Detection of Antibiotics and Evaluation of Antibacterial Activity with Screen-Printed Electrodes

26 Pages·2017·3.18 MB·English

sensors
Review
Detection of Antibiotics and Evaluation of
Antibacterial Activity with Screen-Printed Electrodes
Florentina-DanielaMunteanu1 ID,AnaMariaTitoiu2,Jean-LouisMarty3,*and
AlinaVasilescu2 ID
1 FacultyofFoodEngineering,TourismandEnvironmentalProtection,“AurelVlaicu”UniversityofArad,
ElenaDragoi,No.2,Arad310330,Romania;fl[email protected]
2 InternationalCentreofBiodynamics,1BIntrareaPortocalelor,Bucharest060101,Romania;
[email protected](A.M.T.);[email protected](A.V.)
3 BAELaboratory,UniversitédePerpignanviaDomitia,52AvenuePaulAlduy,66860Perpignan,France
* Correspondence:[email protected];Tel.:[phone]
Received:30January2018;Accepted:15March2018;Published:18March2018
Abstract: Thisreviewprovidesabriefoverviewofthefabricationandpropertiesofscreen-printed
electrodes and details the different opportunities to apply them for the detection of antibiotics,
detection of bacteria and antibiotic susceptibility. Among the alternative approaches to costly
chromatographic or ELISA methods for antibiotics detection and to lengthy culture methods for
bacteria detection, electrochemical biosensors based on screen-printed electrodes present some
distinctiveadvantages. Chemicaland(bio)sensorsforthedetectionofantibioticsandassayscoupling
detection with screen-printed electrodes with immunomagnetic separation are described. With
regardstodetectionofbacteria,theemphasisisplacedonapplicationstargetingviablebacterialcells.
Whiletheelectrochemicalsensorsandbiosensorsfacemanychallengesbeforereplacingstandard
analysis methods, the potential of screen-printed electrodes is increasingly exploited and more
applicationsareanticipatedtoadvancetowardscommercialanalyticaltools.
Keywords: antibiotic;bacteria;antibioticsusceptibility;screen-printedelectrodes
1. Introduction
Pharmaceuticalsareincreasinglyusedworldwidetoensurepublichealth,areasonforwhicha
largenumberofactivecompoundsisusedtoprevent/treathumanandanimaldiseases[1],buttheir
developmentimpliesimplementationofarigorouswastemanagementsystem[2].
Aconsiderableincreasehasbeenrecordedintheuseofantibiotics,whichisgenerallyduetotheir
specificactivityagainstbacteriaorfungiinhumanandanimalhostsand,additionally,duetotheir
abilitytoincreasegrowthratesandimprovefeedefficiency[3]inthefieldofanimalhusbandry. The
groupofantibioticsincludesnaturalmoleculesproducedbybacteriaandfungi(e.g.,benzylpenicillin
andgentamicin),semi-syntheticones,i.e.,chemicallymodifiednaturalantibioticstoincreasetheir
stabilityorsyntheticproducts. Dependingontheirchemicalstructure,antibioticscanbeclassifiedinto
severalgroups: beta-lactams(e.g.,penicillin),macrolides(erythromycin),tetracyclines,quinolones
(ciprofloxacin),aminoglycosides(e.g.,kanamycin),sulfonamides(e.g.,sulfadiazine)glycopeptides
andoxazolidinones[4].
Theirpresenceintheenvironmentiscontributingtotheincreaseinthenumberofmulti-resistant
bacteria,withsubsequentseriousimplicationsforhumanandanimalhealth[5]. Consequently,many
countrieshaveimposedmaximumresiduelimits(MRLs)forantibioticsinfoodstuffsofanimalorigin,
whichvaryintheppb–ppmrange,dependingonthemoleculeandthefoodmatrix. Someantibiotics
arebannedforanimaluseinsomepartsoftheworldwhilestillallowedinothers—asinthecaseof
chloramphenicolintheEUorenrofloxacinintheUS.
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In this context, sensitive analytical methods are required, not only for the quantitative
determination of antibiotics in food and the environment, but also for the detection of bacteria,
screeningcompoundswithantibacterialactivityandforantibioticsusceptibilitytesting.Themostused
methodsforthedetectionofantibioticsarechromatographic[6–9],duetotheautomation,accurate
quantification,simultaneousdetection,andthehighspecificitybasedonthestructuralinformation
oftheanalytes. Othermethodsforantibioticdeterminationincludeelectrophoresis[10–14], diode
array [15,16] or enzyme immunosorbent assay (ELISA) [17–19]. Meanwhile, for the detection of
bacteria,theculture-basedmethodremainsthegoldenstandard,althoughagoodnumberofmethods
basedonnucleicacid(e.g.,basedonpolymerasechainreaction(PCR))orimmunologicreactionshave
beendeveloped.
Despitetheperformancesofthewell-establishedstandardmethodsforantibioticsandforbacteria
detection,thesemethodsaresufferingofsomewell-knowndisadvantagesrelatedtocostlyequipment
andconsumables,aswellaslaboriousworkthatincludesthesamplepreparationandtheneedfora
well-trainedtechnicalstaff[20].
In this context, electrochemical methods have the advantages of allowing a reliable, fast
and portable, in-field analysis to detect a growing range of environmental pollutants, including
antibiotics[21–25]. Aspecialplaceisoccupiedbybiosensors,whichareanalyticaldevicesthathave
the advantage of having high selectivity and rapid detection [26,27]. Biosensors achieve selective
detectionofanalytesincomplexmixtureswithoutresortingtoseparationmethods,bycombininga
specificbiorecognitionelement(antibody,peptide,protein,cell,aptameretc.) withasensitivephysical
transducer. Researchinthefieldofbiosensorsforthedetectionofantibioticresiduesandofbacteria
hasintensifiedinthelastyears,mainlyduetotheuseofnewnanomaterialsfortheconstructionof
thesebiodevices[28,29]andtheselectionofnovelbiorecognitionelementswithincreasedselectivity
andaffinity,e.g.,aptamers[20].
