Consumer-friendly food allergen detection: moving towards smartphone-based immunoassays

19 Pages·2017·1.52 MB·English

AnalyticalandBioanalyticalChemistry(2018)410:5353–5371
https://doi.org/10.1007/s00216-018-0989-7
REVIEW
Consumer-friendly food allergen detection: moving towards
smartphone-based immunoassays
GeorginaM.S.Ross1&MoniqueG.E.G.Bremer1&MichelW.F.Nielen1,2
Received:29December2017/Revised:14February2018/Accepted:26February2018/Publishedonline:26March2018
#TheAuthor(s)2018
Abstract
Inthiscriticalreview,weprovideacomprehensiveoverviewofimmunochemicalfoodallergenassaysanddetectorsinthecontextof
theiruser-friendliness,throughtheirconnectiontosmartphones.Smartphone-basedanalysisiscenteredaroundcitizenscience,putting
analysisintothehandsoftheconsumer.Foodallergiesrepresentasignificantworldwidehealthconcernandconsumersshouldbeable
toanalyzetheirfoods,wheneverandwherevertheyare,forallergenpresence.Owingtotheneedforascientificbackground,traditional
laboratory-baseddetectionmethodsaregenerallyunsuitablefortheconsumer.Therefore,itisimportanttodevelopsimple,safe,and
rapidassaysthatcanbelinkedwithsmartphonesasdetectorstoimproveuseraccessibility.Smartphonesmakeexcellentdetection
systems because of their cameras, embedded flash functions, portability, connectivity, and affordability. Therefore, this review has
summarized traditional laboratory-based methods for food allergen detection such as enzyme-linked-immunosorbent assay, flow
cytometry, and surface plasmon resonance, and the potential to modernize these methods by interfacing them with a smartphone
readoutsystem,basedontheaforementionedsmartphonecharacteristics.Thisisthefirstreviewfocusingonsmartphone-basedfood-
allergendetectionmethodsdesignedwiththeintentionofbeingconsumer-friendly.
Keywords Foodallergen .Immunoassay.Smartphone .Consumer.Multiplex .Citizenscience
Abbreviations FC Flowcytometry
AA AllergyAmulet FDA FoodandDrugAdministration
ABA AllergenBureauofAustraliaandNewZealand FO-SPR Fibreopticsurfaceplasmonresonance
ALARA Aslowasreasonablyachievable GPS Globalpositioningsystem
CCD Chargecoupleddevice HRP Horseradishperoxidase
CIP Cleaninplace Ig Immunoglobulin
EC EuropeanCommission LED Lightemittingdiode
ELISA Enzymelinkedimmunosorbentassay LFIA Lateralflowimmunoassay
FARRP FoodAllergyResearchandResourceProgram LOAEL Lowestobservableadverseeffectlimit
MFC Miniaturizedflowcytometry
MIP Molecularlyimprintedpolymers
PublishedinthetopicalcollectionFoodSafetyAnalysiswithguesteditor
NOAEL Noobservableeffectlimit
StevenJ.Lehotay.
PAL Precautionaryallergenlabelling
Electronicsupplementarymaterial Theonlineversionofthisarticle
PDMS Polydimethylsiloxane
(https://doi.org/10.1007/s00216-018-0989-7)containssupplementary
PoC Point-of-care
material,whichisavailabletoauthorizedusers.
PPE Personalprotectiveequipment
* GeorginaM.S.Ross ppm Partspermillion
[email protected] QD Quantumdots
ROI Regionofinterest
1 RIKILT,WageningenUniversityandResearch,P.OBox230,6700 (i)SPR (imaging)Surfaceplasmonresonance
AEWageningen,TheNetherlands USB Universalserialbus
2 LaboratoryofOrganicChemistry,WageningenUniversity,Helix UV-VIS Ultravioletvisiblespectrophotometry
Building124,Stippeng4,6708WEWageningen,TheNetherlands VITAL Voluntaryincidentaltraceallergenlabelling
5354 RossG.M.S.etal.
Introduction simpletoperform,linkingtheseteststoasmartdetectorwill
make them more accessible for the general public. As the
An allergen is a protein capable of eliciting an immune majority of the population already owns a smartphone, with
response in sensitized individuals. Food allergies repre- thenumberrising,smartphonesrepresentasourceofanalyti-
sent a significant international health problem. calequipmentthatcanreacheventhemostdesolateareasof
Worldwide, allergies toward foods affect 2% of the adult theglobe,makingthemidealforsensors[12].
population and 5%–8% of the children population [1, 2]. Smartphonesareidealtouseasdetectorsystemsbecauseof
There are many existing methods for food allergen de- theirpowerfulinternalcomputers,opticalsensors,globalpo-
tection, which can be split into two general categories: sitioningsystems(GPS),andmostimportantly,theirabilityto
protein-based and DNA-based detection. For a general connecttotheinternet,throughBluetoothandWiFi[13–15].
and in-depth explanation on all in-vivo and in-vitro al- Connectivity is a key benefit of smartphones as results can
lergen assays, the review by Poms et al. can be referred instantaneously be uploaded to Cloud databases and results
to [3]. General and quantitative methods for allergen de- canbedisseminatedasspatio-temporalmapsacrosstheglobe
tection have been reviewed by Kirsch et al. and Walker [16].Sincetheirdevelopmentin1992andfirstuseasanalyt-
et al. [2, 4]. Additionally, an overview on commercially icaldevicesin2008,smartphoneshavealreadybeenusedas
available rapid immuno-analytical allergen detectors has sensors, for light microscopy, single-molecule microscopy,
been presented by Schubert-Ulrich et al. [5]. All immu- cell imaging, bacteria detection, colorimetric detection, en-
nochemical and DNA-based methods were reviewed by zymelinkedimmunosorbentassay(ELISA),andlateralflow
Monaci and Visconti and by Slowianek [6, 7]. Further immunoassays(LFIA),whichexemplifiestheircapabilitiesas
discussion into allergen detection methods with a partic- detectors in rapid diagnostics [12, 17–26] . For an in-depth
ular focus on proteomic mass-spectrometry has been giv- reviewintoallexistingsmartphone-baseddiagnosticdevices,
en by Prado et al. [8]. The most recently published food Quesada-GonzalezandMerkoçicanbereferredto[27].Fora
allergen review [9] focused on the use of biosensors for morefocusedreviewconcerningbiosensorsandbioelectron-
detection, so only limited attention will be paid to them ics on a smartphone see Zhang and Liu [28]. General ap-
in this review. proachestosmartphone-basedfooddiagnosticshavebeenre-
Although analytical methods such as mass spectrometry cently reviewed by Rateni et al. [29] and Choi [12], which
canprovideawealthofinformationwhenusedcomplemen- addressedthenecessityandmarket-gapforuser-friendlyfood
tarilywithimmuno-methods;currentallergenanalysistrends detection. This is particularly important in the field of food
aremovingawayfromlabmethodsandtowardpoint-of-care allergenanalysiswheredetectionmethodsmustbeconsumer-
diagnostics(PoC)andacitizenscienceapproach[10].Point- friendly so that the allergic individuals can apply analysis
of-carediagnosticsallowinstanton-sitetestingforfoodaller- themselvesinthecomfortoftheirhomeand/oratarestaurant.
