SensorsandActuatorsB171–172 (2012) 62–76
ContentslistsavailableatSciVerseScienceDirect
Sensors
and
Actuators
B:
Chemical
jou rn a l h o m e pag e :w w w . e l s e v i e r . c o m / l o c a t e / s n b
Review
Biosensors
for
cardiac
biomarkers
detection:
A
review
Anjum
Qureshi
a,∗, Yasar
Gurbuz
b,
Javed
H.
Niazi
a,∗aSabanciUniversity,NanotechnologyResearchandApplicationCenter,OrtaMahalle34956,Tuzla,Istanbul,Turkey bFacultyofEngineeringandNaturalSciences,SabanciUniversity,Orhanli34956,Tuzla,Istanbul,Turkey
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received10February2012
Receivedinrevisedform22May2012 Accepted25May2012
Available online 2 June 2012 Keywords: Cardiovasculardisease Cardiacbiomarker C-reactiveprotein Electrochemical Opticalbiosensor
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b
s
t
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c
t
Thecardiovasculardisease(CVD)isconsideredasamajorthreattoglobalhealth.Therefore,thereisa growingdemandforarangeofportable,rapidandlowcostbiosensingdevicesforthedetectionofCVD. BiosensorscanplayanimportantroleintheearlydiagnosisofCVDwithouthavingtorelyonhospital visitswhereexpensiveandtime-consuminglaboratorytestsarerecommended.Overthelastdecade, manybiosensorshavebeendevelopedtodetectawiderangeofcardiacmarkertoreducethecostsfor healthcare.Oneofthemajorchallengesistofindawayofpredictingtheriskthatanindividualcan sufferfromCVD.Therehasbeenconsiderableinterestinfindingdiagnosticandprognosticbiomarkers thatcanbedetectedinbloodandpredictCVDrisk.Ofthese,C-reactiveprotein(CRP)isthebestknown biomarkerfollowedbycardiactroponinIorT(cTnI/T),myoglobin,lipoprotein-associatedphospholipase A(2),interlukin-6(IL-6),interlukin-1(IL-1),low-densitylipoprotein(LDL),myeloperoxidase(MPO)and tumornecrosisfactoralpha(TNF-␣)hasbeenusedtopredictcardiovascularevents.Thisreview pro-videsanoverviewoftheavailablebiosensorplatformsforthedetectionofvariousCVDmarkersand considerationsoffutureprospectsforthetechnologyareaddressed.
© 2012 Elsevier B.V. All rights reserved.
Contents
1. Introduction... 62
2. Cardiacbiomarkers... 63
3. BiosensorsforCVDs... 67
3.1. Opticalbiosensors... 67
3.1.1. Fluorescencebasedbiosensorsforcardiacmarkersdetection... 67
3.1.2. Luminescenceandcolorimetricmethodsforcardiacmarkerdetection ... 68
3.1.3. ELISAbasedmethodsforcardiacmarkerdetection ... 68
3.1.4. SPRbasedbiosensorforcardiacmarkerdetection... 69
3.1.5. SPRbasedfiberopticbiosensorsforcardiacmarkerdetection... 71
3.1.6. Otheropticalbasedbiosensors... 71
3.2. Acousticbiosensorsfordetectionofcardiacmarkers... 71
3.3. Electrochemicalbiosensors... 71
3.3.1. Useofnanomaterialsinelectrochemicalbiosensors... 72
3.3.2. Electrochemicalbiosensorswithoutelectronmediators... 73
3.4. Magneticbiosensors... 74
4. Conclusionsandperspectives ... 74
Acknowledgements... 74
References... 74
Biographies... 76
∗ Correspondingauthors.Tel.:+902164832413;fax:+902164839885. E-mailaddresses:anjum@sabanciuniv.edu(A.Qureshi),javed@sabanciuniv.edu (J.H.Niazi).
1. Introduction
Cardiovasculardisease(CVD)isamajorcauseofhumandeathin bothdevelopinganddevelopedcountries.AccordingtotheWorld 0925-4005/$–seefrontmatter © 2012 Elsevier B.V. All rights reserved.
A.Qureshietal./SensorsandActuatorsB171–172 (2012) 62–76 63
HealthOrganization (WHO),anestimated 17.5million(30%) of
allglobaldeathsin2005areassociatedwithCVDanditis esti-matedthatby2015,CVDcanbetheleadingcauseofdeathinthe developingcountries[1].Recently,accordingtothenewEuropean cardiovasculardiseasestatistics2008,astaggeringfigureofover 4.3milliondeathsinEuropealoneand2milliondeathsinEuropean UnionarecausedbyCVD,anditisoverallestimatedtocosttheEU economyD192billionayear[2].Theearlyandquickdiagnosisof cardiovasculardiseaseisextremelyimportantandcrucialnotfor onlypatientsurvivalbutalsosavingcostandgreatdealoftimein successfulprognosisofthediseases.Existingmethodsofdiagnosis forCVDrelyheavilyonclassicalmethodswhicharebasedontests conductedincentrallaboratoriesthatmaytakeseveralhoursor evendaysfromwhentestsareorderedtowhenresultsarereceived [3].Thediagnosisof CVDhasbeenbasedontheWHO criteria, wherebypatientsmustmeetatleasttwoofthreeconditions: char-acteristicchestpain,diagnosticelectrocardiogram(ECG)changes, andelevationofthebiochemicalmarkersintheirbloodsamples [3].Although,ECGisanimportantmanagementtoolforguiding therapy[4,5],butitisapoordiagnostictestforCVD,becauseabout halfoftheCVDpatientswhopresenttotheEmergencyDepartment
shownormalornodiagnosticelectrocardiograms, whichmakes
earlydiagnosisofCVDmoredifficult[4–7].Therefore, measure-mentof cardiac markersis critical inassisting the diagnosisof CVD.Amoresensitiveandrapidtechnologyplatformistherefore neededtofulfilltherapiddiagnosisrequirementsinCVD detec-tion.Theelaboration ofbiosensorsis probablyone ofthemost promisingwaystosolvesomeoftheproblemsconcerning sensi-tive,fastandcosteffectivemeasurements[8].Biosensorcanhelpin rapiddiagnosis,providingbetterhealthcareandreducingthe wait-ingtimeforresultsdisseminationwhichishighlystressfultothe patients.Recently,lab-on-achipandmicrofluidicsbased biosen-sortechnologyisreviewedforthedetectionofcardiacmarkers[9]. Thisreviewprovidedinformationoncommerciallyavailableafew point-of-careimmunosensinginstrumentsandchipbased technol-ogyforthedetectionofdifferentcardiacbiomarkers.Inthepresent
paper,wereviewed thedevelopmentsinapplication of
biosen-sorsoverthepast10yearsforthedetectionofcardiovascularrisk assessment.Thisreviewalsosummarizedthefrequentlytargeted
CVDbiomarkers invariousbiosensor platformsandhighlighted
themajorclinicallyrelevantparameters,suchastheirdetection limit/rangeanddesigningofbioassay.
2. Cardiacbiomarkers
CVDisnotasingledisease,butitisagroupofdifferentdisorders thataffectheartandbloodvessels.CVDincludesatherosclerosis conditionthatdevelopswhenaplaquebuildsupinthewallsofthe arteries.Thisplaquenarrowsthearteriesandmakesitdifficultfor bloodtoflowthroughandcausesaheartattackorstroke.CVDcan becausedbyarangeoffactorsanddisordersthatincludegenetic, gender,age,highbloodpressureandcholesterol,diabetes,obesity andoverweight,smokingandstress.ThecausesofCVDaremore diversethatclinicaltestingbecomesincreasinglycomplex.There areanumberofdiseasesassociatedwithCVDthataffectdifferent partsofthebody.Although,greatprogresshasbeenmadeinthe treatmentofthisdisease,currentmedicalknowledgeisunableto effectivelypredictitsrisk.WithregardstopredictingCVDrisk,one oftheactiveresearchareasrecentlyistheuseofdiagnosticand prognosticbiomarkersthatcanbeidentifiedinblood[10].Onthe basisofdiagnosticandprognosticstandpoint,CVDbiomarkerscan becategorizedintopathogeneticandtherapeutictypes.The diag-nosticandprognosticbiomarkersalsoprovidetherapeuticvalue inmedicalapplications.Thevascularwallreleasesmoleculesinto thebloodstreamthatcanreflectthepathologicalprocessestaking
Fig.1.Mostfrequentlystudiedbiomarkersinrelationtothedifferentmechanism involvedinCVDrisk[10].
place.Intheory,theconcentrationsofthemoleculesinvolvedin differentpathologicalprocessescouldbethebiomarkers.However, notallofthesemoleculesaresuitedtothisaimbutshouldfulfill certainconditions[10].
