmamembrane
integrityandmitochondrialactivity.
Abbreviation Methods/Definitions
Classicalcellculture Exposuretocertaincytotoxicagentscancompromisethecell
membrane,whichallowscellularcontentstoleakout.Viability
MTT3,11,155 3-(4,5-Dimethylthiazol-2-yl)-2,5- testsbasedincludeneutralred.Neutralred,ortoluylenered,is
diphenyltetrazoliumbromide
a weak cationic dye that can cross the plasma membrane by
MTS156 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4- diffusion.The dye tendsto accumulatein lysosomeswithinthe
sulfophenyl)-2H-tetrazolium cell.Ifthecellmembraneisaltered,theuptakeofthedyedecreased
NRU155 neutralred(3-amino-7-dimethyl- and can leak out, allowing discernment between live and dead
amino-2-methylphenazine
cells. Cytotoxicity can be quantified by taking spectrophotonic
hydrochloride)uptake
XCT157 X-rayComputedTomography measurementsoftheneutralreduptakeundervaryingexposure
LDH155 Lactatedehydrogenase conditions.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
SRB158 SulforhodamineBassay
bromide (MTT) assay is among the most versatileand popular
WST-1159 (4-[3-(4-iodophenyl)-2-(4-
nitrophenyl)-2H-5-tetrazolio]- assaysusedforinvitrotoxicology.Mitochondrialactivitycanbe
1,3-benzenedisulfonate) tested using tetrazolium salts as mitochondrial dehydrogenase
CFE160 Colonyformingefficiency enzymescleavethetetrazoliumringandthisreactiononlyoccurs
FCM161 Flowcytometry
inlivingcells.Reductionofwater-solubleMTTsaltbymetabol-
aReaders are referred to the cited reviews and articles for detailed icallyactivecellsleadstotheformationofMTT-formazancrys-
descriptionsoftheadvantagesanddrawbacks.3,11,156–163 tals.TheinsolubleMTT-formazanisdepositedinmitochondria,
in the cytoplasm, and in the regions of plasma membranes.
Assaysandkits
ReductionofMTTinisolatedcellsisregardedasanindicatorof
Cytokineassay162 Abloodtesttodetectinterleukins ‘‘cellredoxactivity’’.Thistechniquehasmanyadvantageswhen
Phagocytocisassay Engulfinganddestroyingoffungi
compared to other toxicity assays because it requires minimal
andbacteria
physical manipulation of the model cells and yields quick,
NitricOxideassay AKitfortheQuantitative
ColorimetricDeterminationof reproducibleresultsrequiringsimpleopticaldensityacquisition.
NitricOxide Othertetrazolium-basedassaysusedtotestthecytotoxicityare
Gluthathioneassay
theMTS,XTTorWSTassay.Thenumberoflivingcellscanbe
TryphanBlueassay
Fluorescenceassay determined similarly by quantifying the production of soluble
formazan.Inassaysthatproduceinsolubleformazandyes(such
Instrumentaltechniques astheMTTassay),exocytosisofthecrystallineproductcanskew
results;thereforeassaysthatproducesolubledyes(suchasMTS,
GPC/SEC GelPermeationChromatography
(GPC)/SizeExclusion XTT or WST-1) are preferred. A summary of colorimetric
Chromatography(SEC) methodsinvitrotoxicologicaltechniquesusedintheassessmentof
GFAAS GraphiteFurnaceAtomic
nanotoxicityhasbeenreviewed.3,11
AbsorptionSpectrometry
Although understanding nanoparticle effects on mitochon-
NMR Nuclearmagneticresonance
XRD X-RayDiffraction drialactivityisimportant,itisjustoneofmanyrelevantcellular
XPS X-rayphotonscattering functions.Themajorproblemwithusingtraditionaltoxicology
spectroscopy
assaysisthatnanomaterialsmayinterferedirectlywiththesignal
TGA Thermogravimetricanalysis
FFF Field-Flowfractionation transduction based on the increased reactivity of their surface
ICP-AES(OES) Inductivelycoupledplasmaatomic sites compared to bulk materials. In one example, Ag nano-
emissionspectroscopy particleswerefoundtointeractdirectlywiththetetrazoliumsalt
TIRF Totalinternalreflectance
of the MTT assay, suggesting greater than 100% viability of
fluorescence
EELS ElectronEnergyLossSpectroscopy nanoparticle-exposed cells based on the ability of the nano-
ICP-MS InductivelyCoupledPlasmaMass particlestogeneratethecoloredformazanproduct.Usingacell-
Spectrometry
free system, it was also reported that polyoxyethylene sorbitan
MALDI MatrixAbsorptionLaser
DesorptionIonization monooleate-suspended SWCNTs interfered less with MTT
EDS EnergyDispersiveSpectroscopy assays than sodium dodecyl sulfate-suspended SWCNTs.