gens by individuals, whilst citizen science centers around The present review specifically focuses on how successful
consumer-friendly devices thatallow users tocarry out their lab-based methods can be based on smartphones to enable
ownPoCallergenanalysis.Itisofparticularimportancethat consumer-friendlyallergendetection.
foodallergendetectiondevicesareconsumer-friendlyasaller- Up until now, the literature has lacked specific focus on
gic individuals will need to carry out testing at home or in consumer-friendly food allergen detection devices. To that
restaurants prior to eating. Many allergic individuals suffer end,literaturehasbeenreviewedfromtheperiodof2002to
from more than one food allergy, due to cross-reactivity, the end of 2017 using the SciFinder, Scholar, Scopus, and
whereantibodiesagainstoneallergenrecognizeastructurally WebofSciencedatabasesandkeywordssuchas:foodaller-
relatedepitopeofanothersimilarallergen[11].Owingtoal- gen, detection, smartphone or cell phone, multiplex, lateral
lergensbeingcross-reactive,itisnecessarytodevelopmulti- flow, immunoassay, cross-flow, microfluidics, strip reader,
plexdevicesthatcandetectarangeofallergenswithinasingle and ELISA. Section 1 of the review will provide a general
sample, saving time and money and making sure that the backgroundoffoodallergensandthelegislationsthatcontrol
consumersareconfidentthattheirfooddoesnotcontainany thelabellingprocedures.Thestudywillthendiscussthecon-
undesiredallergens.Forthepurposeofthisreview,aconsum- ceptofconsumerfriendlinessinSection2.InSection3there
ercanbeconsideredastheend-useroftheassay/detector,and willbefocusontraditionallaboratory-basedmethodsforfood
thusthetermsconsumeranduserareusedsynonymously.The allergenanalysisandhowthesemethodscouldimprovetheir
authors define consumer friendly to mean that any adult of consumerfriendlinessthroughcouplingtoasmartphoneasa
averageintelligencewouldbeabletoperformtheassaysafely detector.Section4willdiscussassays/devicesthathavebeen
andeffectivelywithminimalinstruction.Onewayofmaking designed with the intention of being consumer-friendly, in-
allergentestingmoreuser-friendlyistolinktheassayswitha cluding commercial consumer-friendly allergen detectors.
smart-detectorsuchas:asmartphone,tablet,orwearablede- Finally, the conclusion will summarize the findings of the
vice.Althoughsomeoftheexistingallergenassayformatsare review.
Consumer-friendlyfoodallergendetection:movingtowardssmartphone-basedimmunoassays 5355
Background on food allergens of. Many traditional allergen analysis methods use environ-
mentally harmful reagents, which contain additives that im-
Typesoffoodallergens
Table1 Themainallergenicproteinsinfoodswithinthe‘Big8’plus
‘gluten’
Food allergies can be debilitating, and food requires proper
monitoringtoensuresensitizedindividualsarenotexposedto Food Majorallergenicprotein Reference
allergens.Symptomsoffoodallergycaninclude:itching,di-
Cow’sMilk B-lactoglobulin(Bosd5) [34]
arrhoea,stomachpains,eczema,shortnessofbreathaswellas
Casein(Bosd8) [35]
more significant effects such as loss of consciousness and
α-lactalbumin(Bosd4) [36]
anaphylactic shock, which can be fatal [30]. The prevalence
Egg Ovomucoid(Gald1) [37]
of food allergies is increasing, but awareness of allergies is
Ovalbumin(Gald2) [37]
growing even faster with dedicated events such as ‘Food
Ovotransferrin(Gald3) [38]
Allergy Awareness Week’ in the USA [31]. The Codex
Lysozyme(Gald4) [39]
AlimentariusStandardlistedeightallergenswithinternational
α-livetin(Gald5) [40]
variants, which require mandatory labelling [32]. These are
Crustacean Tropomyosin(Pena1) [41]
referredtoastheBig8andconsistof:peanuts,tree-nuts,milk,
Fish Β-parvalbumin(Lepw1;Pon [42]
eggs,fish,crustacean,soya,andwheat[33].Wheatcontainsa
14;Pon17;Sebm1;Xipg1)
varietyofproteinsthathavebeenimplicatedasallergens(see
Peanut Arah1 [43]
Table 1). In addition to wheat allergy, other wheat-related
Arah2 [44]
disorders include the autoimmune disorder, celiac disease.
Arah3 [45]
Celiac disease is triggered by gluten, a protein mixture of
Arahh4-9 [46]
prolamins and glutelins, which can be found in wheat, rye,
Treenuts
barley,andtheircross-breeds[58].Allergicreactionsarepro-
Hazelnut Cora1;Cora2;Cora8;Cora9; [47]
vokedbymanydifferentproteinswithintheallergenicfoods. Cora11;Cora12;Cora13;Cora14
Those allergenic proteins which have been repeatedly refer- Brazilnut Bere1;Bere2 [48]
enced in the literature and databases (e.g., allergen.org) as Cashew Anao1;Anao2;Anao3 [49]
causing an allergic reaction in the majority of sensitised Almond Prudu3;Prudu4;Prudu5;Prudu6 [50]
individualsaredescribedinTable1below. Walnut(Black) Jugn1;Jugn2;Jugn4 [42]
Allergenic proteins can result in hypersensitivity of the Walnut Jugr1-6 [42]
immunesystem,arbitratedbyallergen-specificimmunoglob- (English)
Pecan Cari1;Cari2;Cari4 [42]
ulin E (IgE) (type I allergies); but allergies can also be cell-
Pistachio Pisv1;Pisv2;Pisv3;Pisv4;Pisv5 [42]
mediated (non-IgE) (type II allergies) [9, 59]. Disruption of
Soybean GlymBd30K [51]
the structure of allergens by food processing can lead to an
GlymBd60K [52]
increaseordecreaseintheirimmunogenicity,alteringhowan
GlymBd28K [52]
allergicindividualmightreacttotheprotein[60].Themodi-
Wheat Tria12 [53,54]
ficationofallergenicproteinsisdependentontheprocessing
Tria14 [53,55]
procedureapplied.Forexample,byhydrolyzingorthermally
treatinganallergen,thestructureisaltered,whichcanresultin Tria18 [53,56]
either a reduction in immunogenicity of the allergen, or the Tria25 [53,56,
57]
formationofaneo-allergen.Themethodusedforprocessinga
Gluten* Gluten(Tria26&Tria36) [42,53]
food will affect the extractability of the allergens from their
Gliadin(Tria19&Tria20) [42,53]
matrix[61].Whenextractinggluten,forexample,itiscrucial
to have a homogenized sample so that particulates can be *Althoughnotanallergen,glutenhasbeenincludedinthistabletoshow
extracted. As ethanol-based extractions result in the incom- the toxic portion of the protein responsible for gluten’s autoimmune
pleteextractionofgluten,itisdesirabletouseacocktailex- effects.