Thereareseveralimportantcharacteristicsthatanidealcardiac biomarkershouldexhibit.Theseinclude:(a)highclinicalsensitivity andspecificity,(b)quickreleaseofbiomarkerinthebloodenabling earlydiagnosis,(c)capabilitytoremainelevatedforlongertimein theblood,and(d)abilitytobeassayedquantitatively[10].Itis diffi-culttoselectaspecificmarkerforthediagnosisofCVD.Therefore,a rangeofbiomarkerscanpotentiallybeanalyzedsimultaneouslyfor theaccuratediseasediagnosis[6,7,10–13].Andersonetal.reported asetof177candidatebiomarkersthatarepotentialplasma mark-ersforCVDandstroke[13].Recently,themostfrequentlystudied biomarkersaresummarizedinrelationtothedifferentmechanisms involved indevelopmentand ruptureof atheroscleroticplaque, suchas endothelialdysfunction,inflammation, oxidativestress, proteolysis,andthrombosis(Fig.1)[10].
Several other cardiac-specific biomarkers have emerged as
strongandreliableriskpredictorsforcoronaryheartdisease,as listedinTable1.Ofwhich,CRPhasbeenthemostfrequentlyused singlebiomarkerforcardiovascularrisk(CVR).TheCVRdefinedby theAmericanHeartAssociation(AHA)andtheCenterforDisease ControlandPrevention(CDC)isregardedaslowriskforaCRP con-centrationbelow1.0mgL−1,moderatefor1.0–3.0mgL−1,andhigh riskforconcentrationsover3.0mgL−1[14].CRPcanriseashighas 1000-foldbecauseofinflammationinducedbyinfectionorinjury, oftenleadingtoCVR[15].Recentresearchsuggeststhatpatients withelevatedbasallevelsofCRPareatanincreasedriskofdiabetes andhypertensionaswellasCVD[15].
Myoglobin,althoughnotaveryspecificmarker,butitisthefirst markerreleasedafterthedamageoccurredtomyocardialmuscle cells.B-typenatriureticpeptide(BNP),cardiactroponinI(cTnI), andCRParereleasedaftermyoglobin,buttheyarespecificmarkers forcoronaryevents.BNPisusefulfortheemergencydiagnosisof heartfailureandfortheprognosisinpatientswithacutecoronary syndromes(ACS)[16].CRPisanimportantprognosticindicatorof CVRandACS.cTnIhasbecomeastandardmarkerforthedetection
64 A. Qureshi et al. / Sensors and Actuators B 171– 172 (2012) 62– 76 Table1
Asummaryofprimaryclinicallyutilizedcardiacbiomarkers,highlightingtheirrespectivecut-offvalues.
Cardiacbiomarker Typeofcardiovasculardiseases involved
Cut-offlevels Specificity(low,
medium,high) MW (kDa) Initial elevation Timeto peak Returnto normal POCtest available TroponinI(cTnI) Detectionofacutemyocardial
infarction(AMI)
0.01–0.1ngmL−1 High 23.5 4–6h 12–24h 6–8days Yes
TroponinT(cTnT) DetectionofAMI 0.05–0.1ngmL−1 High 37 4–6h 12–24h 7–10days Yes
Myoglobin EarlydetectionofAMI 70–200ngmL−1 Low 18 1–3h 6–12h 24–48days Yes
C-reactiveprotein(CRP) Earlydetectionof inflammation/cardiacrisk factor
<103ngmL−1lowrisk
1–3× 103ngmL−1intermediaterisk >3–15×103ngmL−1highrisk(nodefinitive)
High 125 ND ND ND Yes
CreatinekinaseMBsubform (CK-MB)
EarlydetectionofAMI 10ngmL−1 Medium 85 4–6h 12–24h 3–4days Yes
B-typenatriureticpeptide (BNP) Acutecoronary syndromes/diagnosisofheart failure/ventricularoverload High 3.4 ND ND ND Yes N-terminalpro-B-type natriureticpeptide (NT-proBNP) Acutecoronary syndromes/diagnosisofheart failure/ventricularoverload 0.25–2ngmL−1 High 8.5 ND ND ND Yes
Myeloperoxidase(MPO) Detectionofinflammation PatientswithelevatedMPOlevels >350ngmL−1stratificationrisk
Medium 150 ND ND ND Yes
Heartfattyacidbinding protein(H-FABP)
Myocardialnecrosis PatientswithelevatedH-FABPlevelselevated ≥6ngmL−1stratificationrisk Low 15 2–3h 8–10h 18–30h Yes TNF-␣ Inflammation/cardiacrisk factor <0.0036ngmL−1lowrisk ≥0.0036ngmL−1highrisk ND ND ND ND ND ND
Interlukin-6(IL-6) Inflammation/cardiacrisk factor Low<0.0013ngmL−1 Mid0.00138–0.002ngmL−1 High>0.002ngmL−1 ND ND ND ND ND ND Fibrinogen Low<3.58×106ngmL−1 Mid3.58–4.20×106ngmL−1 High>4.20×106ngmL−1 ND ND ND ND ND ND
A. Qureshi et al. / Sensors and Actuators B 171– 172 (2012) 62– 76 65 Table2
Cardiacmarkersdetectionondifferenttransductionplatformsandtheirdetectionrangereportedintheliterature.Thetimerequiredforthedetectionofcardiacmarkersusingthemethodslistedintableisestimatedtobewithin ∼20minto1h.
Target/biomarker Transductionplatform Detectionrange Assaytype Reference
Opticalbiosensors CRP
Myoglobin cTnI
Fluorescence CRP-LOD– 30ngmL−1 Microfluidic
with multianalyte
[22]
NF-␣,IL-8,IL-6,IL-4,IL-2␥,IL-2,IL-1b, IL-12,IL-10,interferon-gamma (IFN-␥)
Fluorescence Allcytokine0.01–10ngmL−1
LOD– 0.01ngmL−1 Multianalyte [23]
IL-6 Fluorescenceusinglabelednanoparticles 0.02–1.25ngmL−1
LOD– 0.007ngmL−1
Singleanalyte [24]
TNF-␣ Fluorescence 1ngmL−1 Singleanalyte [25]
TNF-␣ Fluorescence 1–0.0016ngmL−1 Singleanalyte [93]
CRP Fluorescence 0.1ngmL−1 Singleanalyte [26]
CRP Fluorescence 10–105ngmL−1 Singleanalyte [27]
CRP Fluorescence 20ngmL−1 Singleanalyte [28]
cTnI Fluorescence 0.1–100ngmL−1 Singleanalyte [29]
Heart-typefatty-acidbindingprotein (H-FABP) CRP ELISA H-FABP-LOD<6× 103mL−1 CRP-LOD<103ngmL−1 Multianalyte [30] cTnT ELISA 0.1–100ngmL−1
LOD– 0.027ngmL−1 Singleanalyte [31]
Myoglobin ELISA 20–230ngmL−1
LOD– 16ngmL−1
Singleanalyte [32] cTnI ELISA RattroponinIprotein(5.3× 104to5.3×106ngmL−1)orhuman
troponinIprotein(4.3× 104to4.3× 106ngmL−1) Singleanalyte [33]
CRPandIL-6 Fluorescence/colorimetric 1–1000ngmL−1
LOD-CRP–1ngmL−1 Microfluidicwith
multianalyte
[35]
Myoglobin,creatinekinasemb(CKmb), cTnI,andfattyacid-bindingprotein (FABP) Chemiluminescence Myoglobin– 1.2ngmL−1 CKmb–0.6ngmL−1 TnI–5.6ngmL−1and FABP– 4ngmL−1 Multianalyte [37] CRP Chemiluminescence 50–5× 104ngmL−1 LOD–12.5ngmL−1 Multianalyte [34] CRP Chemiluminescence 10to104ngmL−1 Microfluidic withSingle analyte [14]
cTnI Colorimetric;PDMS–goldnanoparticlecompositefilm 104to5×103ngmL−1 Singleanalyte [36]
Low-densitylipoprotein(LDL) Electrochemiluminescence 0.025–16ngmL−1 LOD–0.006ngmL−1
Singleanalyte [38]
cTnI Electrochemiluminescence 0.002ngmL−1 Singleanalyte [39]
Metalloproteinase(MMP)-2 SPR LOD– 0.036ngmL−1 Single [42]
CRP SPR LOD– 103ngmL−1 Singlemarker [43]
B-typenatriureticpeptide(BNP) SPR 5pgmL−1to100ngmL−1
LOD– 0.005ngmL−1 Microfluidic withsingle analyte [48] CRP SPR 2–5×103ngmL−1