PET PositronEmissionTomography Moreover,dependingonthepurificationprocedureofSWCNTs,
XPS X-rayPhotoelectronSpectroscopy
theywereabletoconvertMTTintoitsMTT-formazaninsoluble
SRT Synchrotronradiationtechniques
formintheabsenceofanylivingsystem.33
ThisjournalisªThe RoyalSociety ofChemistry 2009 J.Environ. Monit., 2009,11,1782–1800 | 1787
In addition, Trypan blue, a diazo dye, is only permeable to appropriate.Conductingmultipletestsisadvantageoustoensure
cells compromised membranes, therefore dead cells are stained that valid conclusions are drawn. It seems clear that in vitro
blue while live cells remain colorless. The amount of cell death cellularsystemswillneedtobefurtherdeveloped,standardized
can be determined via light microscopy. More extensive cyto- and validated (relative to in vivo effects) in order to provide
toxity studies have provided supportive information for nano- usefulscreeningdataabouttherelativetoxicityofnanoparticles.
particles studies such as cobalt, magnetic, TiO , Al O , Silica, In that respect, standard reference materials (SRMs) of engi-
2 2 3
silver, carbon nanotubes12,34–41,11,42 by examining the extent of neerednanomaterialsareneededforvalidation,justasthereare
DNA damage using several methods. This includes flow SRMsforurbanairanddieselexhaustparticles.Currently,only
cytometry,micronucleiassay,cometassay,mutationfrequency, one type of SRM exists: the ‘‘gold nanoparticle standard’’
and 8-ooxo-dG assays as well as DNA microarray studies. developedattheNationalInstituteofStandardsandTechnology
Wholegenomemicroarrayanalysisoftheearlygeneexpression (NIST).
changes induced by 10- and 500-nm particles showed that the
magnitudeofchangeforthemajorityofgenesaffectedcorrelated
4.3 Methodologiesdevelopedforthequalitativeanalysisof
moretightlywithparticlesurfaceareathaneitherparticlemass
nanomaterials
ornumber.17,43
As described in the above section, microscopy techniques and
4.2.2 Fluorescence assays. Optical imaging techniques have traditionalcellculturetechniquescouldbeusedforthecharac-
recentlyattractedalotofinterestformedicalapplicationsdueto terizationofnanoparticleuptakeandlocalization,bio-distribu-
itsnon-invasiveprocedure,hightemporalresolutionandrelative tionandqualitativeanalysisofnanotoxicitywhereinhumanand
lowcost.Fluorescenceimaginginthevisible-wavelengthrangeis environmental samples are exposed to nanoparticles. Quanti-
routinely used for conventional and intravital microscopy.44 fyingtheamountofnanomaterialishoweveranimportantissue
Becausecellularuptakeisanecessaryprerequisite,fluorescence in view of potential toxicity of nanomaterials. Several methods
microscopyhasbeenusedtoqualitativelydeterminethebinding wereaddressedhereincludingelectron-dispersiveX-rayanalysis
and uptake of nanomaterials. One simple cytotoxicity test (EDS),ICP-AES/OES/MS,isotopelabeling,synchrotronradia-
involvesvisualinspectionofthecellswithbrightfieldmicroscopy tiontechniques.Table2providesalistofrelevantinstrumental
forchangesincellularornuclearmorphology.Fioritoetal.first techniquesforquantifyingengineerednanomaterials.