traction solution that contains a reducing agent and alcohol,
whichiscapableofextractingmonomericandpolymericpro- proveallergensolubility/extractabilityandreducebackground
teinsfromgluten[62–64].Extractionprocedureshavebeena interference from the food matrix [66]. It is desirable to use
detriment in the past, where hazardous and environmentally eco-friendly extraction buffers, but these must first be com-
damagingextractionsolutionssuchas2-mercaptoethanol(2- pared and validated against traditional buffers to ensure that
ME) have been applied in food allergen extraction [65]. In theyareaseffectiveinallergenextraction.
order to step toward consumer-friendliness it is necessary to Allassaysanddetectorsneedtobeeffectivelyvalidatedby
haveextractionbuffersthataresafetouseandeasytodispose standardizedprocedures.Certifiedreferencematerialsinraw
5356 RossG.M.S.etal.
andprocessedfoodsneedtobedevelopedforfoodallergens contamination, that pose the biggest risk to the consumer
as well as reference methods for allergen analysis [67, 68]. [76].TheEUdoesnotcurrentlyprovideguidanceonlabelling
Currentlackofstandardizedreferencematerialsforallergens for allergens that may have unintentionally been introduced
in foods means that there is a lack of consistency between intotheproductviasharedfacilities[72].
differentallergendetectionmethodsaseachtestkitiscalibrat-
edinadifferentway.Referencematerialsarecriticalforqual- Precautionarylabellingandthresholds
ityassuranceofallergendetectionmethods,buttheirproduc-
tion is complicated in food allergen analysis owing to the TheEUhasazerotolerancepolicyforallergenlabelling,and
changes in allergen protein structure during food processing anyfoodslistedinthelegislation(seeESMTableS1)mustbe
procedures[6].Whenstandardizedreferencematerialsarede- statedonthe foodpackagingwhentheyareusedasingredi-
veloped, they should be based on a whole protein extract as entsorprocessingaidsinthefood.However,theEUhasno
allergens are a mixture of non-defined proteins in complex obligationtolabelanyallergensthatarenotpartoftherecipe
matrices [69]. Having a set of standards for allergen testing and may have accidentally been introduced by cross-
deviceswillensurethateffectiveandsmartdetectiondevices contamination[67].Somecountrieshavesetthresholdlevels,
canbecreated,validated,andbenchmarkedagainsteachoth- andanyfoodcontainingallergensabovethoselevelsrequire
er, allowing consumer science to be achieved by providing labelling.Forexample,inJapan,anyfoodscontaininganyof
individuals with personalized smart-detection platforms for the legislated allergens (see ESM Table S1) above 10 ppm
foodallergens. mustbedeclaredonthepackaging,meaningthatthemajority
of the allergic population are protected from exposure [70].
Worldwidelegislationandmandatorylabelling However,duetoindividualdifferencesinsensitivitiestoaller-
gens, having such a low labelling threshold may further re-
Worldwide,dietarydifferencesandtheBig8influencewhich strict the diet of individuals who are less sensitive to those
allergensrequiremandatorylabelling.Somecountriesinclude allergens.Switzerlandhastakenanalternative approach,not
additionalmandatoryandrecommendedallergensforlabelling mandating allergen labelling for any product containing less
depending on the staple diet of that particular country [70]. than1000ppm ofallergen [77].TheSwiss approachcan be
Despiteworldwidecommunication,significantvarianceexists detrimental to the allergic individual, with many people
indifferentcountries’regulatorylabellingframework.Thiscan experiencingallergicreactionsatlevelsfarlowerthan1g/kg
beproblematicduetothehighpercentageofinternationalfood forparticularallergens[78].TheSwissallergenlabellingleg-
tradeandindividualpeople’stravellingpatterns[71]. islationilluminatestherequirementforconsumerstobeable
The European Commission (EC) produced legislation in totesttheirownfoodsforallergenpresencesothattheydonot
2003 (Directive 2003/89/EC) covering a list of 14 allergens havetosolelyrelyonlabellinglegislations.
thatrequiremandatorylabelling;thelegislationiscommonly In addition, it is also common practice for food manufac-
referredtoastheBallergen-labelling-directive^[72].Ifaman- turerstoincludeprecautionaryallergenlabelling(PAL)ontheir
ufacturer uses any of the allergens listed, it must be stated, foodsforprotectionagainstunintentionalpresenceofallergens.
with clear labelling, on the packaging [73]. This is a crucial ThereisalackofconsistencyinthewordingofPAL,whichcan
amendment,aslabellingofthepresenceofallergenicingredi- be confusing to the allergic consumer and reduces the con-
ents is currently the only way allergic individuals can effec- sumer’s ability to make informed food choices [68]. Labels
tivelymaintainstrictavoidancediets[74].Properlabellingof such as Bmay contain nuts^ are used if there is any risk the
allergensiscrucialasitinformsconsumerswhatproductsare product may have come into contact with an allergen [77].
safetoeat.In2014,theEURegulationamendment1169/2011 Food manufacturing companies have highlighted their desire
came officially into effect. This amendment stipulated that forstandardisedPALonfoodpackagingtoavoidmisinterpre-
evennon-prepackagedfoodsrequireallergenlabelling,mean- tation [79]. Although advisory labelling is well-intentioned,
ing in practice that all food retailers must provide allergen excessiveuseofwarningscanleadtoindividualstakingrisks
information [72, 75]. Food manufacturers and retailers are with what they eat by ignoring the labels [80–82]. Currently,
responsible for the proper labelling of their products; when most countries’ PAL is not on threshold-based criteria, and
anallergenhasbeenlabelled,itthenbecomestheconsumer’s manufacturersincludelabelsforanypotentialallergen.
responsibility to avoid this food [68]. As a large amount of Thereisanevidentrequirementforthreshold-basedaction
food allergic reactions happen to individuals when they are levels,toproperlyassesstheriskofanunintentionalallergen
abroad,itisvitalthatconsumersareawareofthedifferences beingintroducedtoafood,andtoestablishwhenandwhere
in which allergens require labelling in other countries (see advisory labelling is necessary and beneficial to the allergic
Electronic Supplementary Material (ESM) Table S1). consumer. These action levels should be science-based.