LOD–103ngmL−1 singleanalyte [44]
CRP SPR 1ngmL−1to5× 104ngmL−1 LOD– 1ngmL−1 Singleanalyte [45] cTnT SPR 0.03upto6.5ngmL−1 LOD– 0.01ngmL−1 Singleanalyte [49] cTnT SPR 0.05and4.5ngmL−1
66 A. Qureshi et al. / Sensors and Actuators B 171– 172 (2012) 62– 76 Table2(Continued)
Target/biomarker Transductionplatform Detectionrange Assaytype Reference
MyoglobinandcTnTI SPR LOD–below1ngmL−1 Multianalyte [52]
CRP SPR 104ngL−1 Singleanalyte [46]
TNF-␣ SPR 1–2×103ngmL−1 Singleanalyte [53]
CRP SPR 0.1–200ngmL−1 Singleanalyte [47]
cTnT SPR 100ngmL−1 Singleanalyte [51]
cTnT SPR 0.068ngmL−1 Singleanalyte [18]
Myeloperoxidase(MPO) SPR 50ngmL−1 Singleanalyte [54]
BNP,cTnI,myoglobin,andCRP Opticalfiber BNP–0.1ngmL−1
IcTnI–7×10−3ngmL−1
MG–70ngmL−1
CRP–700ngmL−1
Multianalyte [16]
CRP Opticalfiber 5–12.5×103ngmL−1 Singleanalyte [55]
Nervegrowthfactor(NGF) Opticalfiber 1–200ngmL−1 Singleanalyte [57]
IL-6 Opticalfiber LOD–0.12ngmL−1 Singleanalyte [58]
CRP Opticalfiber/fluorescence/evanescent 4.4×10−11to2.9×10−9ngmL−1and1.3×10−10to
2.29×10−8ngmL−1
Singleanalyte [56] CRP Photonicmicroringresonator 3×10−6ngmL−1 Singleanalyte [59]
IL-6 Photoniccrystalresonant 0.001–0.01ngmL−1 Multianalyte [60]
cTnT Optomagnetic 0.0039–3.9ngmL−1(1–1000pM)
LOD–0.0117ngmL−1
Multianalyte [61]
cTnT Optomagnetic 0.03ngmL−1 Singleanalyte [62]
IL-6 Surfaceacousticwave 20ngmL−1to2×103ngmL−1 Singleanalyte [19,20]
CRP Resonantacousticprofiling 0–231ngmL−1
Directassay– LOD20ngmL−1 Sandwichassay– 3ngmL−1
Singleanalyte [63]
Electrochemicalbiosensors
Myoglobin Faradaic,Impedance/interdigitatedelectrodes 100ngmL−1 Singleanalyte [64]
CRP Faradaic,CNTmodifiedcarbonelectrodes 0.5–500ngmL−1
LOD–0.5ngmL−1
Singleanalyte [65] CRP Faradaic,impedance/goldelectrodes to20ngmL−1
LOD–0.1ngmL−1
Singleanalyte [66] Lipoprotein-associatedphospholipase
A(2)
Faradaic,iridium-modifiedcarbonelectrodes 0–150UmL−1 Singleanalyte [72] IL-6 Faradaic,goldelectrodes LOD–4.1×10−3ngmL−1 Microfluidic
withsingle analyte
[94]
Low-densitylipoprotein(LDL) Faradaic,impedance/AuNPs-AgCl@PANI-modifiedglassycarbon electrode
LOD–3.4×10−3ngmL−1 Singleanalyte [73] CRP Faradaic,magneticbeadswithcarbonelectrodes LOD–5.4×10−11ngmL−1 Singleanalyte [67]
cTnT Non-Faradaic,impedance/Alinterdigitatedelectrodes 0.07–6.83ngmL−1 Multianalyte [81]
CRP Faradaic,impedance/goldelectrodes 1.15×10−13to1.15ngmL−1
LOD–6×10−14ngmL−1
Singleanalyte [68] CRP Faradaic,diamondlikecarbonelectrodes LOD–10−4ngmL−1 Singleanalyte [69]
IL-6 Faradaic,goldinterdigitatedelectrodes LOD–0.005ngmL−1 Singleanalyte [95] CRP Non-Faradaic,goldinterdigitatedelectrodes 25–800ngmL−1 Singleanalyte [70] CRPandMPO Faradaic,Impedance/iridiumoxidemodifiedelectrodes LOD-CRP–1ngmL−1andLOD-MPO–0.5ngmL−1 Multianalyte [71]
cTnIandCRP Faradaic,poly(dimethylsiloxane)–goldnanoparticlecomposite microreactors
cTnI–0.01ngmL−1andCRP–0.5ngmL−1 Microfluidic
withsingle analyte
[74]
CardiacbiomarkerN-terminal pro-B-typenatriureticpeptide (NT-proBNP)
Faradaic,nanostructuralgoldandcarbonnanotubescomposite 0.006ngmL−1 Singleanalyte [96]
CRP Non-Faradaic,impedance/Alinterdigitatedelectrodes 0.1ngmL−1 Singleanalyte [82]
cTnT Faradaic,streptavidin-microspheremodifiedscreenprinted electrodes
A.Qureshietal./SensorsandActuatorsB171–172 (2012) 62–76 67 Table 2 (Continued ) Target/biomarker Transduction platform Detection range Assay type Reference CRP Faradaic, nanotextured polystyrene (PS) electrode 0.001–10 3ng mL − 1 Single analyte [75] Myoglobin Faradaic, nanoparticles modified electrodes 17.8–1780 ng mL − 1 Single analyte [98] cTnI Faradaic, gold nanoparticles modified ITO electrodes 1–100 ng mL − 1 Single analyte [76] Myoglobin Faradaic, nanoparticles modified Fe graphite electrodes 5 ng mL − 1in human plasma Single analyte [77] cTnT Conductance, metal-oxide semiconductor-compatible silicon nanowire In buffer solution – 10 − 6ng mL − 1 Undiluted human serum –3 × 10 − 6ng mL − 1 Single analyte [78] Myoglobin Polyaniline nanowires/conductance 1.4 ng mL − 1 Single analyte [79] cTnI SnO 2 nanobelt FET sensor/IV curve ∼ 2 ng mL − 1 Single analyte [80] Magnetic biosensors CRP and creatine kinase isoenzyme MB fraction (CKMB) Magnetometer sensing coil/coated paramagnetic particles CKMB – 1–15 ng mL − 1 CRP – 1–40 ng mL − 1 LOD-CKMB –2 ng mL − 1 LOD-CRP –3 ng mL − 1 Multianalyte [86] CRP Streptavidin-coated magnetic beads and avidin-coated polystyrene microspheres/beads 10 − 5ng mL − 1 Single analyte [87] CRP Solid phase (polyclonal anti-CRP conjugated silica microparticles), labeling agent 2 × 10 − 10 ng mL − 1 Single analyte [88] CRP Antibody conjugated dextran iron oxide nanoparticles (70 nm) as superparamagnetic labels 3 × 10 3ng mL − 1 Single analyte [89] CRP Magnetometer sensing coil/coated magnetic beads 25 ng mL − 1to 2.5 × 10 3ng mL − 1 LOD –2 5 ng mL − 1 Single analyte [90] cTnI Magnetic tweezer/magnetic beads 0.368 ng mL − 1 Single analyte [20]
ofacutemyocardialinfarction(AMI).Duringtheheartinfarction, thetroponinT(TnT)isimmediatelyreleasedtothebloodstream, abiosensorabletomonitorthisbiomarkerinashorttimecould improvepatientcarebyallowingadefinitediagnosisofmyocardial infarctioninrealtime.
Elevatedconcentrationsofthesecardiacmarkersinserumare
associated with recurrent CVD events and higher death rates.
Simultaneousquantificationofthesebiomarkersallowsclinicians todiagnoseCVDquicklyand/ortoaccuratelydesignapatientcare strategy.Afastandreliabledetectionoftheseproteinswillalsohelp medicalprofessionals differentiate diseasesamong those show-ingsimilarsymptoms.Theclinicallysignificantsensingrangesof myoglobin,BNP,cTnI,andCRPareextremelylow(pMtonM),and therefore,assaymethodsforthesebiomarkersneedtobehighly sensitive.Inrecentyears,differentbiosensorplatformshavebeen designed for detection of available cardiac disease biomarkers. Here,wesummarizedthemostprominentlyusedcardiac biomark-ersandtheirdetectionbydifferentbiosensorplatforms.