Among various types of electrodes used as transducers in electrochemical biosensors,
screen-printedelectrodes(SPE)[30,31]haveattractedspecialattentionbecausetheyallowsensitive
and selective analysis at a low cost that affords their use as disposable devices, are appropriate
for the analysis of low sample volumes, can be integrated in different fluidic setups (e.g., in flow
injectionanalysis)andareconvenientlyusedincombinationwithmagnetic-basedseparation[32].
The commercial success of glucose biosensors stands as a solid confirmation of the feasibility
of electrochemical devices based on SPEs. In addition, SPEs have the advantage that they can
be mass-produced easily, without any preliminary laborious preparation steps or requirement
for highly qualified personnel. SPEs are versatile devices that can be used in conjunction with
simple, portable electrochemical equipment and with various electroanalytical methods such as
amperometry[33–36],voltammetry(differentialpulsevoltammetry(DPV),squarewavevoltammetry
(SWV),cyclicvoltammetry(CV)andlinearsweepvoltammetry(LSV)[37–47]),potentiometry[48]
or EIS [49,50]. This review addresses recent advances made available by using screen-printed
electrodes for detection of antibiotics as well as for the evaluation of antibacterial activity. The
assessmentofantibacterialactivityofdifferentcompoundstypicallyreliesonculture-growingtests
andcolonycountingtoestimatetheproportionofviablebacteria.However,alternativebiosensor-based
approacheshavealsobeendescribedforthedetectionofviablebacteriaandscreen-printedelectrodes
havebeenemployedwithsomeofthesensingconcepts. Antibioticsusceptibilitytestingisanother
areawherescreen-printedelectrodeshavebeensuccessfullyemployedandisalsodiscussedbelow.
2. Screen-PrintedElectrodes
Recent years have witnessed the emergence of a wide variety of screen-printed electrodes
modified with different materials and printed in various configurations. The current needs for
reproducibility,linearrange,sensitivityandselectivityrequirefabricationofchemicalsensorsand
biosensorsthataresimpletobeproduced,withlowpowerconsumption,cost-effectivenessforthe
analysis,andversatilefordifferentanalytes. Moreover,thereareseveralsituations(e.g.,on-spot,in
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situmonitoring,etc.) whenthesensorsshouldbedirectlyusedwithoutanypre-treatmentorcleaning
betweenmeasurements.
SenSsocrrse 2e0n18-,p 1r8i, nx tFinOgR PtEeEchR nRoElVoIEgWy a llows the fabrication of a wide range of screen-printed elect3r oofd 25e s
Screen-printingtechnologyallowsthefabricationofawiderangeofscreen-printedelectrodes
that are nowadays commercially available [51]. Moreover, this technology enables immobilization of
thataSrcerneeonw-padrianytisncgo mtecmhenroclioagllyy aavllaoiwlasb ltehe[5 f1a]b.rMicoartieoonv eorf, ath wisitdeec hrnanogloeg oyfe sncarbeelens-pimrimntoedbi leilzeacttiroondoefs
biomolecules onto the electrode surface for selective and disposable biosensors [52]. Compared to
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other types of electrodes, SPEs have the advantage that many electrodes can be modified
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simultaneously in a reproducible manner using inks that include nanomaterials, mediators, etc., to
roetphreord utycpibelse mofa nenleerctursoidnegsi,n kSsPtEhsa thinavcleu dtehen aandovmaantteargiael s,thmaetd imataonrsy, eetcle.,cttoroadcehsi evceanh igbhe semnosidtiivfiietdy
achieve high sensitivity in biosensing. While other simple electrodes are investigated for fabrication
isnimbuioltsaennesoinugs.lyW inh ail ereoptrhoedrusciimblpel emealnencterro udseisnga rienkinsv tehsatti giantceldudfeo rnafanbormicaatteiorinalos,f mfleexdiibalteo,rslo, wetcc.,o tsot
of flexible, low cost electrochemical devices, e.g., hand-drawn pencil electrodes [53], SPEs have the
ealcehciterovceh heimghic saelndseivtiivcietsy, ien.g b.,iohsaennds-idnrga.w Wnhpileen octilheelre scitmropdlees e[l5e3c]t,rSoPdEess ahraev ientvheestaidgvataendt afgoer ofafbbraictcahti-oton
advantage of batch-to batch reproducibility [53]
boaf tfclhexriebplero, dlouwci bcoilsitty e[l5ec3t]rochemical devices, e.g., hand-drawn pencil electrodes [53], SPEs have the
SPEs fabrication consists in the deposition of thin layers of ink on a solid substrate by forcing
advaSnPtaEgsef aobf rbiactacthio-tno cboantscihs trsepinrothdeucdibepiloitsyi t[i5o3n] ofthinlayersofinkonasolidsubstratebyforcing
thet hinekin StkhPrtEhosru ofgauhbg rahi cmaatmeiosehns h(cFo(iFngisugisruters e 1i)n1.) L.thLaeya yedreesrp sooofs fiintiinkokn o ofo fdfd itfihfffienerr eelnanytt emmrsaa toteefrr iiianallkss occaannn a bb seeo ddlieedpp osoussibittesetdrdac ctoeon nbsseyec fcuoutritvciivenleygly
usiutnhsgein isgnckrse cterhenreson uosgfo hcf uacs umtsoetmoshmiz (ieFzdieg dduderese is1gi)gn. n Laaanynded rmsm oaanf niyny keel loeecfc tdtrrioofdfdeeerses naatrr eem pparrtoeorddiauulccsee cdda naat tb ttehh edees spaamomseiette itdmim ceoe.n.T sThehicsuismt imvoedoldey e
of opufsreipnprgea prsaacrtrieaoetnino san lolafol lwcouwss tasonma naizuaetudotm odmaestaeitgdend p aprnordoc cemesssas nfofyor r etlthehecet prporrdooeddsuu accrtteiioo pnnr ooodff uflflceeexxdiibb allete tddheeess isigagnmnssew wtiimtihthea .a Tv vhereisyr ymg ogoooddoed
reproerfop pdroruedcpuiabcriialbititilyoit ny[5 a[45l]l4.o ]w. s an automated process for the production of flexible designs with a very good
reproducibility [54].

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Figure 1. Schematic diagram of the basic screen-printing process for electrodes manufacturing.
ReRpreipnrtiendte fdrofrmom [5[45]4, ]w,withit hpepremrmisissisoionn frfroomm EEllsseevviieerr..