However,itisundeclaredfoodallergensthatareaccidentally Clinical information regarding minimum eliciting doses has
introduced into non-allergenic foods, through cross- been translated into lowest-observed adverse effect levels
Consumer-friendlyfoodallergendetection:movingtowardssmartphone-basedimmunoassays 5357
(LOAEL) and no observed adverse effect levels (NOAEL) Criteriaforconsumer-friendliness
[78,83,84].DevelopingeffectivethresholdsusingLOAELs
isasafetyassessment-basedapproachthatprotectsthemajor- Astheworldmovestowardspersonalizedtestinganddiagno-
ity of the allergic population. The Allergen Bureau of sis,theneedforuser-friendlydevicesbecomesmoreapparent.
AustraliaandNewZealand(ABA)isagloballeaderinregu- Whilstmanyproductsclaimtobe‘fortheconsumer’,inreality
lationoflabellingandhasalreadyestablishedvoluntarylabel- onlyalowpercentageofthesedevicesactuallyare.Itisuseful
ling thresholds for the major allergens, based on LOAELs, to consider the parameters that make an assay usable for the
which protect 95% of allergic population from severe reac- generalpopulation.Recently,stakeholderguidanceintothede-
tions[82,85].VoluntaryIncidentalTraceAllergenLabelling velopment of consumer-orientated allergen analytical devices
(VITAL)aimstolimittheuseofexcessive,unnecessaryPAL hashighlightedtheneedforstandardizationofinstructionsfor
infoods;andhasalsoincorporatedreferencedoseinformation assayuseandfortransparencyinvalidationproceduresincon-
intotheLOAELactionlevelsforallergenlabelling[82,86]. sumerassays[95].Foratrulyuser-friendlyassay,themajority
ThereferencedoseinVITALisdefinedasmilligramsoftotal oftheadultpopulationshouldbeabletoperformitsuccessful-
protein from an allergenic food from which only the most ly;usingthedeviceshouldbeself-explanatoryorrequiremin-
sensitiveindividualwouldbelikelytoexperienceanadverse imalinstruction.Whenlinkinganassaytoasmartphoneappit
reaction[87].Iftheindividualreferencedoseisexceededinan is possible to include safety information and instructions for
unlabelledfood,VITALrecommendsprecautionarylabelling applicationwithintheapp,limitingtheneedforaninstruction
[67].In2011,ascientificexpertcommitteeincludingthefood manual.Alongsidebeingeasy-to-use,theassayshouldbesafe
allergy research and resource program (FARRP), revised and not contain toxic chemicals; it should also not be able to
VITALtodevelopVITAL2.0,whichusesactionlevelsbased staintheuser/damageclothingandthereforeshouldnotrequire
on reference doses [88]. The action levels provide a clear theuseofpersonalprotectiveequipment(PPE).Thereshould
indication on when Bmay contain^ labelling should be ap- benotoxicwasteproduced,andpreferablytheassayshouldbe
plied. Despite Australia and New Zealand being at the fore- environmentally friendly and recyclable; there should be in-
front ofallergen labelling regulation, further implementation structions on how to dispose any waste that does come from
andstandardizationinPALisrequired[85]. theassay[95].
Regardless of dedicated labelling procedures, presence of The assay/detector should require minimal external equip-
undeclaredallergensstillremainsthegreatestcauseforfood- ment.Byhavingtousescientificequipmentsuchasprecision
basedrecallsglobally[31,89].Largescalerecallscanhavea pipettesandcentrifuges,themanufacturerintroducestheneed
significant socio-economic burden on a country [90]. The forfurthertraining/explanationtonegatehumanerror.Inaddi-
Rapid Alert System for Food and Feed (RASFF) is a tion, requiring basic laboratory skills (such as pipetting), pre-
Europeanfoodsafetyriskassessmentsystemthathasexperi- vents individuals with no scientific background from being
enced an increased volume of notifications regarding unde- abletousethedevice.Externalequipmentincreasestheoverall
clared allergens in recent years [91]. When an allergen has cost of the assay, and affordability is a prerequisite for user-
beenmislabelled,itmustbereportedtothecompetentauthor- friendlyassays.Pre-containingreagentswithintheassayelim-
ity as well as recalled in the notifying country and then inates pipetting steps and allows waste to be minimized and
RASFF issuesanalert informing thatthe product containsa costreduced.Astheconsumercannotrelysolelyonthevisual
mislabelled allergen [92]. It is an option to notify RASFF readoutofascreeningassay,anothermajorcostinmanyassays
aboutallergensthatmayhavebeenunintentionallyintroduced istherequirementforaspecializeddetector/reader[95].Next-
into a product by cross-contamination; however, this is not generation citizen science detectors such as smartphones re-
mandatoryasitisnotregulatedbytheEU.Riskcommunica- duce cost significantly, as most people already own at least
tionisexpectedwithinthefoodindustry,butitisnotmanda- onesmartphone.Oftentheassaycanbeperformedwithrelative
tory, so providing the industry with sensitive tests that can ease(e.g.,LFIA)butitistheresultinterpretation,suchasdif-
detectallergensatconcentrationsaslowasreasonablyachiev- ferentiatingbetweenlighteranddarkerlines,whichisdifficult
able (ALARA) is the best way to ensure that unintentional for the consumer and can be negatively affected by personal
allergenpresenceinfoodismonitored.Inorderforconsumers bias.Ingeneral,LFIAreadersareexpensiveandarenotsome-
tobeentirelyconfidentthattheirfoodisfreefromallergens,it thingthatconsumerswouldownandcarryaroundwiththem,
is necessary to manufacture easy to use assays to detect un- whereassmartphonesareuniversallypresentacrosstheglobe.