3. BiosensorsforCVDs
Abiosensorisadevicedesignedtodetectandquantifytarget moleculesthatiswidelyusedasapowerfulanalyticaltoolin med-icaldiagnostics[8].Itincludesproteinsdetection,nucleicacidsor monitoringantigen–antibodyinteraction.In principle,it is gen-erallyfabricatedbyimmobilizing abiological receptormaterial, forinstance,antibody,DNA,orRNAonthesurfaceofasuitable transducerthatconvertsthebiochemicalsignalintoquantifiable electronicsignals.ArangeofsensorshasbeendevelopedforCVD markersdetection.Here,asummaryofavailabledifferentsensor
platformsforthe detectionofCVDand mostprominentlyused
CVDmarkers is presentedand summarized in Tables 1 and2).
Thissignalcanbeelectrochemical,optical,masschange (piezo-electric/acousticwave)ormagneticinnature[17–20].
3.1. Opticalbiosensors
Opticalbiosensorsareconsideredasthemostsensitive
tech-niques that are based on the change in the phase, amplitude,
polarization,or frequency of theinput lightin response tothe biorecognitionprocesses.Opticalbiosensorcanbeclassifiedinto followingcategories,suchascolorimetric,fluorescence, lumines-cence,surfaceplasmaresonance(SPR)andfiberoptics/bio-optrode basedbiosensors.In colorimetricand fluorescence-based detec-tion, either target or biorecognition molecule is labeled with chromogenic/fluorescenttag,suchasdyes.Thechangeinthe inten-sityofthecolor/fluorescencesignalindicatesthepresenceofthe targetmolecules,whichisextremelysensitive,withthedetection limitdowntoasinglemolecule[21].Inlabel-freedetection meth-ods,thetargetmoleculesarenotlabeled,andaredetectedintheir nativeforms.Thistypeofdetectionisrelativelyeasyandcheap toperformthatalsoallowsquantitative/kineticmeasurementof molecularinteractionsbysurfaceplasmaresonanceoropticalfiber biosensors[reviewedin21].Although,opticalbiosensorsarehighly sensitive,theyarebulky,expensiveandrequirededicated person-neltoperformthetests.Additionally,colorimetric,fluorescence andluminometrictypeofsensorsrequiredifficultlabeling proce-duresthatdependonindirectindicatorbasedsignalschemes. 3.1.1. Fluorescencebasedbiosensorsforcardiacmarkers detection
Arangeofdifferentcardiacbiomarkershasbeendetectedon differentopticalbasedbiosensorplatforms.Currentresearchhas
morefocusedonthemethodologiesfor multi-analytedetection
andtheintegrationofimmunosensorsintochipbased
68 A.Qureshietal./SensorsandActuatorsB171–172 (2012) 62–76
based immunosensor is designed for the multianalyte cardiac
markerdetectionthat includesCRP,Mband cTnI [22].
Fluores-cencemicrosphereimmunoarraybasedplatformwasemployed
usingantibodysandwichassayforthedetectionofcytokinessuch as,TNF-␣,IL-1,IL-6,andIL-8[23].Insuchtypeofmulti-analyte detectionplatform,simultaneousdetectionofmultiplemarkers, therapidavailabilityofresultsandthesimplicityofthe experimen-talsetupmakesthisapproachanoptionforpoint-of-caretesting
devices.AnovelsandwichfluoroimmunoassayforIL-6detection
withfunctionalizedRubpy-encapsulatedfluorescentcore–shell sil-icananoparticleslabelshasbeendeveloped [24].Here,IL-6was measuredbasedonthespecificinteractionbetweencapturedIL-6 antigensandfunctionalizedfluorescentcore–shell nanoparticles-labeledanti-IL-6antibodies.Thereportedmethodofferedpotential advantages of sensitivity, simplicity and reproducibilityfor the determinationofIL-6inserumsamples.Inanotherapproach,a
20-foldenhancementofemittedlightbyfluorescentmoleculeswas
achievedbyemployingphotoniccrystalscoupledwithcolloidal quantumdotemittersthatresultedinhighsignal-to-noiseratiofor cardiacmarker(TNF-␣)detection[25].Improvementsinphotonic crystalperformancecanbemadetofurtherlowerthedetection limitsthatisusefulindetectingnewbiomarkersassociatedwith thedisease.WhileJungetal.developedacompetition-basedtagged internalstandardassaytosensitivelymeasurethesub-nanogram levelsofCRPinhumanserum[26].
Inanotherapproach,onchipsandwichassayforthedetection ofCRP,whichissimilartomicroarraysusingfluorescenttagsis
reported[27]. Thismethodwassuperior interms of CRP
mea-suringrange,butlowerindatareproducibilitycomparedtoother methods.Arelativelynewmethodwithoutemployingantibodyfor theestimationofCRPinserumwasdeveloped[28].Thismethod
centered on the variation of fluorescence intensity of
copoly-mers,containingafluorophore(Fluoreseinamineisomer1)anda CRPligand,O-phosphorylethanolamine(PEA).Here,thecopolymer
containing fluorophore quenches the fluorescence in the
pres-enceofPEAthat eventuallydeterminestheCRP levels.Further, anantibody-taggedfluoro-microbeadguidingchipintegratedwith microchannelsforsimultaneouslyfiveidenticaltestswas devel-opedforthedetectionofcTnIinhumanplasma[29].Design,picture andschematicdiagramofthesandwichassayonfluoro-microbeads guiding chipis shown in Fig. 2. This chip contained five gold functionalsurfacestoperformfiveidenticaltestssimultaneously throughcTnIantibody-linkedfluoro-microbeadsasthedetection component(Fig.2c:System1).Toamplifytheantigen–antibody bindingsignal,theavidin–biotinaffinityinteractionwasemployed. Afterimmobilizationofthecaptureantibodyandbindingofthe target antigen cTnI, biotin-conjugated cTnI detection antibody wasloadedintothechipandreactedfor30min.Afterwashing
the micro-channels withPBS, 0.005% avidin-conjugated
fluoro-microbeadswereinjected(Fig.2c:System2).Thentheamplified signalfromtheavidin–biotinreactionwasdetectedundera
flu-orescencemicroscope.Humanplasmasampleswerespikedwith
varyingconcentrationofcTnIfortestingintheSystems1and2 forsensitivelydetectingcTnIincomplexmixtures(Fig.2c). How-ever,additionofmicrofluidiccomponentscanminimizeprocedural stepssuchassamplepreparations.
3.1.2. Luminescenceandcolorimetricmethodsforcardiacmarker detection
Recently,luminescencebasedbiosensingplatformsand
meth-ods were examined for the early cardiac biomarker detection
[14,30–35].Theluminescencemethodsarebroadlyclassifiedinto twotypes,namelychemiluminescenceandelectroluminescence.In chemiluminescence,luminescentsignalisgeneratedbytheaction ofanenzymaticantibodylabelinthepresenceofluminogenic sub-stratewhileinthecaseofelectroluminescence;theluminescent
signalisinducedfollowinganelectrontransferreactionofa lumi-nescentcompoundimmobilizedneartheproximityofanelectrode surface.
Acardiac chipwasdeveloped byexploitinga geometrythat
allowsforisolationandentrapmentofsinglepolymericspheres inmicro-machinedpits, whileprovidingtoeachbeadtherapid introductionofaseriesofreagents/washesthroughmicrofluidic structures.Opticalsignalsderivedfromsinglebeadsareusedto completeimmunologicaltestsforthesimultaneousdetectionof thecardiacriskfactors,suchasC-reactiveproteinand interleukin-6(IL6)inhumanserumsamples[30].Inanotherapproach,PDMS
surfacewasdirectlymodified witha randomlyorientedprotein
antibodyspotsduringthepolymerization[31]reliesontheability ofthePDMSpolymertoentrapmacromoleculesatitssurfacewhile
thepolymerization process occurs.This methodwasemployed
todevelopasensitivesandwichimmunoassayforCRPdetection.
The main advantage of this concept is to be able to combine
boththemacromoleculeimmobilization,withouttheneedof addi-tionalchemicals,andtheeasy3Dstructuredplatformforsandwich assays.CRPmeasurementswerealsoperformedbyusingan inte-gratedmicrofluidicchipincorporatedwithmagneticbeads[14].
ThemagneticbeadscoatedwithCRP-specificDNAaptamers
rec-ognize, purifyand enrich thetarget CRP from thesample that
wassandwichedbybindingwithacridiniumester-labeled
CRP-antibodyforchemiluminescencesignal.Thedevelopmentofthis microfluidicsystemispromisingforfast,accurate,andsensitive detectionofCRP.