used this technique when evaluating the cytotoxicity of single- First, qualitative elemental analysis techniques can be per-
walled carbon nanotubes (SWNTs).45 Quantitative assessment formedonbiologicalsamplesexposedtonanoparticlesusingan
canbeachievedinamannersimilartoICP-AESthroughuseof electron microscope coupled with a microanalysis system to
bulk fluorescence or on a cell-to-cell basis using confocal fluo- identifythechemicalcompositionofthenanoparticlespresentin
rescence.Totalinternalreflectancefluorescence(TIRF)hasgreat thesample.50Forexample,EDSwasusedtoconfirmthepresence
potentialforreal-timeimagingoffluorescentnanoparticlesasin of silver nanoparticles within cells,27 and electron energy loss
the work by Lee et al. examining uptake and localization of spectroscopy (EELS) was used in conjunction with TEM for
dendrimer nanoparticles for gene delivery.46 Real-time NIR elementalconfirmationofcarbonnanotubeuptake.51Inductively
fluorescence imaging has been developed for the quantitation coupledplasmaatomic/opticalemissionspectroscopy(ICP-AES/
and biodistribution of semiconductor quantum dots.47 Two OES) is widely regarded as a powerful technique for the quan-
importantfluorescencedyes(calceinacetoxymethyl(calceinAM) tification of elemental nanoparticle (NP).52 The first complete
and ethidium homodimer) have been used to test the live/dead quantitative in vivo pharmacokinetics study on QDs was ach-
viabilitytestwhenexposedtothenanomaterials,suchasfuller- ieved by using ICP-AES to quantify quantum dot distribution
enesandgoldnanoshells.Whenexcitedat495nm,calceinAM showingthemononuclearphagocytesystem(MPS)uptakewith
andethidiumhomodimeremitdistinctfluorescencesignaturesat noexcretion,ausefuloutlineoftechniquesapplicabletotracking
515nmand635nm,respectively. QDsinvivo.53Subsequently,theiruseinthequantitativeanalysis
Alamar Blue has been recently applied to nanotoxicological of nanomaterials was reviewed by Marquis et al.3 Recently, it
studies by assaying cellular redox potential.48 Alamar Blue is was reported that the toxicity of nanoparticles increases with
reducedtoasolublefluorescentproduct,resorufin(lem590nm), decreasing particle size on a mass basis or production of ionic
providing simpler sample preparation compared to the MTT metals.54–56,153 This observation may be true for certain type of
assay.However,interpretationofAlamarBlueresultsisdifficult nanoparticles,buttheredoesnotappeartobeevidencethatthis
becausethebiochemical mechanismsofAlamarBluereduction is true for all types of nanoparticles. To understand this
havenotyetbeenexplored.Additionally,nanoporoussiliconhas phenomenon, inductively coupled plasma mass spectrometry
beenfoundtoreactwithAlamarBlueintheabsenceofcells.49In (ICP-MS) techniques were carried out to explore how they
the case of cytotoxicity, it is important to recognize that cell producetoxicityinvivo.Theresultssuggestthatwhenthesizesof
cultures are sensitive to changes in their environment such as particlessuchascopperdecreasesdowntoananoscale,copper
fluctuationsintemperature,pH,andnutrient,inadditiontothe becomesextremelyreactiveinasimulativeextracorporealenvi-
concentration of the potentially toxic agent being tested. ronment. The underlying chemistry relies on the enhanced
Therefore, controlling the experimental conditions is crucial so chemical reactivity at the nanoscale. Chemical reactions occur-
as to ensure that the measured cell death corresponds to the ring between a solid and liquid phase always initiate at the
toxicityoftheaddednanomaterialsversustheunstableculturing interface of the two phases. Hence, the surface molecules can
conditions.Inaddition,asnanoparticlescanabsorbdyesandbe directly influence chemical reactivity. In accordance with
redox active, it is important that the cytotoxicity assay is the collision theory, large surface area must lead to a high
1788 | J.Environ. Monit.,2009, 11, 1782–1800 Thisjournal isªTheRoyalSociety ofChemistry2009
probabilityofeffectivecollision,whichdeterminestheultrahigh their predicted environmental concentrations (PEC). Mueller
reactivity during molecular interaction. Certain chemical reac- etal.describedastudyutilizingalife-cycleperspectivetomodel
tions are thermodynamically possible but are too slow kineti- thequantitiesofengineerednanoparticles(theflowsofTiO ,Ag
2
cally.However,whentheparticlesizereducestothenano-scale, and CNT) released into the environment.63 The method was
thelargespecificsurfaceareawillsharplyspeedupthechemical applied to the engineered titanium dioxide nanoparticles,
reactionandmayeventuallycausenanotoxicitythatmicro-scale including the nanoparticles of silver, carbon nanotubes, fuller-
substance does not allow. Hence in this case, the nanosized enes, ZnO and carbon black. The quantification was based on
copperparticlesconsumethehydrogenionsinthestomachmuch a substance flow analysis from products to air, soil and water.