Reprinted from [54], with permission from Elsevier.
Various electrode geometries and microelectrode arrays can be realized by screen printing by
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simply adapting the screen design. The typical format of screen-printed electrochemical devices
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corresponds to a classic three-electrode cell, comprising working, counter and reference electrodes
ccoorrrreessppoonnddss ttoo aa ccllaassssiicc tthhrreeee--eelleeccttrrooddee cceellll,, ccoommpprriissiinngg wwoorrkkiinngg,, ccoouunntteerr aanndd rreeffeerreennccee eelleeccttrrooddeess
printed on the same substrate in different configurations, as illustrated in Figure 2 [55–60].
pprriinntteedd oonn tthhee ssaammee ssuubbssttrraattee iinn ddiiffffeerreenntt ccoonnfifigguurraattiioonnss,,a assi illlluussttrraatteeddi innF Figiguurree2 2[ [5555––6600].].

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conccofionngfifiuggrauutrriaaottniioo 1nn. 11R..e RRpeerppinrriitnnetdtee ddfr fofrrmoomm [6 [[066]00,] ],w, wwitiihtthh p ppeeerrmrmmiisisssssiioioonnn f ffrrrooommm EEElllssseeevvviiieeerrr...
Two-electrode systems are used in potentiometric sensors or in some amperometric ones where
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the reference electrode also plays the role of counter electrode. However, with respect to detection by
thet hreefreerfeenrecnec eeleecletrcotrdoed ealaslos oplpalyays sththee rorolele oof fccoouunntteerr eelleeccttrrooddee.. HHoowweevveerr,, wwiitthh rreessppeeccttt otod deetetectcitoionnb yby
voltammetry sensors, three-electrode systems are required; moreover, they have inherent enhanced
voltammetry sensors, three-electrode systems are required; moreover, they have inherent enhanced
sensitivity compared to two-electrode ones [61]. Multi-electrode systems and electrode arrays are
sensitivity compared to two-electrode ones [61]. Multi-electrode systems and electrode arrays are
currently available commercially. For a more extensive discussion of designs and materials of SPEs,
currently available commercially. For a more extensive discussion of designs and materials of SPEs,
as well as their applications in electrochemical biosensors, the reader is referred to several reviews
as well as their applications in electrochemical biosensors, the reader is referred to several reviews
[32,54,56,62–64].
[32,54,56,62–64].
Sensors2018,18,901 4of26
voltammetrysensors,three-electrodesystemsarerequired;moreover,theyhaveinherentenhanced
sensitivity compared to two-electrode ones [61]. Multi-electrode systems and electrode arrays are
currently available commercially. For a more extensive discussion of designs and materials of
SPEs, as well as their applications in electrochemical biosensors, the reader is referred to several
reviews[32,54,56,62–64].
Traditionally,thereferenceelectrodeconsistsofaAgorAg/AgCllayer[65],whilethecounter
electrode is usually C or Au. With regards to fabrication of working electrodes, the most used
inkandtheleastexpensiveiscarbon,presentinglowbackgroundcurrent,widepotentialwindow,
chemicalinertness,andmultiplefunctionalizationpossibilities[56,66]. Furthermore,carbon-based
electrodes can be easily modified with different nanomaterials or mediators by mixing these in
the printing inks. Different carbon based SPEs were fabricated for determination of natural and
synthetic antibiotic using: multi-walled carbon nanotubes for determination of sulfonamides [35]
andgentamicinsulfate[48];carbonforsimultaneousdetectionoftetracyclinesandsulfonamides[34],
penicillins[36,49]orgemifloxacin[39,43];graphene/polyanilinenanocompositeforsulfonamides[35];
orgoldnanoparticles(AuNPs)forthedetectionofsulfamethoxazole[33]andepirubicin[45].
Besidescarbon,goldandplatinuminksarealsoemployedtoprintworkingelectrodes,being
attractive,despitethehighercost,fortheircatalyticproperties,highconductivity,highmechanical
strength[55,67]andofferingsomeuniquefunctionalizationopportunities(e.g.,chemisorptionofthiols
onAu,widelyemployedinbiosensing). SPEsfordetectionofantibioticsthatarebasedonworking
electrodesmadeofnoblemetalsincludepre-treatedplatinumscreen-printedelectrodes(SPEs)for
thequantificationofthestrengthofgarlic,anaturalantibiotic[68],andgoldSPEsforthedetectionof
fluoroquinolones[40],streptomycin[41],tetracyclines[42]andcefixime[47].
OneaspectoftheversatilityofSPEsistheireaseofmodificationwithenzymes,polymers,metals,
complexingagents,mediators,nanomaterials,etc.,eitherbyincorporatingthemintheprintinginkor
bymodifyingtheprintedelectrodes. Amongthevariousmodifiers,nanomaterialsaremostfrequently
considered when developing new ink compositions, as nanomaterials are positively affecting the
sensitivity, selectivity, and stability of the electrodes [50], enhancing the number of catalytic sites
andtheactivesurfacearea,conductivity[69,70]aswellastheloadingcapacitywithbiomolecules.
Forthisreason,aspecialattentionwaspaidtotheuseofmaterialssuchascarbonnanotubes[48,71],
metallicnanoparticles[72,73],nanostructuredconductivepolymers[56,74],nanocomposites[35,52,75]
orsemiconductorquantumdots[76].
3. DetectionofAntibioticswithScreen-PrintedElectrodes
Screen-printedelectrodeswereusedinnumerousapplicationsforthequantitativedetermination
ofantibiotics(Table1). Directelectrochemicaldetectionofantibioticswasachievedwithconveniently
modifiedelectrodes(e.g.,withnanomaterials)forabettersensitivityoftheassay[77]comparedto
bareelectrodes.