Currently,electroluminescence(ECL)andcolorimetric biosens-ingplatformshavebeenlessextensivelyexaminedbyresearchers forcardiacmarkersdetections[34,35].BasedontheECLofCdS nanocrystals,anovellabel-freeECLbiosensorforthedetectionof low-densitylipoprotein(LDL)hasbeendevelopedbyusing self-assemblyandgoldnanoparticleamplificationtechniques[34].The
LDL concentration was measuredthrough the decrease in ECL
intensityresultingfromthespecificbindingofLDLtoapoB-100 (ligand of LDL receptor).The ECLpeak intensity ofthe
biosen-sor decreased linearly with LDL concentration in the range of
0.025–16ngmL−1withadetectionlimitof0.006ngmL−1.
Colorimetric method using poly(dimethylsiloxane)
(PDMS)–gold nanoparticles (AuNPs) composite film as basis
withsilverenhancementhasbeenreportedforthedetectionof cardiactroponinI(cTnI)[30,32].InthisstudyPDMSmaterialwas usedbecauseitsadvantages.Firstly,thepolymermatrixofPDMS
couldprotectAuNPsfromaggregation,sothisPDMS–AuNPs
com-positefilmcouldbewellstoredat4◦Cforseveralmonthswhich enhancedthestabilityandprolongshelf-lifeofthedevice. Exper-imentalprocedureforsilverenhancementcolorimetricdetection ofcardiactroponin I,isshownin Fig.3.ForcTnI detection,the monoclonalantibodyagainstcTnIwasfirstlyimmobilizedonthe
PDMS–AuNPscompositefilm,followedbyblockingsolutionand
cTnIundertheprocedureasshowninFig.3.Themechanismof goldbasissilverenhancementmethodisthatAuNPsplayarole ofcatalystduringreactionsofsilverreduction,andthiscatalytic abilitycouldbeinhibitedwhentherewereproteinscoveringthe surfaceofAuNPs,whichinfluencetheamountofreductionsilver metal,whichledtothecolordifferenceinthereactionmixture. Thisinhibition effect couldbedistinct due todifferentspecies, quality and/or quantity of covering proteins. The results were
consistentwiththatfromEnzyme-linkedimmunosorbent assay
(ELISA),whichisconsideredtobeoneofthemostsensitiveand standardtechniques[30].
3.1.3. ELISAbasedmethodsforcardiacmarkerdetection
ELISA typically relies on optical properties of chromogenic reporterstogiverisetoasensitivesignalintoeither colorimet-ric,fluorogenicorluminescentforms,respectively.ELISAhasbeen
A.Qureshietal./SensorsandActuatorsB171–172 (2012) 62–76 69
Fig.2.(a)Design,(b)photographand(c)schematicdiagramofthesandwichimmunoassayonthefluoro-microbeadsguidingchip[29].
widelyutilizedforthedetectionofcardiacmarkers[36–39].Darain etal. reported a simple and highlyefficient sandwichELISA in
whichin-channelpolystyrenewasimmobilizedwithantibodies,
andfabricatedplasticbasedmicrofluidicchipforthedetectionof myoglobin[38].Cardiacmultianalytes,suchasheart-type fatty-acidbindingprotein(H-FABP)andC-reactiveprotein(CRP)were detectedin asimple, rapidand“digital-style” semiquantitative lateralflowassay usinganin-houseELISA[36].Thisassaywas abletosimultaneouslydetectcardiacmarkers andenables pre-dictingearlyCVDrisk,simplybycountingthenumberofredlines inthetestwithoutanyexpensivereadinginstruments.Choetal. reportedachemiluminometricbiosensorsystemforpoint-of-care
testingusinganimmuno-chromatographicassaycombinedwith
anenzyme(e.g.,horseradishperoxidase)tracer,whichproduces alightsignalmeasurableonasimpledetector[37].Inthiswork, toenhancethesensitivity,biotin–streptavidincapturetechnology
wasemployedinpreparinganimmuno-stripthatwasthen
inte-gratedontothechipinordertogeneratetheELISA-on-a-chip(EOC) biosensor,andsensitivelydetectedcTnIaslowas0.027ngmL−1 detectionlimit.However,furtherminiaturizationofsensor plat-formhastobeprovidedwithoutsacrificingthedetectioncapability.
A relatively different method involving specific peptide
sequence of cardiac marker detection has been reported. This
methodutilizespolyvalentphagedisplaytoisolateuniquelinear peptidemotifswhichrecognizeboththehumanandrathomologs oftroponinI[39].ThepeptidespecificforhumantroponinIhasa
sequenceofFYSHSFHENWPS,whileFHSSWPVNGSTIfortherat
tro-poninIthatlaterdetectedbyELISA.Itwasshownthatthebinding affinitiesofthephagedisplayedpeptidesweredecreasedbythe presenceofcomplextissueculturemedia,andtheadditionof10% calfserumfurtherinterferedwiththebindingofthetargetproteins.
Inthisstudy,kineticindirectphageELISAsrevealedthatboth tro-poninIbindingpeptideswerefoundtohavenanomolaraffinities forthetroponinproteinswhileattachedtothephageparticles[31]. Thesenewpeptidesmayhavepotentialutilityinthedevelopment ofnewclinicalassaysforcardiacinjuryaswellasinmonitoringof cardiaccellsgrowninculture.
3.1.4. SPRbasedbiosensorforcardiacmarkerdetection
Surfaceplasmonresonance(SPR)isauniqueoptical
transduc-tionmethod,whichhasbeencommerciallyemployedforoptical
biosensors [40]. SPR biosensors exploit special electromagnetic waves–surfaceplasmontoprobechangesintherefractiveindex (RI)atsurfacesofmetals.SPRbiosensorscanthereforebeusedto monitortheinteractionbetweenananalyteanditsbiospecific ele-mentimmobilizedonthemetalsurfacewithouttheuseoflabels. ThebasicSPRapparatusisreferredtoastheKretschmanprism arrangement[40].
In the SPR, a thin film of metal (usuallya 400–500 ˚A thick goldorsilverfilm)iscoatedontheprismontowhicha biosens-inglayeriscoatedbyimmobilizationofbiorecognitionelements
(proteins/antibodies/DNA/RNA). When an incident light of an
appropriatewavelengthstrikesonthedielectric–metalinterface ataparticularangle,itinducesSPRphenomenonatthemetal sur-face.Depending upon thethicknessof amolecularlayeratthe metalsurface,SPRresultsina gradedreductionintheintensity ofthereflectedlight[41].Thus,onecanobserveasharpminimum oflightreflectancewhentheangleofincidenceisatthisproper resonantangle.Sincetheresonanceangledependsonseveral fac-tors(e.g.,thewavelengthoftheincidentlight,themetal,andthe natureofthemediaincontactwiththesurface)thenatureofthe mediacanbesensedbymeasuringthisresonanceangle.Further,
70 A.Qureshietal./SensorsandActuatorsB171–172 (2012) 62–76
Fig.3.ExperimentalprocedureforsilverenhancementcolorimetricdetectionofcardiactroponinI:(a)PDMSchipwithHAuCl4solution,(b)photoofPDMS–AuNPscomposite filmand(c)schematicdiagramforcolorimetricdetection[32].
inadditiontotheprismcoupling,SPRsensorscanalsobebasedon opticalfibersorintegratedopticalwaveguides.Biomedical appli-cationstakeadvantageoftheexquisitesensitivityofSPRtotheRI ofthemediumnexttothemetalsurface,whichmakeitpossible tomeasureaccuratelythecapturingoftargetbiomarkersonthe metalsurface[41].
SPRbasedsensingplatformshavebeenextensivelystudiedand appliedforthedetectionofcardiacmakersthatinclude metallo-proteinase(MMP)-2[42],CRP[42–47],B-typenatriureticpeptide (BNP)[48],cTnT[49–51],myoglobin[52],cTnI[18,52],TNF-␣[53],
andMPO[54].Aone-stepSPRbasedsandwichassayformatfor
thequantificationofMMP-2hasbeenreportedwithBiacore2000, usingcolloidalgoldwithaparticlediameterofabout20nmfor signalamplification[42].Thereportedassayhasadetectionlimit
belowtheleveldocumentedbyestablishedmethodsforMMP-2
detection(Table2).Amodified CRP(mCRP)wasdetectedusing SPRbasedsensorbecausemCRPisregardedasapowerfulinducer
thanpentamerCRP(pCRP),therefore,mCRPisconsideredtobe
importantindicatorforassessingtheriskofdevelopingCVD.Three
monoclonal antibodies (Mabs), C8,8D8, and 9C9, were
immo-bilizedona proteinGlayerfor subsequentCRP detection.It is
shownthatdetectingpCRPusingMabC8,theSPRbioassay
pro-videdsufficientsensitivity toevaluatewhetheror notapatient isatriskofdeveloping CVD[43].Aminiaturizedimmunosensor is designed todeterminetracelevelsof cardiacmarker,B-type natriureticpeptide,usingamicrofluidicdevicecombinedwitha portableSPRsensorsystemthatshowedadetectableconcentration range5pgmL−1to100ngmL−1bymonitoringtheSPRangleshift, whichcoverstherequireddetectionrangefortheB-typenatriuretic peptideconcentrationsfoundinblood[48].