wanted allergen presence so that consumers do not have to Thesmartphoneasareadoutsystemmakesmostassaysmore
relyonrecallornotificationdatatomaintaintheiravoidance consumer-friendlyasthemajorityofpeopleareaccustomedto
diets [93, 94]. A consumer-friendly allergen test that can be using smartphone applications. A significant benefit of using
basedona smart-detector could provideconsistent,essential thesmartphoneisthattheresultscanbeinstantlyuploadedto
informationfortheallergicindividual,regardlessofthequal- Cloud databases/sent to relevant stakeholders, which can be
ityofproductlabelling. particularly useful for remote quality control. Conversely, it
5358 RossG.M.S.etal.
shouldbeconsideredthatwhenusingasmartphone-basedan- It is critical for user-friendly assays to be reproducible so
alytical device in a low resource setting, the wireless system thatthe userisconfidentinthe result.Inorder for thistobe
maysufferwithlowconnectivity,andsothesmartphoneappli- achieved, assays should be validated by intra- and inter-
cationmustbeabletosupportasynchronousdatatransmission laboratory testing and benchmarked against successful com-
[12]. Linking an assay to a smartphone detector goes a long mercialallergenassays.Bypropervalidation,thereliabilityof
wayinmakingtheassaymoreportable.Portabilitymeansthat the assay can be proved and consumer confidence can be
the assay can be taken anywhere and applied under in-field attained.Poppingetal.suggestthatconsumerdevicesshould
conditions,suchasinarestaurant. firstgothrough single laboratoryvalidation, followedbyin-
Anotherkeycomponentofauser-friendlydeviceisthatit dependentlaboratoryvalidationandproficiencytestinginpar-
shouldprovideresultsquickly.Consumersdonotwanttowait allel, including being tested by untrained personnel/
for extended periods for results, so rapid tests are desirable. consumers[95].Itwouldimprovetheaffordabilityoftheas-
The assay should provide results as quickly as reasonably sayiftheassaywerereusablesuchaswhenusingaSPRchip;
possible without compromising the sensitivity or reliability however,iftheassaycannotbereused(LFIA)thesmartphone
of the test. The speed of an assay can be optimized by first attachmentandappshouldbeabletobereusedforanumber
carryingoutdetailedkineticsstudiestoselectantibodieswith ofcyclesandtheassayshouldberecyclable.Theidealdevice
rapid association rates and high affinities to the allergen of forconsumerswouldthereforebe:easytouse,safe,recycla-
interest, for use in the assay. The reaction rate can also be ble, affordable, a smartphone-based detector or other smart
increased byproperorientation ofthe antibodies, so that the device with connectivity possibilities, portable, rapid, sensi-
relevant binding sites are directed away from the surface, tive, multiplexed where appropriate, and properly validated
wheretheycanbetterinteractwiththetargets.Assayscanbe andbenchmarked.
further sped up by using internal microfluidics, which also
limits the necessity for excessive sample handling/
preparationasmixingcanbeachievedinthefluidicssystem. Methods for food allergen detection using
Microfluidicsoftenincreasethespeedoftheassayasmixing, a smartphone readout system
pumping, and directional flow can be carried out at precise
locations in the assay itself, limiting the need for operator Immunochemicalmethodsforallergendetectionfocusaround
interaction [96]. Proper mixing can also speed up the assay the complementary interaction of an allergen-specific anti-
by increasing the rate of diffusion of the sample. The assay body and an allergen. An overview of commercial
shouldnot havesignificant cross-reactivitywithdifferental- laboratory-based allergen assays is provided in ESM
lergens,sothatuserscanbecertainthattheirresultsarecor- Table S3. Lab-based methods are highly sensitive, selective,
rect. Proper characterization of antibodies ensures that the and accurate. However, lab-based methods require trained
assayisselectivefor thetargetallergen.Inadditiontobeing personnel, scientific knowledge, and often expensive equip-
selective, the assay should be sensitive and able to detect ment. By linking traditional lab-based methods with a
allergensattheirLOAEL. smartphonereadout system, theybecomemoreuser-accessi-
Multiplexingallowsmultipleallergenstobedetectedina ble.A comparisonoflab-and smartphone-basedmethods is
singlesample,whichisdesirable,savingtimeand moneyin giveninESMTableS4.Themostpopularopticalapproachto
comparison with using several singleplex assays [97]. smartphonedetectorsisbasedoncolorimetricreactionssuch
Furthermore, a proportion of the allergic population suffers as in LFIA or ELISA [28]. Colorimetric smartphone-based
frommorethanoneallergy,duetocross-reactivitywithsim- sensing conventionally reliesonthe phone’s complementary
ilarlystructuredallergens,soitisattractivetotestmorethan metal oxide semiconductor (CMOS) filters to assign red,
oneallergenatatime[96–99].Anindividualwhosuffersfrom green, blue (RGB) values to light. Therefore, smartphone-
multipleallergiesshouldbeabletotestforallofthemusinga basedsensorsareabletodetectchangesinopticaldensityor
singular device. As allergens are structurally different pro- intensityofanalyte–reagentcomplexesoverarangeofwave-
teins,theymayrequiredifferentextractionprocedures;when lengths[12].Themajorityofthepopulationhasandisfamiliar
testing formultipleallergenstheextractionbufferwilllikely with smartphones, so interfacing a scientific method with a
beacompromisebetweenmaximumextractionefficiencyand simplesmartphoneappimprovesconsumerfriendliness.
the ability to co-extract different allergens from the matrix.
Truly personalized allergen testing where consumers select Lateralflowimmunoassays
theallergenpaneltheywantincludedintheassaywouldcome
at an expense, but this could be lowered if companies start Lateral flow immunoassays (LFIA) are immuno-chromato-
including more allergens in multiplex assays. The current graphic test strips designed to be easy to use, as has
proof-of-concept allergen multiplex assays are displayed in been exemplified by their success as pregnancy tests [100].
ESMTableS2. Many food manufacturers utilize LFIA to test their clean-in-
Consumer-friendlyfoodallergendetection:movingtowardssmartphone-basedimmunoassays 5359
place(CIP)proceduresandtoensurethattheirproductionlines to-use, safe, affordable, portable, rapid, sensitive, and can be
are free from allergens. Cross-contamination can be monitored quantitative when linked with a dipstick reader such as a
forinstanceusingLab-2-go,auser-friendlytesttoolkitdeveloped smartphone.
byZeulab(Zaragoza,Spain)toprovegoodmanufacturingprac-
tice(GMP)[101].ThestandardcomponentsofaLFIAare:the Smartphonelateralflowimmunoassayreaders
samplefilterpad,theconjugatepad,themembrane,theabsorp-
tionpad,andthetest/controllines[102]. AlthoughLFIAresultscanbevisuallydetectedwiththenaked
Ina sandwichformatLFIA, the conjugate pad containsa eye,byintegratingLFIAwithasmartphonedetectorsystem,a
pre-sprayedantibodythatisspecifictotheallergenofinterest. quantitativeresultcanbeachieved.Owingtotheirsimplestruc-
Thisspecificantibodyislabelledwithcoloredorfluorescent ture,LFIAsarefairlysimpletointerfacewithsmartphones,asthe
moieties. The test line contains an immobilized allergen- results can be easily detected via the phone’s camera.
specific antibody, which binds to a different epitope on the Smartphone dipstick readers can be categorized based on their
allergenthanthelabelledantibody.Thecontrollinecontains lightsource;somerelyonLEDastheexternallightsourcewhilst
an antibody raised against the animal species of the labelled othersutilizetheinternalflashinthephone.