Alternatively, to achieve selectivity of the assay, the electrodes were modified with chemical
receptorssuchascalixarenes[48]orwithbiorecognitionelementssuchaspenicillin-bindingprotein[36]
or with aptamers [41,46,78]. Aptamers, short oligonucleotides selected invitro to bind with high
affinityandspecificityatargetanalyte,areincreasinglyresearchedforantibioticdetection. Aptamers
havebetterstabilitythanantibodiesandtheirproductionismorereproducibleandinvolveslower
cost than for antibodies, which recommend them as replacements for antibodies in commercially
availablekitsanddevices. Theelectrochemicalmethodsusedtodetermineantibioticsinfoodand
biologicalsamples(Table1)includedamperometry[33–36],variousvoltammetrytechniques(DPV,
LSV, and SWV) [37–47], potentiometry [48], electrochemical impedance spectroscopy [49,50] and
electrochemiluminescence[76,79].
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Table1.Examplesof(bio)sensorsbasedonscreen-printedelectrodesfordetectionofantibiotics.
LinearRange
WorkingElectrode1 Antibiotic Matrix Reference
(LR)/DetectionLimit(DL)
Amperometry
LR:20–200µM
SPCE/AuNP/tyrosinase Sulfamethoxazole Buffer [33]
DL:22.6µM
LR:1.92–454nM
DualSPCE/ProteinG Sulfapyridine
DL:0.39nM
Milk [34]
LR:6.4–385nM
Tetracycline
DL:1.93nM
LR:0.01–10µg/L
Sulfaguanidine
DL:1.162µg/L
LR:0.01–10µg/L
Sulfadiazine
DL:1.601µg/L
LR:0.01–10µg/L
Sulfamerazine
Graphene/polyaniline DL:2.900µg/L
modified LR:0.01–10µg/L
Sulfamonomethoxine
screen-printed Buffer DL:2.467µg/L [35]
electrodecoupledwith LR:0.01–10µg/L
Sulfadoxine
UPLC DL:2.995µg/L
LR:0.01–10µg/L
Sulfamethoxazole
DL:2.513µg/L
LR:0.01–10µg/L
Sulfisoxazole
DL:3.287µg/L
LR:0.01–10µg/L
Sulfadimethoxine
DL:6.127µg/L
Affinity LR:2.3–57.3ngmL−1
Penicillin-Binding Penicillin Milk DL:0.93ngmL−1 [36]
ProteinMagnetosensor
Differentialpulsevoltammetry
LR:0.1–10.0µmolL−1
Sulfamethoxazole DL:38nmolL−1
SPCE/MWCNT/PBNC Urine LR:0.1–10.0µmolL−1 [37]
Trimethoprim DL:60nmolL−1
LR:N.D.
Screen-printed Erythromycin DL:0.2ngmL−1
graphite Bovinemuscle [38]
LR:N.D.
electrode/antibody Tylosin DL:2ngmL−1
LR:0.5–10.0µM
SPCE Gemifloxacin Buffer [39]
DL:0.15µM
LR:0.8–400nM
Milk
DL:351pM
LR:0.8–400nM
Aptamer Ciprofloxacin Serum [40]
DL:336pM
LR:0.8–400nM
Water
DL:261pM
LR:30–1500nM
Au/aptamer/cDNA
DL:11.4nM
strands,arch-shaped/ Streptomycin Buffer [41]
14.3nM(milk)
exonucleaseI
15.1nM(serum)
aptamer/cDNA LR:1.5nM–3.5µM
Tetracycline Buffer [42]
strands(M-shaped) DL:0.45nM
LR:10–120µM
Graphene-SPCE Tetracycline Milk,Serum [43]
DL:3µM
LR:1µM–5mM
SPCE/aptamer Tetracycline Buffer [44]
DL:0.6nM
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Table1.Cont.
LinearRange
WorkingElectrode1 Antibiotic Matrix Reference
(LR)/DetectionLimit(DL)
Electrochemiluminescence
RatiometricECL LR:0.1–120nM
Chloramphenicol Buffer [79]
aptasensor DL:0.03nM
“Dual-potential”ECL LR:0.2–150nM
Chloramphenicol Buffer
aptasensor DL:0.07nM
[76]
“Dual-potential”ECL LR:0.1–100nM
MalachiteGreen Buffer
aptasensor DL:0.03nM
ElectrochemicalImpedanceSpectroscopy
LR:0.4–1000µg/L
DNAaptamer Penicillin Buffer [49]
DL:0.17µg/L
LR:1.2–75ngmL−1
DNAaptamer Kanamycin Milk DL:0.11ngmL−1 [50]
Linearsweepvoltammetry
thiolatedaptamer
/SPCE/AuNPs/magnetic
LR:0.07–1.0µM
double-charged
Epirubicin Buffer 3.0–21.0µM [45]
diazoniabicyclo[2.2.2]
DL:0.04µM
octanedichloride
silicahybrid
Potentiometrictitration
Calixarene/carbon LR:10−7–10−2µM
nanotubes GentamicinSulfate Water [48]
75nM
screen-printedsensors
Squarewavevoltammetry
bssDNA LR:10nM–10µM
Tetracycline Buffer [46]
aptamer/SA-SPAuE DL:10nM
LR:4–800µM
Urine
0.42µM
Tetracycline
LR:4–700µM
Serum
DL:0.54µM
LR:4–700µM
Milk
SPCE/AuNPs/cysteine DL:0.52µM
[47]
SAM LR:2–700µM
Urine
DL:0.32µM
Cefixime
LR:2–500µM
Serum
DL:0.38µM
LR:2–500µM
Milk
DL:0.35µM
1SPCE:screen-printedcarbonelectrode;AuNP:goldnanoparticles;SPAuE:screen-printedgoldelectrode;SAM:
self-assembledmonolayer; ECL:electrochemiluminescence; MWCNT/PBNC:multi-walledcarbonnanotubes
decoratedwithPrussianBluenanocubes;cDNA:complementaryDNA;bssDNA:biotinylatedsinglestrandDNA;
SA:streptavidin;UPLC:ultra-performanceliquidchromatography.