A carboxymethyldextran hydrogel planar sensor chip was
employedforthedetectionofcTnT[49].Thissensorwascoupled
to the SPR AutoLab Spirit® system (Eco Chemie, Netherlands)
attachedat thebottom of theprism withcedar oiland a flow
cellwasinstalledonthechip.Inthiswork,thewell-established
biotin–streptavidinchemistry wasusedtoincrease theamount
ofimmobilizedantibodiesbyhighlyspecificinteractionbetween thebiomolecules.Further,twomyocardialinfarctionbiomarkers, myoglobin andcardiac troponinI,were quantifiedatbiological
levels and in undiluted serum without sample pretreatment
using SPR sensors and the detection limits for both markers
A.Qureshietal./SensorsandActuatorsB171–172 (2012) 62–76 71
sensorsurface for CRPdetection is developedin which
poly(3-(2-((N-succinimidyl)succinyloxy)ethyl)thiophene) (P3SET), a
poly(thiophene)withpendantNHSestergroupswaspreparedand usedfortheformationofaself-assembledmonolayer(SAM)ona goldsurfaceforCRPbiosensorfabrication[46].Improvementsin
thefabricationofSPRsensorshavebeenattemptedand
demon-stratedtheenhancementofsensitivityforanSPRinterfaceusing
Kretschmann’s configuration, through nano-gratings combined
with nano-patterned immobilization of surface bioreceptors
[53]. This configuration resulted in high electromagnetic field intensityfor thedetectionofTNF-␣.Inaninteresting approach
Escherichiacoli outer membrane withautodisplayed Z-domain
wasusedasamolecularrecognitionlayerforthedetectionofCRP usingSPRbiosensor[47].Fromthisstudy,theLODofSPRbiosensor
wasestimatedtoimprovemorethan100-foldcompared tothe
SPR biosensor with the antibody-layer by physical adsorption.
ThemainproblemofSPRsensorcouldbemainlybecauseofits
foulingability, lowaffinityand specificitythat affectsensitivity ofbiosensingtransducers.Onestudyattemptedtoimprovethese
factorbyutilizingamodifiedmethodofSAMformationusinga
homogeneousmixtureofoligo(ethyleneglycol)(OEG)-terminated
alkanethiolateandmercaptohexadecanoicacid(MHDA)ongold
surfacefordetectingcardiactroponinT[51].
3.1.5. SPRbasedfiberopticbiosensorsforcardiacmarker detection
Recently,SPRbasedonopticalfibershavebeenutilizedforthe detectionofcardiacspecificbiomarkersBNP[55],cTnI,Mg[55], CRP[16,55,56],nervegrowthfactor(NGF)[57],andIL-6[58].The detectionprincipleofalargeclassoffiberopticbiosensors(FOBs)is basedontheprincipleoftotalinternalreflectionfluorescence(TIRF) [55,56].Anevanescentwaveformswhenthelightpropagatesinthe fibercoreatanincidentanglegreaterthanthecriticalangle,which resultsintotalreflectionofincidentlight.Thisisduetothechange inhightolow refractiveindicesofthemedia,respectively.The evanescentwavecanbeusedtoexcitefluorophoresofthe immo-bilizedfluorescentlylabeledspecies,resultingintheevanescent waveexcitationoffluorescence.Theintensityoftheevanescent wavedecaysexponentiallyalongthedirectionperpendicularto theinterface(typicalpenetrationdepthof100–200nmrange).This characteristicenablesreal-timemonitoringofthekinetics behav-iorbetweenreceptorsandtargetanalytesduringthemeasurement. Moreover,thedetectedsignalintensityofanFOBdependsonthe amountoffluorophoresinthecapturedtargetmolecules,andthe relationshipbetweenthesignalresponseandtheconcentrationof targetmoleculescanbedeterminedaccurately.Studiesinvolving useofnovelFOBhasbeenreportedtodeterminethe mCRP-anti-CRPbindingkinetics[55].Inthis FOBplatformthefluorescence signalexcitedbyevanescentwaveinthenearfieldregionoffiber coresurfaceandaphotomultipliertube(PMT)facingthefiberwall, andperpendicularlytothefiberaxiswasfixedthatenabledto col-lectthefluorescencesignalmoreefficientlyintheFOB.Thereported FOBwascapableofdetectingCRPatphysiologicalconcentration.
Sandwichimmunoassay hasalsobeen performedusing FOB in
whichfiberopticprobewasusedasadipprobeforthesensitive detectionofIL6(5pMor0.12ngmL−1)[58].
3.1.6. Otheropticalbasedbiosensors
Refractive index based other optical biosensors have been
reported.Theseinclude,resonantbiosensortodetectCRPmarkers andsiliconphotonicmircroringresonatorsusingoptofluidic plat-formtodetectIL-6[59,60].Optomagneticbiosensorshavebeen appliedforthedetectionofcardiactroponincTnTandI[61,62]. Whileaprototypehandhelddevicewasdevelopedactuated mag-netic nanoparticle labels (500nm)bound tothe sensor surface
viaasandwichimmunoassayfor theoptomagnetic detectionof
cTnT [61]. Optomagnetic detection of cardiacbiomarkers using functionalizedmagneticparticleshashighpotentialinanalytical performanceand ease-of-usethat issuitable forapplicationsin point-of-carediagnosis[61,62].
3.2. Acousticbiosensorsfordetectionofcardiacmarkers
Acousticbiosensorsallowlabel-freedetectionofbiomolecules andanalysisofbindingeventsmainlyforCRPandinterleukin fam-ilyofproteins.Thedetectionofthesemarkersisbasedonthemass ofcapturedanalytebyanimmobilizedreceptormoleculeonthe quartzcrystalresonatorsurface,whichisproportionaltothe res-onantfrequency[63].Theirdetectionhasalsobeendemonstrated byusingsurfaceacousticwavebasedbiosensorplatform[19,63]. ItwasfoundthatZnO/SiO2/SiLovemodesurfaceacousticwave (SAW)biosensorforthedetectionofinterleukin-6(IL-6)hasbeen reportedwithhighsensitivity.Thissensorconsistedoffully inte-gratedCMOSSichipsforportablerealtimedetectionofinterleukin familyofproteinsinhumanserum[19].However,performances ofacousticbiosensorsdependonfactors,suchasitstemperature dependence,attenuationandtheRaleighwavesaresurface-normal waves,depositionofnon-specificmolecules,dustorother contam-inantsonsensorcrystalsmaycontributetolargebackgroundsignal noisesthatmayaffectsensitivityandspecificityofthesensors. 3.3. Electrochemicalbiosensors
Electrochemicalbiosensorsareaffinitybasedbiosensorswhere theyuseanimmobilizedrecognitionelement(probe)thatbinds thetarget/analytemoleculeselectively.Thedetectionofbinding atargettoprobeinsolutionisgovernedbydetectingchangeata localizedsurfaceintermsofchangeincurrentsand/orvoltages. Based ontheir operating principle,the electrochemical
biosen-sorscanemploypotentiometric,amperometricandimpedimetric
transducersconvertingthechemicalinformationintoameasurable amperometricsignal.Thus,thiscategoryexcludessensorswhich require light (e.g., surface plasmon resonance or fluorescence), mechanicalmotion(e.g.,quartzcrystalmicrobalanceorresonant cantilever),oruseofmagneticparticles.Duetotheirlowcost,low powerandeaseofminiaturization,electrochemicalbiosensorshold greatpromiseforapplicationswhereminimizingsizeandcostis crucial,suchaspoint-of-carediagnostics and bio-warfareagent detection.