antibody. When a sample containing the target allergen is Mudanyali et al. described a smartphone readout system
added to the sample pad, the target binds with the labelled termedrapiddiagnostictestreader(RDS)[25].Thereaderis
antibody in the conjugate pad, forming a labelled complex. madeupofa3D-printed65gmechanicalattachment,which
Thelabelledcomplexflowsviacapillaryaction,drivenbythe consists of: a LFIA strip holder, an inexpensive lens, three
absorptionpad,laterallyupthemembrane.Whenthetestline LEDs,andthreeAAAbatteries.Thedevicecapturedimages
is reached, the complex is captured by the immobilized oftheLFIA,whichweredigitallyprocessedwithintherelated
allergen-specific antibody. The target analyte is sandwiched smartphone app. The linked central database received and
between the labelled and the captured antibodies, which re- stored the processed results in a world map through geo-
sultsintheappearanceofacoloredlineinthetestregion.The timestamping. This devicewas validatedbyusing commer-
remaininglabelledantibodybindswiththeimmobilizedanti- cially available malaria, tuberculosis, and HIV LFIA [25].
speciesantibodyatthecontrolline,resultingintheappearance Another example applying LED as an external light source
ofasecondcoloredlineinthisregion.Inasandwichassay,the was described by Lee et al. for using a smartphone-based
color intensity of the test line is directly proportional to the readoutsystemintegratedwithaLFIAreaderforthedetection
concentrationofthetargetallergeninthesample.Whilstthe of aflatoxin B1 [23]. The device described a LFIA reader
test line informs the user of the relative concentration of the consisting of: a close-up lens, white LEDs, and batteries. A
allergeninthesample,thecontrollineprovesthattheassayis smartphonecamerawaspositionedoverthelensoftheLFIA
functioningcorrectly. readerwherethecamerarecordedimagesoftheopticalden-
sityoftheLFIAtestandcontrollines.Leeetal.furtherrefined
Multiplexdipsticktests this LED-based format of LFIA reader and smartphone app
forimagecaptureanddataacquisitionforSalmonelladetec-
Lateral flow immunoassays can also be multiplexed through tion [24]. This format of LFIA strip readers utilize LEDs as
theadditionofmultipletestlines.Eachtestlinecorrespondsto lightsources,whichrequiresexternalbatterypacksforpower.
the target analytes to be detected [103]. Detecting a range of Another format of smartphone LFIA readers utilizes the
allergensinasampleisattractiveasitreducesanalysistimeand smartphone’s embedded camera flash as the light source.
reagent waste, as multiple analytes can be assessed under the Oncescu et al. developed a smartphone readout system for
same conditions. Structures other than simple strip tests can thecolorimetricdetectionofchangesofpHinsweatandsaliva
alsobeappliedinmultiplexing.Fentonetal.haveshownthat [106].Thedeviceuseda3Dprintedphonecase,whichhoused
two-dimensionalshapingofcapillarydrivenmembraneassays aslotfortheindicatorpHstrip,areferencestrip,androomto
intocandelabraorotherstructurescanimprovethespatialdis- store up to six spare pH test strips. The attachment applied
criminationoftheassay[104].Assaysfordifferentanalytescan PDMSlightdiffuserstoallowreproducibleilluminationfrom
be positioned on separate arms of the device, which can be thecameraflash.ThestripswerephotographedandtheRGB
directlylabelledtominimizeuserconfusion.Currently,much (red,green,blue)valueswereanalyzedandconvertedtoahue
of the attention of multiplex flow assays has been focused spectrum. Hue moreappropriately fitsthe range of color for
towards mycotoxin analysis [105]. It is expected that future pHstrips.Inanotherstudy,Oncescuetal.advancedtheuseof
research will focus on incorporating multiplex into the food theinternalflashofaphonecameraforreadingofLFIAfor
allergen field in order to make food allergen analysis more cholesterol testing [107]. This device is referred to as the
user-friendly. When multiplex dipsticks are constructed for smartCARDanditmonitorsthecolorimetricchangeresulting
food allergens, they should be designed to fit the criteria of from a cholesterol enzymatic interaction on a test strip. The
consumer-friendliness. Lateral flow immunoassays are easy- phoneflashandcameraarethenusedtorecordimagesofthe
5360 RossG.M.S.etal.
colorimetricreaction,whichisthendigitallyprocessedinthe ELISA
relatedapp.Theattachmenthasaslotfortheteststripanda
PDMS light diffuser. The device converts recorded RGB Enzyme linked immunosorbent assays (ELISA) is the most
values to hue, luminosity, and saturation values within the routinelyusedmethodofallergenanalysisinthefoodindustry
appandiscapableofquantifyingcholesteroloverallphysio- [5,114].CommerciallyavailableallergenELISAsarelistedin
logicalvalues[106,107].Afurtherexampleofanembedded ESMTableS3.ELISAsexistinbothcompetitiveformat(suit-
flash-based LFIA smartphone reader for screening thyroid ableforlowmolecularweightproteins)andsandwichformat,
stimulating hormone (TSH) was described by You et al. which is the prominent choice for food allergens [83]. Both
[108].Thisdeviceusedanopto-mechanical3Dprintedattach- formatsofELISAarebasedontheinteractionofanenzyme
ment that directed the light from the phone camera, via an labelled allergen-specific antibody with an antigen. An anti-
optical fiber, to a collimating lens to illuminate the LFIA. bodyislabelledwithanenzyme,whichinitiatesameasurable
The study emphasized the importance of minimizing the colorimetric change upon the addition of the substrate. The
Mie scattering of the nitrocellulose membrane particles and reactionismeasuredbyanELISAplatereader[115].Insand-
maximisingtheRaleighscatteringofthegoldnanoparticlesof wichELISA,themeasuredresponseisdirectlyproportionalto
the test/control lines, increasing the signal in these regions. the concentration of allergen in the sample. Owing to the
The improved signal to noise ratio allowed a very sensitive laboratory-basednatureofELISA,whichinvolvesfollowing
LODtobeachievedwiththisreadoutsystem.Althoughthese astandardoperatingprocedureandtechnicalinstructions,the
examples have not yet been applied to allergen testing, the requirementforscientificequipment/trainedpersonnelandthe
technologycouldeasilybetranslatedforallergenanalysis. longincubationsteps,ELISAcannotbeconsideredconsumer-
Commercial companies are now finding ways to advance friendly [116]. Nevertheless, a few smartphone interfaces
their traditional LFIAs by interfacing them with smartphone havebeendesignedforuseinresourcelimitedsettings.