The research results summarized above reflect the utility of screen-printed electrodes for the
detectionofantibioticsindifferentsensingconfigurations,startingfromthedirectelectrochemical
oxidationofantibiotics[37]tocomplexbiosensorsincorporatingcomplicatedarch-shapedorM-shaped
DNA constructs [41,42], with emphasis on novel electrode materials and nanomaterial modifiers
to improve selectivity and sensitivity. Another simple approach where screen-printed electrodes
keep a good advantage over other electrodes is the coupling of electrochemical detection with
immunomagneticseparation. Finally,variousaptamer-basedsensingconceptsweredevelopedwith
differentcomplexityand,giventhecurrenttrendtowardsdevelopingnewaptamersforantibiotics
andtherecentemergenceofaptamer-basedtestingkitsinthefoodanalysisfield(e.g.,formycotoxins,
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availablefromNeoVenturesBiotechnologyInc.,London,ON,Canada),thefuturewillbringnovel
analyticaltoolsforantibioticswhereantibodieswillbereplacedbyaptamers. Inthedevelopment
of such biosensors, the possibilities for functionalizing the screen-printed electrodes in a manner
compatiblewithmassproductionwillplayacentralrole.
3.1. DetectionofAntibioticsbyAmperometry
Theamperometricmethodisperformedataconstantpotentialappliedtotheworkingelectrode,
and, subsequently, the current generated by the oxidation/reduction of the electroactive species
ismeasured.
Oneofthedevelopedamperometricsensorsfordeterminationofsulfamethoxazoleisbasedon
the cross-linking of tyrosinase on screen-printed carbon electrodes that were previously modified
withgoldnanoparticles[33]. Theobtainedbiosensorwasusedatanappliedpotentialof+500mVvs.
Ag/AgClandcanbealsousedforthedetectionofthisantibioticfromwatersamples. Thedetection
limitforsulfamethoxazolewasdeterminedtobe22.6±2.1µM[33].
Conzueloetal.designedanamperometricimmunosensorobtainedbycovalentbindingofProtein
GontheSPEsurfacethroughapeptidebond,aftergraftingafilmof4-aminobenzoicacid(4-ABA)onto
thecarbonworkingelectrodesurface. Theobtainedimmunosensorwasusedforthedeterminationof
tetracyclineandsulfapyridine[34]. Theamperometricsignalwasmeasuredat−200mVvs. asilver
pseudo-referenceelectrode,andthedetectionlimitsforbothantibioticsweredeterminedtobeinlow
ppblevelsinuntreatedmilksamples.
Another application of the screen-printed electrodes was the coupling of the electrochemical
detection with a chromatographic separation step to ensure selectivity. For example, this was
demonstratedusinganin-houseworkingcarbonelectrodemodifiedwithgraphene/polyanilineby
electrospraying[35]. Thisexperimentwasperformedfordetectionofeightsulfonamidesinshrimps.
The amperometric response was measured at an applied potential of +1.4 V, a potential that was
experimentallydeterminedfromhydrodynamicvoltammetry. Thedetectionlimitsfortheanalyzed
sulfonamideswereintherange1.162–6.127ng/mL.Ithastobepointedoutthatpriortheamperometric
detection,theshrimpsampleswerecleaned-upthroughsolid-phaseextractionandpreconcentratedby
UPLCseparation[35].
Gamellaetal.[36]proposedanamperometricmethodforthedetectionofresiduesofβ-lactams
frommilkusinganaffinitydisposablemagnetosensor,basedonrecombinantpenicillin-bindingprotein
immobilizedonmagneticbeadsthatwerekeptatthesurfaceofaSPEs[36]withamagnet. Thesensor
displayedabroadselectivitytoβ-lactamantibioticsbutwasinsensitivetootherantibioticsthatmay
befoundinmilksuchassulfapyridine,enrofloxacinandtetracycline. Theassayprinciplereliedon
thecompetitionbetweenfreepenicillinGfromthesampleandpenicillinGmarkedwithhorseradish
peroxidase(HRP),whichcompetedforbindingtothePBPonthemagneticbeadsattheelectrode
surface. TheamountofpenicillininthesamplewascorrelatedwiththeactivityofthelabeledHRP,
measuredviatheenzymaticsubstrateH O ,andtheelectrochemicalmediatorhydroquinone. The
2 2
current intensity due to the reduction of hydroquinone was thus correlated with the amount of
antibioticinthesamples. Theobtaineddetectionlimitsforsixantibioticswereinlowppblevelsinthe
caseofuntreatedmilksamples.
3.2. VoltammetryDetectionofAntibiotics
Involtammetry,thepotentialoftheworkingelectrodeisvariedacrossaspecifiedrangeandthe
currentintensityismeasuredtoobtaininformationaboutthenatureandamountoftheelectroactive
speciespresentintheelectrochemicalcell,eitherinthesolutionorattheelectrodesurface.Thedifferent
techniques—CV,LSV,SWV,andDPV—differinthemannerofvaryingthepotential;numberofscans
performed,i.e.,singleorrepetitive;andthewaytheintensityofelectricalcurrentismeasured.
Inasimpleapproachamenabletothedetectionofelectroactiveantibiotics,electrochemicalsensors
havebeendevelopedbasedonSPEswithoutusinganybiologicalmaterialsandrelyingexclusively
Sensors 2018, 18, x FOR PEER REVIEW 8 of 25
3.2. Voltammetry Detection of Antibiotics
In voltammetry, the potential of the working electrode is varied across a specified range and the
current intensity is measured to obtain information about the nature and amount of the electroactive
species present in the electrochemical cell, either in the solution or at the electrode surface. The
different techniques—CV, LSV, SWV, and DPV—differ in the manner of varying the potential;
number of scans performed, i.e., single or repetitive; and the way the intensity of electrical current is
measured.