more rapidly than the micron-sized coppers leading to massive The PEC-values were then comparedto the predicted no effect
generationofcupricionswhicharehighlytoxicinvivo.54–56 concentrations (PNEC) derived from the literature to estimate
However, there are a few recent examples of nanoparticle- apossiblerisk.Theresultsofthisstudymakeitpossibleforthe
enabled MS that, though not explicitly linked to nanoparticle first time to carry out a quantitative risk assessment of nano-
toxicity studies, demonstrate the potential of this approach. particles in the environment and to suggest further detailed
Kong et al. used carboxylated/oxidized diamond nanoparticles studiesofnano-titaniumdioxide.Thefollowingparameterswere
to extract proteins from blood, centrifuged the nanoparticles, usedasmodelinputs:estimatedworldwideproductionvolume,
mixedthemwithaMALDImatrix,andperformedMSanalysis allocation of the production volume to product categories,
with 2 orders of magnitude improvement in sensitivity over particle release from products and flow coefficients within the
analysisdonewithoutnanoparticles.57 environmental compartments. To cope with uncertainties con-
In addition, isotopic labeling is a technique for tracking the cerningtheestimationofthemodelparameters(e.g.transferand
passage of a sample of substance through a system. Wang partitioningcoefficients,emissionfactors)aswellasuncertainties
et al.labeled the water-soluble hydroxylated carbon single-wall about the exposure causal mechanisms (e.g. level of compound
nanotubes with radioactive 125I atoms, and then the tracer was production and application), probabilistic methods, sensitivity
usedtostudythedistributionofhydroxylatedcarbonsingle-wall anduncertaintyanalysiswasapplied.
nanotubes in mice. This study, for the first time, affords InthetoxicitystudyofnanoscaleTitania,whatdidcorrelate
a quantitative analysis of carbon nanotubes accumulated in strongly to cytotoxicity was the phase composition of the
animaltissues.58Twootherdifferentnuclearimagingmodalities nanoscaleTitaniainsteadofsamplesurfacearea.AnataseTiO ,
2
have been used for the quantitative analysis of quantum dot forexample,was100timesmoretoxicthananequivalentsample
including the single photon emission computed tomography of Rutile TiO . The most cytotoxic nanoparticle samples were
2
nuclear imaging (SPECT) and positron emission tomography alsothemosteffectiveatgeneratingreactiveoxygenspecies;ex
(PET). For SPECT, the most commonly used gamma-emitting vivo Reactive Species (RS) generation under UV illumination
radionuclidetracerincludes99-metastable-technetium(99mTc), correlatedwellwiththeobservedbiologicalresponse.Thesedata
iodine-125 (125I) or indium-111 (111In).47 Recently, Colvin et al. suggest that nano-TiO samples optimized for RS.15 Also, in
2
revealed an important fact that the bioactivities of fullerene a study entitled ‘‘Nano–C cytotoxicity is due to lipid perox-
60
derivativeswerealteredlargelywiththechangeofoutermodified idation’’, by changing the number of hydroxyl groups on the
hydroxylgroups.59Sincetraditionalmeasurementmethodssuch fullerene surface resulted in a reduction of toxicity by several
asXPSandNMRwerenotpreciseenoughtodeterminetheexact orders of magnitude.59 Also as discussed earlier, functionaliza-
number of hydroxyl groups, a further measurement of the tion, which often change part or the entire structure of nano-
hydroxyl number was performed using synchrotron radiation materialscangreatlyinfluenttheirtoxicity.Thesestudiesshowed
X-rayphotoemissionspectroscopy.Throughthebindingenergy thatthetoxicityofnanomaterialscouldbehighlycorrelatedwith
spectraofC1selectronsforC]CandC–OHinGd@C82(OH)x, their structures. In addition, potential toxicity of the fullerene
intensitiesforthenon-functionalizedandhydroxylatedcarbons nanoparticles was lowered significantly by using these in vitro
wereobtained.60 assays to target chemical aspects of the nanomaterials that
contribute to toxicity, indicating that in vitro testing provides
a cost-effective means for such studies, and high-throughput in
4.4. Molecularmodelingtechniques
vitro assays59,64,65 are well suited for developing mechanistic
Acomputerassistedpredictionapproachcouldhelptoidentify modelstoinformmaterialdevelopment.