technology. R-Biopharm’s (Darmstadt, Germany) RIDA
QUICK lateral flow assays are compatible with the RIDA Smartphone96-wellmicroplatereaders
SMARTApp, which acts as an embedded flash smartphone-
based lateral flow strip reader. Currently, the mycotoxin strip Microplate readers are one of the most used instruments in
test range has been converted for use with the app but it is routineimmunochemicalanalysis.However,theyarerelative-
expected that soon all RIDA QUICK assays (including the lyexpensive,requiremaintenance,andarenon-portable,mak-
extensive allergen range) will be compatible with the app ingtheminaccessibleforin-fieldtesting[117].Itispossibleto
[109]. Once a sample has been tested with the LFIA, a strip create smartphone-based spectrophotometers using the
cover withthecolorcalibrationrequiredbytheapptodistin- smartphonecamera[25,117–119].Ina2016study,Fuetal.
guishthedifferencesintest/controllineintensity,isplacedover describedthedevelopmentofasmartphone-basedmicroplate
thestrip.Thestripandcoverareplacedinacardboardenclo- reader capable of detecting biomarkers in the absorbance
sure;thisboxistocontrolambientlightconditionsandensure range of 340–680 nm [120, 121]. This research relied upon
that consistent results are achievable. The app uses the establishedcommercialELISAandcomparedtheresultswith
smartphonecameratocaptureaphotoofthestrip.Theresults microplatereaderSynergyH1HybridMulti-modeMicroplate
areautomaticallystoredwithintheappdatabase/andorcanbe Reader(BioTekInstruments;Winooski,VT,USA)forvalida-
exportedto e-mail orprinted viaa WiFi connected/Bluetooth tion. Once the assay was complete, the 96-well plate was
printer.Themajorbenefitoftheappistheabilitytoquantify introducedtothesmartphone-basedmicroplatereader,which
results; however, when testing for food allergens, a semi- wasattachedtothecameraofthesmartphone.Therelatedapp
quantitative result would be sufficient as there are currently storescalibrationcurvesthatconvertthetransmittedlightin-
no set threshold levels for allergens EU legislation. Although tensity to absorbance values and then to analyte concentra-
the company also makes quantitative readers, using a tions[120].Theresultsobtainedwereslightlylowerthanwith
smartphone is significantly more affordable and user-friendly thecommercialmicroplatereader.
forthegeneralconsumer.Amajorlimitationforthisset-upis AnotherexamplewasdescribedbyBergetal.fromOzcan’s
thatitiscurrentlyonlysuitableforusewiththeAndroidplat- groupofUniversityofCalifornia,LosAngeles(UCLA),which
form (5.1-8.0 OS) and on a limited number of smartphone describes a microplate reader based on a Windows phone
models(GoogleNEXUS6,NEXUS6P,andPixelXL)[109]. (Lumia 1020, Nokia) with 3D printed attachment and a data
Lateral flow fits the criteria of being affordable, portable, processorconnectedtotheCloud[117].Thecolorimetricreader
disposable,andrapid.Thepopularityofusingsmartphonesas useda3Dprintedopto-mechanicalattachmentwithalightemit-
LFIAreadershasalsobeenhighlightedbycommercialcom- tingdiode(LED)toilluminate96-wellplates.Thelightfromthe
panies, such as Novarum and Mobile Assay, which develop LEDistransmittedthrough96individualopticalfibersthatre-
bespokesmartphoneappsforthereadingofestablishedLFIAs direct the light to a collection lens, which then transmits the
[110–113]. capturedimagesofthesamplestothecustom-designedappfor
Consumer-friendlyfoodallergendetection:movingtowardssmartphone-basedimmunoassays 5361
signalreading.Theprocessingalgorithmfocusesonfindingtwo appthatconvertstransmissionimagesreceivedfromthecam-
centroids to use as references in the 96-well plate and pixel era torelativeabsorptionvalues,whichcanberelatedtothe
intensitythresholdingtoseparatewellsforindependentanalysis. concentrationofallergenpresentwithinthesample[122].The
The device was successful and was able to match the perfor- attachment weighs around 40 g and is made up of: a plano-
mance of a Food and Drug Administration (FDA) approved convexlens,twoLEDs,twolightdiffusers,andcircularaper-
microplatereader[117]. tures to allow control of the field of view. Once the peanut
The use of smartphones as microplate readers will make assayhasbeenperformed,transmissionintensitiesarerecord-
ELISAtechnologiesmoreaccessible;bymakingthemporta- edusingthesmartphonecamera,andtheimagesaredigitally
ble,abletoconnecttoWiFi,anduploadresultstotheCloudin processed. The digital processing in the app occurs by
real-time. This adaptation will be significantly beneficial in converting the transmission images of the light through the
low resource settings such as in developing countries. As testtubesintobinarymaskimages.Thedetectorissemi-quan-
ELISA requires multiple reagent handling steps, it is neces- titative, giving a positive signal for samples containing over
saryfortheusertobeabletoutilizeapipette.Longincubation 1 ppm peanut and negative results for lower concentrations.
steps and multiple washing steps prevent the method from Anotherexampleofasingle-strip3Dprintedsmartphonemi-
beingconsumer-friendly.Evenifasmartphoneapphadastep croplatereaderwassuccessfullyexploredbyWangetal.for
bystepguideshowingwhichreagentstouseateachinterval, the detection of herbicide 2,4 dichlorophenoxyacetic acid,
the method would still not be that consumer-friendly. The which further clarifies that in some situations only a limited
detectionmethodonthesmartphoneis,however,moreuser- number of samples require analysis [119]. Like most
friendly in the sense that it is affordable, portable, and can smartphone-basedanalyticaldevices,theiTubehastheability
connectwirelesslysoitissuitableforin-fieldconditions. touploadresultstoserversthroughitsapp.Thismeansthata
personalizedallergentestingdatabasecanbeconstructedand
users can monitor tests they have carried out on different
Smartphone8-wellstripmicroplatereader foods,invariedlocations,creatingaspatio-temporalallergen
map.Usinganonymized‘bigdata’inthiswaynotonlyassists
Insomescenarios,theusermayonlywanttoanalyzeasmall allergic sufferers, but also helps those involved in food
numberofsamplesratherthanawhole96-wellplate;inthese manufacturing,productdesign,andofficialregulatorstobet-
circumstances a smartphone detector that analyzes a strip of terunderstandallergensfromaconsumerpointofview.
eight microwells may be more appropriate. The iTube is a
novel allergen testing platform also developed by Ozcan’s
laboratory at UCLA. The device is a 3D printed opto- FlowCytometry:BeadSuspensionArray
mechanicalattachmentthatisconnectedtotheexistingcam-
eraofasmartphone(Fig.1)[122].Theapproachisbasedona Flow cytometry (FC) in suspension array format uses
8 well strip of the commercial Neogen peanut ELISA. The microbeadsassolidphasesupportsystemsforcaptureantibod-
platform consists of a 3D printed attachment that holds the iestobeimmobilizedonto.Thebead–antibodycomplexcanbe
microwells and the smartphonereader, and a related‘iTube’ identifiedbyitsfluorescent/coloredprofilebyaflowcytometer
Fig.1 (a)AnimageoftheiTube
platform,usingaNeogenPeanut
ELISA8-wellstripanda
smartphone-baseddigitalreader,
isdisplayed.(b)The3Dprinted
opto-mechanicalattachment,
whichisconnectedtotherear-
facingcameraonthesmartphone.