In a simple approach amenable to the detection of electroactive antibiotics, electrochemical
Sensors2018,18,901 8of26
sensors have been developed based on SPEs without using any biological materials and relying
exclusively on the intrinsic electroactivity of the antibiotics themselves. Such an example is the sensor
monodthifieeidn twrinitshi cmeulelctit-rwoaacltleivdi tcyaorbfothne naanntoibtuiobteicss dtehceomrasteeldve ws.iSthu cPhruasnsieaxna mblpulee nisanthoecusebness o(rMmWoCdiNfiTed-
PwBiNthCm) u[3lt7i-]w ilalullsetdractaerdb oinn nFaignuorteu b3eAs,d feecaotruartiendg,w bitehsiPdreus stshiaen MblWueCnNaTn-oPcBuNbeCs (wMoWrkCinNgT e-PleBcNtroCd)e[,3 7a]
ciallrubsotrna tceoduinnteFri geulerect3roAd,ef eaantudr ina gs,iblveesrid/seilsvtehre cMhlWorCidNe Tr-ePfBerNeCncwe oerlekcintrgodelee.c tTrhoed ef,aabrciacrabtioonnc oouf nthteer
weloercktriondg eelaencdtroadsei livnecrl/usdielvde trhceh mloarniduealr defreorpe-nccaesteinlegc torfo tdhee. MThWeCfaNbTri-cPaBtiNonC ocofmthpeowsioter koinn gtheel esccrtreoedn-e
pinricnlutedde dcatrhbeonm ealencutaroldder oaps -ac afisntianl gstoepf.t hTeheM nWanCoNcoTm-PpBoNsiCte ccoomnspisotss iitne MonWthCeNsTcsr euenni-fporrimntleyd cocvaerbreodn
beyle c5t0r ondme aPsBa nfiannaolcsutbeeps. (TFhigeunrea n3oBc)o. mCpomospitaerecdo ntsoi sbtsarien cMarWboCnN eTlsecutrnoidfoersm, tlyhec oMvWereCdNbTy-P5B0NnCm-
mPBodniafineodc ounbeess a(Fffiogrudreed3 Ba )n.eCgoamtivpea rsehdiftto obf athree coaxribdoantioelne cpteroakd epso,ttehnetiMalW foCr NsuTl-fPamBNetCh-omxaozdoilfiee (dSMonXe)s
aanffdo rdtreidmaetnheogpartiimve s(ThiMftPo)f tohfe othxrideea tiaonndp etawkop otitmenetsia lhfiogrhseur lfcaumrerethnot xianztoelnes(iStiMesX, )raensdpetcritmiveetlhyo. pTrhime
e(TleMctrPo)coaftathlyrteiec aenffdecttw woatsi mduese htoig ah ecromcubrirneanttioinnt ebnestwitieeesn,r tehsep ehcitgihv esluy.rfTahcee ealreecat roofc MataWlyCtiNcTef faencdt wthaes
pdrueesetoncaec oofm nbuinmaetiroonubs ectawtaeelyntitch esihteigs hosnu rthfaec eedargeeas ooffM PWBNCCN Tthaant dfathcieliptarteesden tcheeo ffonrummateiroonu socf aFtael(yIItIi)c
csoitmespolenxtehse weditghe SsMofXP BanNdC TtMhaPt.f aTchilei tsaetendsotrh ealfloorwmeadt iosenleocftFivee( IdIIe)tceocmtiopnle oxfe sSwMiXth aSnMd XTManPd iTnM bPin.Tarhye
mseinxstuorreasl laonwde dspsiekleedc tsivyentdheetteicc tuiorninoef, SwMitXh adnedtecTtMionP ilnimbiitns aorfy 3m6 inxtMur aesnda n6d0 snpMik,e rdesspyenctthievteilcyu. rTihnee ,
mweitthhodde tiesc rteiolenvlainmt iftosro afn3a6lynsMis aonf dsu6c0hn aMnt,irbeisoptiecc ctiovmelbyi.nTahtieonmse tthhaotd arise ruesleevda tnot bfoorosatn tahley stirseaotfmsuencht
eafnfitcibaicoyt iacncdo mwbhiincaht iaornes ktnhoawtanr ebuefsoerdethoanbodo. sTthteh eoxtriedaattmioenn tpeoftfiecnatciaylsa nodf SwMhXic hanadre TkMnoPw anreb seefopraerhaatendd .
eTnhoeuogxhi dtoa taioffnorpdo tseuncthia alsnaolfySsMis X(FaignudrTe M3CP).a reseparatedenoughtoaffordsuchanalysis(Figure3C).

FFiigguurree 33.. ((AA)) IImmaaggee ooff tthhee ssccrreeeenn--pprriinntteedd sseennssoorr iinncclluuddiinngg aa ccaarrbboonn wwoorrkkiinngg eelleeccttrrooddee mmooddiiffiieedd wwiitthh
MMWWCCNNTT--PPBBNNCC ((iinn tthhee cceenntteerr)),, aa AAgg//AAggCCl lrerefeferreennccee (l(eleftf)t )aanndd aa ccaarbrboonn ccoouunnteterr eelelecctrtrooddee (r(rigighht tssididee).) .
((BB)) TTrraannssmmiissssiioonn eelleeccttrroonn mmiiccrroossccooppyy ((TTEEMM)) iimmaaggee ooff MMWWCCNNTT--PPBBNNCC ccoommppoossiittee uusseedd ffoorr wwoorrkkiinngg
eelleeccttrrooddee mmooddiiffiiccaattiioonn.. ((CC)) DDPPVV ooff MMWWCCNNTT--PPBBNNCC--SSPPEE iinn 00..0044 mmooll LL−−11 BBrriittttoonn RRoobbiinnssoonn bbuuffffeerr ppHH 77..00
iinn eeqquuiimmoollaarr mmiixxttuurreess ooff SSMMXX aanndd TTMMPP wwiitthh iinnccrreeaassininggc coonncceenntrtaratitoionns:s:( a(a))0 0µ µmmoollL L−−11;; ((bb)) 11 µµmmooll LL−−11; ;
((cc)) 22 µµmmooll LL−−11; ;((dd)) 44 µµmmooll LL−−11; ;((ee)) 66 µµmmooll LL−−1;1 ;(f()f )88 µµmmooll LL−−1;1 ;aanndd (g(g) )1100 µµmmooll LL−−1.1 .AAddaapptetedd frfroomm [[3377]]
wwiitthh ppeerrmmiissssiioonn ffrroomm EEllsseevviieerr..