the effects of physicochemical factors of nanoparticle and how
thesefactorsplaytheirroleontoxicology,andcorrespondingly 5. Sensors for quantitation and nanotoxicity of
inthesystematicanalysisandpredictionofbiologicaleffectsin
engineered nanomaterials
various media. Physiologically based pharmacokinetic (PBPK)
modelinghasplayedasignificantroleinguidingandvalidating Nanomaterials offer new possibilities for the development of
invivostudiesformolecularchemicalexposureandcanserveas novelsensingandmonitoringtechnologies.Nanosensorscanbe
asignificanttoolinguidingsimilarnanotoxicitystudies.Shelley classified under two main categories: (I) sensors that are them-
etal.hasdirectedthefirstattempttomodeltheinvitroeffectsof selvesnanoscaleorhavenanoscalematerialsorcomponents,and
ananoparticleexposure,inthiscase,aluminium(80nm)andits (II) sensors that are used to measure nanoscale properties. The
impact on a population of rat alveolar macrophages61,62 was first category can eventually result in lower material cost,
evaluated. reduced weight and power consumption. The second category
Anotherelementarysteptowardsaquantitativeassessmentof can enhance our understanding of the potential toxic effects of
the risks of new compounds to the environment is to calculate engineered nanomaterials. This is an area of critical interest to
ThisjournalisªThe RoyalSociety ofChemistry 2009 J.Environ. Monit., 2009,11,1782–1800 | 1789
detection and risk assessment, as well as for monitoring of expression of cell determinant markers on the cell surface, and
environmental exposure.66,67 This section provides an overview other studies illustrating the utility in understanding the bio-
of the use of engineered nanomaterials for sensing (Category I logical response to exposures, potentially in whole organism
sensors)andquantitation,nanotoxicityassessmentofengineered studies,96,97 significantly reduces the application of toxic indi-
nanomaterialsusingcategoryIIsensors. cator and reference dyes to the cells.98 Nanomaterials offer
unique properties that can be exploited in environmental
sensors99,100todetectenvironmentaltoxinsincludingpesticides,
5.1 Category1-Nanotechnology-enabledsensorsorsensors
hazardousindustrialchemicals,toxicmetals,pathogenicbacteria
thatarethemselvesnanoscaleorhavenanoscalematerialsor
and undesirable vapors and liquids at the particle’s surface.
components
Recently, application of advanced nanomaterials for environ-
5.1.1 Nanomaterials and nanotechnology for sensing. As mental monitoring have been reviewed systematically by
nanotechnology has come to prominence for a wide range of Andreescu.101 Nanobiosensors based on individual olfactory
devices, it also brings new possibilities for chemical and receptors are developed,102 which are used to mimic the way
biosensor construction. The use of nanoscale materials such as human and animal noses respond to different odors and allow
nanoparticles, nanowire, nanoneedle, nanosheet, nanotube, for rapid and noninvasive assessment of VOCs, that can
nanorod, nanobelt and nanocomposites for sensing have seen constituteasignatureofmetabolicstatesordiseases,participate
explosive growth in the past 5 years, since the report for low- inaromasinfood,andbeassociatedwithdrugsandexplosivesor
potential detection of NADH using carbon nanotube-modified todomesticandenvironmentalpollutants.