(c)AschematicoftheiTubeis
shown.Reproducedfrom[122]
withpermissionofTheRoyal
SocietyofChemistry
5362 RossG.M.S.etal.
[123].Flowcytometrycanbeusedforbothin-vivoandin-vitro ambientlight[133].Thesmartphonecamerawasusedtore-
quantitativeallergenanalysis[124,125].Garberetal.andCho cord images of the fluorescence emitted from the QD. This
etal.haveshownthatbyusingmagneticbeadsetsitispossible assay still takes a substantial amount of time to carry out
todetect14foodallergens(andgluten)in12differentsamples, owingtoincubationstepssoitcannotbeclassifiedasarapid
within6h,withasimilarLODtoexistingELISAmethods(<5 assay.Anevenmoresophisticatedmultiplexsmartphoneap-
ng/mL)[97,126].However,theirmethodsrequiredtwoextrac- proach based on the original rbST microsphere assay was
tion procedures, so although the assay could be multiplexed, presentedforbiomarkersinmilk(Fig.2)[134].Althoughthis
the extraction could not. Otto et al. combined a competitive technologyhas currently onlybeenappliedtofooddiagnos-
formatELISAwithflowcytometry(BDAccuriC6apparatus, tics,focusingthisapproachcouldallowittobeappliedmore
Becton-Dickinson, Vianen, The Netherlands) to develop an specificallytofoodallergens.
assay capable of detecting five different allergens in a cookie
matrix[127].Theassaycoulddetectintherangeof2–10ppm Multiplexsurfaceplasmonresonance-basedfood
all the allergens in the test. Cho et al. further described the allergenbiosensor
usefulness of FC for cross-reactivity profiling between 23 le-
gumesand12treenuts[128]. Thisreviewhasavertedbiosensors,duetothein-depthreview
on using biosensors for food allergen analysis published in
Miniaturizedflowcytometers 2016 and another 2016 review focusing specifically on
smartphone-basedbiosensors[9,28].Onlybriefattentionwill
Despitetheirsuccess,flowcytometers(FCs)arenotportable, bepaidheretobiosensors.Surfaceplasmonresonance(SPR)
are relatively expensive, require trained personnel, and are monitorschangesintherefractiveindexbasedonthedielec-
thereforenotsuitableforin-fieldanalysis.Inresponsetothis, tric properties of a thin layer of sample containing solution,
FCwasminiaturized.MiniaturizationofFCinvolvesfocusing nearthegoldmetalsurfaceofthesensorregion.The energy
the flow of the particles to be analyzed within a microfluidic transfer from polarized light to surface plasmon results in
channel, reducing the size of both the microfluidics and the characteristic reflected light patterns that can be monitored
optics,andintegratingthemwithasignalreadoutdevice[129]. label free, in real-time through a sensorgram (the angle at
The portability of miniaturized flow cytometry (MFC) which the dip is observed versus time) [135]. Analyte-
makes it an attractive technique for in-field routine analysis. specific antibodies are immobilized onto the metal layer of
Connecting MFC to a smartphone readout system further thesensorchip,mountedontoaglassprismwithanintegrated
strengthensitsportability.Ozcan’sUCLAgrouphaveworked flowcellthatisthenplacedintheinstrument.Whenpolarized
since2008todevelopon-chipcytometersthatarecapableof light shines through the prism, the light is reflected by the
interfacingwithsmartphonesasthedetector[130].Zhuetal. metal layer, resulting inanangle ofincidence capable ofin-
have further substantiated the ability to combine MFC and ducing surface plasmon resonance and causing a dip in the
optical microscopy with a smartphone interface [131]. The reflected light intensity [136]. The refractive index near the
studyintegratedamicrofluidicchipwithasyringepumpthat metal surface will change as proteins are adsorbed onto the
controlledthetransportofsampletotheimagingfield,where metalsurfaceandtheamountofadsorbedproteincanthenbe
aphotowascapturedbyasmartphonecamera.Thisexample determined. Unfortunately, current ‘portable’ SPRs still re-
usesanopto-mechanicalattachment,featuring:simplelenses, quirealaptoporsmallcomputertooperate[137].
plasticcolorfilters,LEDs,andbatteries.Furtherdevelopment ImagingSPR(iSPR)hasthebenefitofbeingabletosimul-
on this study yielded a smartphone-based MFC interfaced taneously detect multiple analytes in a single sample. Raz
with an optical-microscope for the counting offluorescently etal.describedaniSPRlinkedwithanallergen-antibodyarray
labelled blood cells [132]. Despite these examples being for forthedetectionof12foodallergenswithin12min[138].The
thehealthcaresector,theyprovideanexcellentbasisforfuture rapid, multi-analyte method is quantitative and detects food
designofsmartphonebasedcytometersforapplicationtofood allergensat2mg/kg.Theprocedureallowedfortotalallergen
allergen analysis. Similarly, MFChas beenusedincontami- profilingwithinfood,providingauniquefingerprintforwhich
nantandresiduemonitoringinmilksamples[133].Anassay allergens each commercially available food contained. The
wasdesignedtodetectgrowthpromotorbovinesomatotropin opticaldeviceslaboratoryofLinköpingUniversity(Sweden)
(rbST). Biomarker-specific antibodies (anti-rbST) were described a smartphone-based angle resolved localized SPR
coupled with quantum dots (QD), which were immobilized device [139]. The device used the phone screen as the light
on paramagnetic microspheres. The device relied on an source,thephonecameratorecordimages,andadisposable
optical-mechanical attachment consisting of a phone holder optical coupler made of PDMS/epoxy, which matched the
(for alignment of optics), a sample tray to hold the cover refractiveindexofglass[139].Thepolymersurfacecontained
slides,12UVexcitationLEDs,whiteLEDs,anopticalfilter, glass coated with a layer of gold, as the thin metal surface,
a de-magnifying lens, and a lid to prevent introduction of withwhichsimpleormorecomplexmicrofluidicsystemsare