However, for unknown samples, there are many compounds oxidizing in the same potential
However, for unknown samples, there are many compounds oxidizing in the same potential
range that could potentially interfere in the assay and this is the general limitation of electrochemical
rangethatcouldpotentiallyinterfereintheassayandthisisthegenerallimitationofelectrochemical
sensors lacking recognition elements. A similar strategy was used for the quantitative determination
sensorslackingrecognitionelements. Asimilarstrategywasusedforthequantitativedeterminationof
of gemifloxacin in pharmaceutical tablets by DPV, by direct electrochemical oxidation on a screen-
gemifloxacininpharmaceuticaltabletsbyDPV,bydirectelectrochemicaloxidationonascreen-printed
printed carbon electrode [39]. The pharmaceutical excipients have not interfered in the assay.
carbonelectrode[39]. Thepharmaceuticalexcipientshavenotinterferedintheassay. Nonetheless,for
Nonetheless, for other types of samples (food and biological) with complex matrices, appropriate
othertypesofsamples(foodandbiological)withcomplexmatrices,appropriatesamplepre-treatment
sample pre-treatment or separations will have to be devised to avoid interfering compounds that
orseparationswillhavetobedevisedtoavoidinterferingcompoundsthataffecttheaccuracyofthe
affect the accuracy of the measurements. A detection limit of 0.15 µM for gemifloxacin was reported.
measurements. Adetectionlimitof0.15µMforgemifloxacinwasreported.
The necessity to determine the presence of antibiotics in some edible tissues lead to the
developmentofacompetitiveELISAassayforthedetectionoftylosinanderythromycinfrombovine
musclewithelectrochemicaldetectionbyDPVonscreen-printedelectrodes[38]. Inthisexperiment,
theantibodiesfortylosinanderythromycinwerelabeledwiththeenzymealkalinephosphatase(ALP).
Thequantitativedetectionofantibioticsisultimatelyrelatedtotheoxidationcurrentofp-aminophenol
(PAP),resultingintheconversionofp-aminophenolphosphate,catalyzedbyALP.Thedetermined
detectionlimitsforerythromycinandtylosinwere0.2ng/mLand1ng/mL,respectively.
Sensors2018,18,901 9of26
Theselectivedetectionofciprofloxacininfoodstuffandenvironmentalsamples[40]wasachieved
with a competitive assay based on an aptasensor. This aptasensor was based on an aptamer as
molecularrecognitionprobeimmobilizedonagoldelectrodeandasingle-strandedDNA-binding
proteinthatbindstotheaptamer,thusformingaphysicalandnegativelychargedbarrierfortheaccess
ofredoxprobe([Fe(CN) )]3−/4−)tothesurfaceofthegoldelectrode. Inthepresenceofciprofloxacin,
6
theaptamerbindstoitstarget,which,consequently,isnolongeravailableforinteractionwiththe
single-strandedDNA-bindingproteinandthecurrentrecordedviacyclicvoltammetryofferricyanide
ishigher. Thedevelopedsensorwassuccessfullyusedforthedetectionofciprofloxacinbydifferential
pulsevoltammetryfromspikedmilk,serum,andwater,withdetectionlimitsof351,336and261pM,
respectively[40].
Filiketal.[43]arereportingareliable,modestcost,portable,easytooperateandsmallsystemthat
isbasedongraphenemodifiedscreen-printedcarbonpasteelectrode. Thesensorcanbeusedforthe
voltammetricdetectionoftetracyclinefrommilkandhoneysamples. Thedetectionlimitwasfoundto
be0.08µM,andthetestedmilkandhoneysamplesdidnotcontaintetracycline. Therecoveriesofthe
antibioticfromspikedsampleswerebetween98%and102%,confirmingtheaccuracyofthesensor.
Another aptasensor for detection of tetracycline was obtained through the immobilization of
biotinylatedssDNAaptameronastreptavidin-modifiedscreen-printedgoldelectrode[46]. Thesensor
ischaracterizedbyalowdetectionlimit(10nM)andthepossibilitytobeusedfordeterminationof
tetracyclineindrinkingwater,foodproducts,andpharmaceuticalpreparations.
Zhanetal.[44]describedascreen-printedcarbonelectrodemodifiedwithacompositeconsisting
ofreducedgrapheneoxide,magnetite,andsodiumalginateontowhichwasboundthetetracycline
aptamer. Detectionoftetracyclinethroughdifferentialpulsevoltammetryledtoaverygooddetection
limitof600pM.Thedevelopedsensorwasprovedtobeanappropriateplatformfortheanalysisof
tetracyclinefromrealsamples.
A screen-printed gold electrode modified with cysteine self-assembled monolayers on gold
nanoparticleswasusedforrapidandsimultaneousdeterminationoftetracyclineandcefiximefrom
milk, honey and serum by means of square wave voltammetry [47]. This sensor combined with
chemometrictoolsisagoodcandidatefordetectionofthetargetedantibioticsfrombiologicalfluids.
Thedetectionlimitsfortetracyclinewerefoundtobebetween0.42and0.52µM,whileforcefiximethe
limitsarebetween0.32and0.38µM.
Gold screen-printed electrodes were modified with a M-shaped DNA construct made from a
tetracycline(TCN)aptamerpartiallyhybridizedwiththreecomplementarystrands(CS).Thebinding
eventwithtetracyclinereleasedtheaptamerfromtheDNconstruct.Moreover,theenzymeexonuclease
I was added to the medium to degrade the complementary sequences CS1 and CS2 remaining on
electrodesurface,thusfacilitatingtheaccessoftheredoxprobe,ferricyanide,totheelectrodesurface.
Theneteffectisanincreasedelectrochemicalsignal,proportionalwiththeamountoftetracyclinein
thesample[42](Figure4).
Thissensordesignenabledmaximizingthedifferencesinthecurrentintensityofferricyanide
in the absence and the presence of the target antibiotic. The aptasensor was applied for detection
ofTCNfromserumandmilk,andthemeasurementaccuracyforsamplesthatcontainproteinsand
otherinterferingmaterialswasdemonstratedbyrecoveriesof93.1%and103.8%fromspikedsamples.
Despite the complexity of these matrices containing proteins and other interfering materials, the
sensitivityofthesensorwassimilartothesensitivityobtainedfordeterminationsinthebuffer;only
thedetectionlimitsinmilkandserumweresomewhathigher,around0.7nM,comparedto0.45nMin
thebuffer[42].
Sensors 2018, 18, x FOR PEER REVIEW 10 of 25
the detection limits in milk and serum were somewhat higher, around 0.7 nM, compared to 0.45 nM
Sensors2018,18,901 10of26
in the buffer [42].