Thefirstscientificallyproposedandsuccessfully commercial-izedbiosensorswerethosebasedonelectrochemicalsensorsfor multipleanalytes.Currently,transducers basedon semiconduc-torsandscreenprintedelectrodesrepresentatypicalplatformin electrochemicalbiosensorfordetectionofCVRmarkers. Electro-chemicalbiosensorsforthedetectionofcardiacmarkers,including cardiac troponin I or T (cTnI/T), myoglobin, CRP, Lipoprotein-associatedphospholipaseA,IL-6,LDLandMPOhavebeenpublished inthelast fewyears. Mostnotably,thedetectionof myoglobin wasperformedbyaminiaturizedpoint-of-caresensor,whichwas basedonimpedimetricsensingofcardiacenzymecapturedbyan antibodylayerimmobilizedonaplanargoldelectrodesensor[64].
Gold/Ti-on-glasssubstratehasbeenusedonwhichtheworking
electrodewasimmobilizedwithantibodiesthatareinturn
inte-gratedwithamicrofluidicsystem.Thepresentedbiosensor was
abletodetectthemyoglobinantigenconcentrationof100ngmL−1. Anumberofstudiesondevelopingelectrochemicalbiosensorsfor thedetectionofCRP havebeenpublishedin thelastfew years [65–71].
Athree-dimensionalordered macroporous(3DOM)goldfilm
modifiedelectrodeshavebeenemployedforthedevelopmentof
72 A.Qureshietal./SensorsandActuatorsB171–172 (2012) 62–76
Fig.4. Procedureforpreparationof3DOMgoldfilm[66].
Theprocedure forpreparation of 3DOMgold filmelectrodes is
showninFig.4.
Theelectrodeusedinthissensorwaselectrochemically fabri-catedwithaninvertedopaltemplatethatenabledenhancedsurface
areaofthe3DOMgoldfilmupto14.4timeshighercomparedto
classicalbare flatelectrodes.The presentedCRP immunosensor
wasdevelopedbycovalentlyconjugatingCRPantibodieswith
3-mercaptopropionicacid(MPA)onthe3DOMgoldfilmelectrode
andschematicispresentedinFig.5.TheCRPconcentrationwas
measuredthroughtheincreaseof impedancevaluesinthe
cor-respondingspecificbindingofCRPantigenandCRPantibody.This enhancedsensorsurfaceareaof3DOMgoldfilmafterimmobilizing CRPantibodiesincreasedtheelectron-transferresistance(Ret) val-uesthatwereproportionaltotheCRPconcentrationsintherange of0.1–20ngmL−1.ThelowestdetectionlimithereforCRPantigen wasaslowas100pgmL−1[66].
A possibility of using a range of dc and ac electrochemical
techniques to probe associative interactions of CRP with CRP
antibody immobilized ona gold electrode surface was
investi-gated by Hennessey et al. [68]. It was demonstrated that the
investigated electrochemical techniquescan be used efficiently toprobetheseinteractionsoverawideCRPconcentrationrange, from1.15×10−5 to1.15mgL−1.Themeasuredsensitivityofthe techniques was in the following decreasing order: differential pulsevoltammetry,charge-transferresistanceobtainedfrom
elec-trochemical impedance spectroscopy (EIS), cyclic voltammetry,
chronoamperometry,anddouble-layercapacitancededucedfrom
EISmeasurementswhichgavethepoorestsensitivity.Thelowest detectionlimitofCRPantigenconcentrationwas6×10−6mgL−1.A
capacitivebiosensorbasedonchronoamperometrywasdeveloped
by using diamond-like carbon (DLC) film as an electric
isolat-ing layer and applicability of the biosensor was demonstrated
bydetectingCRP[69].ThelowestdetectionlimitofCRPantigen concentrationinthismethodwas0.01pgmL−1.Centietal. devel-opedelectrochemicalsandwichassayforthedetectionofCRP[67].
Thescreen-printedcarbon electrodesand magneticbeadswere
employedinthisassaytodevelopelectrochemicalassaythathada limitofdetectionof0.054mgL−1CRPinserum.
Electrochemical biosensors based detection offer sensitivity, selectivityandreliability, makingthemvery attractivetoolsfor
biomarker protein detection. However, these biosensors suffer
fromperturbationsonthesensorsurfacethatareinfluencedby differentpH,ionicstrengthandco-existingmoleculesin biologi-calfluids.Thebiologicalrecognitionelementsusedinamajority ofelectrochemicalbiosensorsareantibodies.Thefactthatthese antibodiesaregenerated,isolatedandpurifiedafterthe adminis-trationofadesiredantigenintotheanimalmodelsmakingthem inherentlytrained tofunctioninnormalphysiologicalsolutions. Thismakesantibodiesdifficulttocontainspecificbindingability outsideofthephysiologicalenvironmentsorsyntheticbuffers.The functionalityofantibodiesmayfurtherdeclineinpresenceof exter-nalredoxmediatorsthat areessentiallymixedwiththesample solutionforelectrochemicalsensorplatforms.
3.3.1. Useofnanomaterialsinelectrochemicalbiosensors
Useofnanomaterialsinelectrochemicalbiosensorshasreceived
considerable attention by the research groups over the past
decade.Thedemonstrationofnovelanduniquedetectionplatform for cardiac marker detection by utilizing nanoparticles, nano-tubes,nanowiresandnanocompositeshasbeenreported.Thereis muchversatilityintheselectionofnanomaterialstomeasurethe detectablesignalbywhichanalyteconcentrationscanbe deter-mined.Recently,asensitive,disposable,andeasytooperatesensor basedonheterogeneoussandwichimmunoassayforhigh sensitiv-ityCRP(hs-CRP)measurementwasdeveloped[65].Thisbiosensor utilizedscreen-printedelectrodesmodifiedwithmulti-walled
car-bonnanotubes(SPE/CNTs)and proteinAtoensuretheoriented
immobilizationofanti-CRPantibodiesandsensitivelydetectedCRP at500pgmL−1levels.Inanotherstudy,detectionof lipoprotein-associatedphospholipaseA2(Lp-PLA2)isreportedinwhichcarbon pastedopedwithiridiumnano-particlesasworkingelectrodeswas usedasbiosensor[72].Here,thedetectionofLp-PLA2wasinthe range0–150UmL−1withasensitivityof1.45nA/U.Thedetection oflow-densitylipoprotein(LDL)usingsilverchloride@polyaniline
(PANI)core–shellnanocomposites(AgCl@PANI)combinedwithAu
A.Qureshietal./SensorsandActuatorsB171–172 (2012) 62–76 73
Fig.5. Schematicoftheimmunosensorfabricationprocess[67].
hybridmaterialwasusedtoprovidesurfaceforhighantibody load-ingduetoitslargesurface-to-volumeratio.Themethodwasbased onadsorptionofantibodytoapolipoproteinB-100(aopB-100)on
anAuNPs-AgCl@PANI-modifiedglassycarbon(GC)electrode.The
biosensorexhibitedahighlysensitiveresponsetoLDLwitha detec-tionlimitof0.34pgmL−1.
In a study by Zhou et al. developed an electrochemical
immunoassayforthesimultaneousdetectionofcardiactroponinI (cTnI)andCRPinwhichCdTeandZnSequantumdotswereutilized forsandwichimmunoassay.Here,Cd2+andZn2+weredetectedby
square-waveanodicstrippingvoltammetrytoenablethe
quan-tificationofthe2 biomarkersin 20humanserumsamples[74]. Similarly,Kunduruetal.designednanotechnology-based
electro-chemicalbiosensorusingmicro-and nanotexturedpolystyrene
polymerstructures.Theresultsdemonstratedthatscalingdown thesurfacetexturingfromthemicro-tothenanoscaleenhances thesensitivityofthisdetectionmethodforCRP[75].Intheworkby Ahammadetal.,goldnanoparticleshavebeenelectrodepositedon indiumtinoxide(ITO)andappliedtodetectmolecularinteraction
betweenhumancardiaccTnIandspecificantibodybymeasuring
opencircuitpotential(OCP).Inthiswork,anewstrategyhasbeen adaptedtoobtainanelectrical signaldue totheenzyme-based immunecatalyticreactionanddetected1–100ngmL−1IcTnI[76].
Electrochemicalimmunosensorbasedonmetalnanoparticlesfor
cardiac myoglobin detection in human blood plasma hasbeen
reported[77].Inthiswork,cardiacmyoglobindetectionwasbased ondirectelectrontransferbetweentheFe(III)-hemeandthe elec-trodesurfacethatwasmodifiedwithmetalnanoparticles(gold,
silver, and copper) stabilized by didodecyldimethylammonium
bromideandantibodies.Themethodproposeddoesnotrequire
labeledsecondaryantibodiesthatresultedinadetectionlimitof 5ngmL−1withabroadrangeofworkingconcentrations.