electrodes by Wang et al.68 and the first use of gold nano-
particles as labels for electrochemical immunosensors by 5.1.3 Detection of biotinylated fullerenes and carbon nano-
Limoges et al.69 Dozens of reviews are available which partly tubes.DetectionoffullereneandcarbonnanotubesusingSurface
dealwiththeuseofnanomaterialsfornanobiosensors,70–72more Plasmon Resonance (SPR) biosensor, was recently reported by
detailed reviews on carbon nanotube-based sensors73–83 and Kirschner et al.118 SPR exists when polarized light reaches the
metal oxide and quantum dots nanoparticles-based interfacebetweenathinmetalfilmandahighdensitymediumin
biosensing84–87 can be found. Nanowires as sensing materials Kretschmann geometry. At this point, the alternating electric
have also been reviewed.88 fieldwithinthelightcausesoscillationofthefirmlyheldelectrons
Due to the electrical, magnetic, optical properties of these inthedielectricsubstance.Thisoscillationproducesevanescent
materials, the nanomaterials-based biosensors has been catego- wavesthatarenon-propagatingspatiallydecayingfieldswhichin
rized into electrochemical,89 optical or photo-electrochemical, turncausesoscillationsinthefreedelocalizedelectrondensityof
magnetic and mechanistic and surface plasmon resonance the metal–the so called ‘‘surface plasmons’’.118,119 In aqueous
enhanced sensing types etc. Their dimensions are on the same solution, fullerene andcarbon nanotubesinteractwith proteins
scaleasbiomolecules,whichunveilsexcitingpossibilitiesfortheir insolutionandbindtothesurfacewhiletheirstructuresremain
interactionwithbiologicalspecies,suchasmicrobial,tissue,cells, unaltered. This is the basis of Kirschner et al.’s118 biosensor
antibodies,DNAandotherproteins.Extensiveresearchpapers protocolasshowninFig.3.Firstthegoldsurfacewascleanedby
and reviews using nanomaterials for electrochemical bioassays removal of macroscopic dirt particles. Next streptavidin was
have since been published and this continues to show an adsorbed to the gold sensor and a biotinylated C -fullerene
60
increasingtendency.Theyhavebeenusedfortheconstructionof derivative was attached. Phosphate buffer solution was flowed
enzyme sensors, immunosensors and genosensors to achieve beforethesensorwasprimedtodetectbindingproteinstoC .
60
direct wiring of enzymes and relative components to electrode Proteins with fullerene affinity, monoclonal anti–C antibody,
60
surface, to promote spectroelectrochemical reaction, to impose wasflowedacrossthesurfacecausingchangeinbindingindexof
barcodeforbiomaterialsandtoamplifysignalofbiorecognition refraction118 which was then correlated to the quantity of
event. fullerenes.Itisimportanttonotethatforthesensorsdescribedin
this section, the antibodies directly bind to the biotinylated,
5.1.2 Application of nanomaterial-based chemical and watersolublefullerenes.Itappearsthatthissensorcanonlysense
biosensors. Thedevelopmentofportablenanotechnology-based
sensors has the potential to meet the needs for low cost, rapid,
high-throughput,andultrasensitivebioassaysforbiomonitoring
an array of chemical markers.90 The resulting nanobiosensors
havebeenappliedinthedetectionofglucose,91foodpathogens,92
biomedicine, antioxidant capacity assessment93 etc. Nano-
structuredmembranehasbeendevelopedforthefabricationof
implantablenano-biosensorandappliedinclinicaldiagnostics94
etc. Recent advances in chemical analyte detection and optical
imagingapplicationsusinggoldandsilvernanoparticleshasbeen
reviewed by Murphy et al.95 The successful coupling of the
functionalgrouponthequantumdotshavemadeitpossibleto
increase the power of a number of biochemical, molecular bio-
logical and physiological experiments, including tracking the Fig. 3 Schematic of biosensor developed for fullerene from reference
movements of proteins within the cell, characterizing the 118.
1790 | J.Environ. Monit.,2009, 11, 1782–1800 Thisjournal isªTheRoyalSociety ofChemistry2009
biotinylatedfullerenes.ThusisitbettercategorizedasCategoryI
nanosensor.
5.2. Category2-Nanoproperty-quantifiablesensorsorsensors
thatcouldbeusedfordetectingnanoscaleproperties
As described in the previous section, category II sensors repre-
sent a developing area of critical interest to detection and risk
assessment, as well as for monitoring of environmental expo-
sure66,67duetotheexplosiveapplicationofengineeredmaterials.
However,comparedtothelargenumberofpublicationsonthe
sensors in the first category, sensors in the second category are
very few and/or some of them are not currently available. This
section provides a quick overview of on-going work in this
category.