Figure F4ig. uSrceh4e.mSachtiecm ialltuicsitlrluastitoranti oonf otfetteratrcaycyclcilninee ddeetteeccttiioonnb absaesdedon oenle cetlreoccthreomchiceamlmiceathl omd.etIhnothde. In the
absencea bosfe ntceetroafctyectrlaincyec, litnhee, tMhe-sMh-asphaep setrsutrcutcuturere ooff AApptt--CCSSssc ocmomplpexlerxem reaminsaiinntsa citnatancdt raednodx rperdoobex probe
couldnothaveaccesstothesurfaceofelectrode,leadingtoaweakelectrochemicalsignal(a).Inthe
could not have access to the surface of electrode, leading to a weak electrochemical signal (a). In the
presenceoftetracycline,AptbindstotetracyclineandleavestheCSs. ExoIdegradesCS1andCS2,
presence of tetracycline, Apt binds to tetracycline and leaves the CSs. Exo I degrades CS1 and CS2,
resultinginthefreeaccessofredoxmediatortothesurfaceofelectrodeandastrongelectrochemical
resulting in the free access of redox mediator to the surface of electrode and a strong electrochemical
signal(b).Reprintedfrom[42]withpermissionfromElsevier.
signal (b). Reprinted from [42] with permission from Elsevier.
Anelectrochemicalaptasensorforthedetectionofstreptomycin[41]wasbasedonanequally
An electrochemical aptasensor for the detection of streptomycin [41] was based on an equally
interesting DNA construct. Thiolated streptomycin aptamer and a complementary strand were
interestiimnmg oDbiNlizAed coonnastgruolcdt.e lTechtirooldaeteadn dsptraerptitaollmy yhycibnri daipzetdaminersu achnda ma acnonmerptolefmoremnttaorgye thsterraannd were
immobialriczhe-dli koens tar ugcotuldre etlheacttraoctdsea sanadba prrairetriatolleyl ehcytrbornidtriaznesdfe irnf rsoumchth ae mredaonxnsepre tcoie fsofremrri ctoyagneitdhee,ra dande adrch-like
in solution. When streptomycin is added the aptamer is displaced from this structure to bind its
structure that acts as a barrier to electron transfer from the redox species ferricyanide, added in
target. Furthermore,asthearch-likestructureisdestroyed,theadditionofexonucleaseItodegrade
solution. When streptomycin is added the aptamer is displaced from this structure to bind its target.
thecDNAremainingontheelectrodesurfacefurtherenablesthediffusionandelectrontransferof
Furthermore, as the arch-like structure is destroyed, the addition of exonuclease I to degrade the
ferricyanideattheelectrodesurface. Asinthepreviousexample,anenhancedelectrochemicalsignalis
cDNA remaining on the electrode surface further enables the diffusion and electron transfer of
observed,proportionaltotheconcentrationoftheantibiotic. Theconceptwasverifiedforthedetection
ferricyaonfisdtree patto tmhyec einlefcrtormodmei lskuarnfadcbel.o Aods siner uthme tphrroeuvgiohudsi fefexraemntipallep, ualnse evnohltaanmcmedet reyl,ewctirtohcdheetemctiicoanl signal
is obselrivmeidts, opf1ro4.p1oarntdio1n5.a3l ntMo ,trhesep eccotnivceelynt[4ra1]t.ion of the antibiotic. The concept was verified for the
detection of streptomycin from milk and blood serum through differential pulse voltammetry, with
3.3. PotentiometricDetection
detection limits of 14.1 and 15.3 nM, respectively [41].
Potentiometricdetectionisasimplemethodthatpresumesmeasuringthedifferencesinvoltage
betweenanindicatorelectrode,whosepotentialiscorrelatedwiththeconcentrationofionsinsolutions
3.3. Potentiometric Detection
and a reference electrode, typically in the absence of electrical current flow in the electrochemical
Pocteelnl.tTioomenesturriec sdeeletcetcitviiotyn, tihse ai nsdimicaptloer meleectthroodde tihsamt opdriefiseudmweisth maeioansouprhinogre t,hseel edcitfifveerfeonrctehse iinon voltage
betweeonf iannte rienstd.icator electrode, whose potential is correlated with the concentration of ions in
Khaledetal. developedasimple,selectiveandsensitivedisposablepotentiometricsensorfor
solutions and a reference electrode, typically in the absence of electrical current flow in the
detectionofgentamycinsulfate,bymodifyingSPCEswithmulti-walledcarbonnanotubes–polyvinyl
electrochemical cell. To ensure selectivity, the indicator electrode is modified with a ionophore,
chlorideinpresenceofcalix[4]areneasionophore[48]. Calixareneisthekeyconstituentofthesensor
selective for the ion of interest.
toachieveselectivity,asitformsaninclusioncomplexwiththegentamicincation. Thedetectionlimit
Khfoarlegde netta mali.c idn-esvuelflaotpeewdi tah tshimisppolete, nsteiolemcetitvriec SaPnEd wseasnsfoituinvde tdoisbpeo7s.5ab×le1 0p−o8tmenotli/oLm.Aetrnioct asbelnesor for
detectiofena toufr egeonfttahme dyecvinel ospueldfamtee, tbhyo dmroesdidifeysiningt ShPeCfaEcts twhaitthth me auvlteir-awgealrleecdo vcearryboofng nenatnaomtuycbinesf–ropmolyvinyl
chloridsep iinke pdrwesaetenrcsea omfp claesliixs [c4o]m apraernaeb laest oiovnaolupehsorerpeo [r4te8d]. wCiathlitxhaersetnane disa rtdhem ketehyo dcosn[4s8t]i.tuent of the sensor
to achieve selectivity, as it forms an inclusion complex with the gentamicin cation. The detection limit
for gentamicin-sulfate with this potentiometric SPE was found to be 7.5 × 10−8 mol/L. A notable feature
of the developed method resides in the fact that the average recovery of gentamycin from spiked
water samples is comparable to values reported with the standard methods [48].
3.4. Detection by Electrochemical Impedance Spectroscopy
Electrochemical impedance spectroscopy (EIS) [80] is a label-free, sensitive method, widely used
to assess the changes in the electrical properties at the sensor–sample interface. In faradaic EIS, a
redox probe, typically [Fe(CN)6]3−/4−, is added in the solution and, over a fixed potential value,