5.2.1 Detectionofmetalandmetaloxidenanoparticles
5.2.1.1 Silvernanoparticles(AgNPs).Arecentreportinthis
category focuses on the detection of metal nanoparticles (NPs)
Fig. 4 (A) Ag+ Promoted Spirolactum Ring Opening of Probe
suchassilvernanoparticles(AgNPs).AgNpsareknownfortheir
(1).107Fig. 4(B): Schematic illustration of the sensing mechanism
antimicrobialactivityandforthatreasontheyhavebeenusedin
promotedbyAg+-coordinationtotheiodideoftheprobe.107
watertreatment103andotherapplicationssuchasbabypacifiers
and food storage containers.101 Their bactericidal activity is
shapeandsizedependent,withparticlesofsizeslessthan100nm
showing optimal antibacterial activity.101 Knowledge about sil- workdiscussedhereisthatthesilverionsensingwasappliedto
ver’s ability to kill harmful bacteria104 has made it popular in silver particles upon silver oxidation with hydrogen peroxide.
creatingvariousconsumerproducts.Despitetheseusefulappli- Themostseriousproblemlimitinguseofion-selectiveelectrodes
cations,AgNPshavebeenreportedtobetoxic.105Forexample, andotherexistingsensorsisinterferencefromotherundesirable
Asharani et al. synthesized AgNPs using starch and bovine ions.Someofthesesensorsarenotcompletelyion-specific;allare
serumalbumin(BSA)ascappingagentstostudytheirdeleterious sensitivetootherionshavingsimilarphysicalproperties.
effects and distribution pattern in zebra fish embryos (Danio
rerio).105 These authors observed concentration-dependent 5.2.1.2 Gold nanoparticles (AuNPs). Recently, detection of
increase in mortality and hatching delay for the Zebra fish AuNPs was reported using Surface Plasmon Resonance (SPR)
embryostreatedwithAgNPs.105 technique.Plasmonresonancesinmetallicnanoparticlesaredue
There are many excellent chemical sensors for silver ions tothecollectiveoscillationofconductionelectronsagainsttheir
including ion-selective electrodes, optodes, and fluorescent matrix.108 Such resonances play a central role in the optical
sensors. Plasma emission spectroscopy, Atomic absorption propertiesofmetallicnanoparticles109andthereforeareusefulin
(AAS), and anodic stripping voltammetric methods have been detection metal nanoparticles such as AuNPs. Lindfors et al.
usedtomeasuretracelevelsofsilver.106However,noneofthese reporteddetectionandspectroscopyofgoldnanoparticlesusing
have been applied for AgNPs detection. As of now, only one super continuum white light confocal microscopy.109 They illu-
articlehasappearedreportingtheirapplicationforAgNPs.The minatedthesamplewithsupercontinuumlaserlightgeneratedin
article published by Chatterjee et al. on ‘‘selective fluorogenic a photonic crystal fiber (PCF) through a cascade of nonlinear
and chromogenic probe for detection of silver ions and silver effectsthatgaverisetoaspectrumextendingfromthevisibleto
nanoparticles in the aqueous media’’.107 The chemistry of their thenearinfrared.Usingthistechnique,thesescientistswereable
sensor was based on Rhodamine B derivative 1 as the fluoro- to detect a single gold particle down to a nominal diameter of
genicandchromogenicprobeforAg+/AgNPsinaqueousmedia D ¼ 5 nm. The authors pointed out that this was the first
(Fig. 4). detection of individual gold nanoparticles below 10 nm using
Probe 1 forms colorless and non-fluorescent solution in 20% afullyopticaltechnique.
ethanolic water. With the oxidation of AgNPs by hydrogen AnotheropticaltechniquefordetectingAuNPsisthephoto-
peroxide, silver ions were generated. The presence of Ag+ ion thermaldetectionofgoldnanoshellsusingphase-sensitiveoptical
leads to the development of a pink color (lmax: 558 nm) and coherencetomography(OCT)asreportedbyAdleretal.110OCT
astrongorangefluorescence(lmax:584nm)ofProbeI(Fig.4B), isahigh-resolutionbiomedicalimagingmodalitythatproduces
indicating that the Ag+-promoted ring opening takes place cross-sectional and three-dimensional images of tissue micro-
readily107(Fig.4B).Thefluorescenceincreasedlinearlydepending structure by interferometrically measuring the amplitude and
ontheconcentrationofAgNPsdemonstratingtheusefulnessof echo time delay of backscattered light.111 Typically, at low
probe1fortheindirectquantificationofAgNPs.Thedetection temperaturegradientsthetechniqueissuitableforinvivouse110
limit(LOD)wasreportedas14ppb.Thisprotocolwasappliedto andrepresentsanewmethodfordetectingAuNPcontrastagents
quantifyAgNPsin sanitizergel andfabricsofteners containing with excellent signal-to-noise performance at high speeds using
unspecifiedamountofAgNPs.107Whatismostuniqueaboutthe OCT.
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