The iridium oxide (IrOx) nanowires based electrochemical
biosensorwaspresentedforthedetectionCRPandMPO
cardio-vascular disease biomarkers [71]. This biosensor wasbased on electricaldetectionofproteinbiomarkerswhereinan
immunoas-say was built onto the iridium oxide nanowires that in turn
undergoesspecificelectricalparameterperturbationsduringeach
binding event associated with the immunoassay. The study
demonstratedthattheiridiumoxidenanowireshasanabilityto detectverysmallchangestothesurfacechargeandthiscapabilityis utilizedforachievingtheperformancemetricsandformsthebasis ofthekeyinnovationsofthistechnologytoimproveselectivityand sensitivityofdetection.
3.3.2. Electrochemicalbiosensorswithoutelectronmediators Anotherclassofelectrochemicalbiosensorsthatdonotutilize
redoxmediatorsknownasnon-Faradaic electrochemicalsensor
hasbeenemployedforthecardiacmarkerdetectioninbufferand complexserumgreatlyvariesbecauseoftheinterferenceofthe non-specificmoleculesandions.Capacitiveelectrochemical (non-Faradaic)immunosensorhasrecentlybeenpresented[78]forthe detectionofTnTbasedontheuseofimmobilizedantibodies spe-cificforTnT.ThedevicewasabletodetectTnTlevelsintherange 0.07–6.83ngmL−1inhumanserumfrompatientswithcardiac dis-easesandintherange0.01–5ngmL−1forTnTinphosphatebuffer saline.IntheworkbyQureshietal.[79],goldinterdigitated capac-itorarrays wereemployedforthedevelopmentofreagent-less, label-freecapacitiveelectrochemicalbiosensorsforthedetection ofCRPthatisbasedonchargedistributiononthesurfaceof capac-itors.ThesensitivedetectionofCRPcapturingoncapacitorswas
observed in a dynamic range 100–500pgmL−1 under standard
conditionsthatincludesthedefinedgeometryofelectrodesand specifiedbiochemicalassayconditions.
Electrochemicalcapacitiveimmunoassaysarepromising
alter-natives to existing immunochemical tests for the development
of hand-held devices for point-of-care applications.The attrac-tionofaffinity-based capacitivesensorsisthattheyareableto determinetheanalytedirectlyin a samplewithnoorvery lit-tle sample preparation. The sensing principle of these sensors isbasedonchangesindielectric properties,charge distribution, and/orconductivitychangebroughtonbybioreceptor–target ana-lytecomplexesformedonthesurfaceoftheelectrodes.Capacitive affinitybiosensorscanbeconstructedbyimmobilizing
recogni-tionelementsontheelectrodes,and measuringchanges inthe
dielectric/surfacepropertieswhenananalytebinds.Itcanthenbe correlatedtotheboundtargetproteinmoleculesandtheamount capturedbybioreceptorsimmobilizedonthesurface.Forproviding
74 A.Qureshietal./SensorsandActuatorsB171–172 (2012) 62–76
largersensor surface area, conductorscanbe madeinto a pat-ternofmicrotonano-interdigitatedfingers.However,non-specific and largebackground signals affectthese typesof sensors and reducesignal-to-noiseratioandtherefore,furtherimprovements isrequiredforimprovingtheselabel-freebiosensors.
3.4. Magneticbiosensors
Magneticparticles/beadshavebeenestablishedinlifesciences andusedfor manyyears inbiological assays.Awide varietyof biologicalspecies,suchascells, proteins,antibodies,pathogens, toxins,andDNAcanbelabeledbyattachingthemto
superpara-magnetic microbeads. These particlesrange in size from a few
nanometerstoafewmicrons,andthenormalmagneticbead struc-tureconsistsof manyiron oxide(magnetite)crystallites, which providetheparamagneticattractionoftheparticlestoamagnet andtheyareencapsulatedinplasticorceramicspheres.Thebeads arecoatedwithachemicalorbiologicalspecies(e.g.,DNAor anti-bodies)thatselectivelybindstothetargetanalyte.Todate,several authorsdescribedtheinteractionofmagneticbeadswitha mag-neticfieldandtheensuingvisualizationofbindingeffects[80],or usethebeadsasaseparationandmagneticimmobilization plat-form[81,82]althoughmagneticbeads/particlesbasedbiosensing platformshavebeenlessextensivelyexaminedbyresearchersfor cardiacmarkersdetections.
A rapid immunoassay system based on antibody-coated
micrometer-sizedparamagneticparticlewasreportedbyLuxton
etal.[83]forthedetectionofcardiacmarkersincludingCRPand creatinekinaseisoenzymeMBfraction(CKMB).Thisassaydonot requiresamplepreparation orwashingstep,and resultscanbe obtainedinlessthan3minafterintroducingthesampleintothe vesselwithsensitivitiesinthenormalclinicalrange.
The use of magnetic beads in CRP detection has also been
describedbyotherresearchgroup[84]wheremagneticbeadwas
used only for separation of bound and unboundaggregates of
sandwichassay.ThequantificationofmagneticbeadsfortheCRP detectionhasalsobeendescribedbyKrizetal.[85].Inthis publi-cation,theauthorsdescribethesedimentationofmagneticbeads complexedwithsilicamicroparticlesuponappearanceofCRPin
the sample. The content of magnetic beads in the sedimented
phaseofthemeasurementisthenanalyzedbyasinglemagnetic
resonantcoil.Themeasurementneedsonly11.5min,butshows
detectionlimitsof0.2mgL−1 anda coefficientofvariation(CV) of11%.Thesameresearchgrouppublishedarapiddetection sys-tembasedonthesametechnique[86]whichprovidesresultsafter 5.5minandhasadetectionlimitof3mgL−1andaCVof10.5%.In anotherapproach,CRPdetectionwasreportedbyanotherresearch group[87]wherethemostsignificantdifferencebetween previ-ouslymethodsisthemeasurementhead.Thisreportedsystemuses twodifferentexcitementcoilsinafrequencymixingmode.This enablestheverylowdetectionlimitof25gL−1,accompaniedby thelowCVof4.2%.
AnewstrategyforthedetectionofcardiactroponinIthatrelies oncompetitiveexchangeinteractionsofananalytewasreported [20].Inthismethod,useofsuperparamagneticbeadsandof mag-netictweezersapparatusallowsasmallforcetobeappliedtothe tethers, providing a drivingforce for thecompetitiveexchange reactionandselectively detectedproteinin asensitive reagent-lessfashion.Although,toobtainthereproducibleresultsfromthe device,furtheroptimizationinthefabricationofsensorwouldbe necessaryforbettersensitivity.
4. Conclusionsandperspectives
It is criticallyimportant to diagnose CVD at early stages of itsprogression,whichallowssuccessfultreatmentandrecovery
ofpatients.Therefore, itisessentialtodevelopsimpleand sen-sitive CVDdiagnosticmethods thatcan detectmultiplecardiac biomarkers atvery lowconcentrations inbiological fluids. Cur-rentbiosensing platformscan fulfillthesedemands but require sophisticatedlaboratoryequipmentandtraining. Inthisreview, availablebiosensingplatformsforCVDbiomarkersdetectionare summarized.MostfrequentlyusedCVDbiomarker/sforbiosensing areintheorderofCRP>cTnI>myoglobin>IL-6>TNF␣onoptical,
electrochemical and magnetic transductionformats. A majority
of cardiac biomarkers are also markers of common
inflamma-tion,makingitdifficulttodistinguishthecardiovascularrisk.An inflammatoryprocessinpatientscanoccurwithouttheexistence ofcardiovascular disease,which mayincurahighrateof false-positives.Therefore,amultiplemarkerdetectionstrategy needs tobeadaptedusingacombinationofestablishedandnewcardiac markers,whichhelpsinmakingcorrectclinicaldecisions.Future innovationinbiosensortechnologycanbedirectedtoward(a)
iden-tification ofnewandnon-inflammatorycardiacbiomarkersand
theirvalidation,(b)developmentofnovelbiorecognitionelements in placeof classicalantibodies,(c) directdetection of biomark-ersin biologicalfluids(blood)withoutsamplepreparations,(d) miniaturization ofelectronictransducersfor portability,and (e) patterningarraysandmicrofluidicssuitableformultiplebiomarker detection.Combinationsofalltheabovefeaturesareessentialto makingdevicesofhighpotentialforearlyCVDdiagnosiswith pre-cision.Improvingsensitivity,specificity,andmakingbiosensorsa low-costandpoint-of-carecapabilityisanotherchallenge.
Inrecentyears,nanomaterialshaveshownwideapplicationsin biosensing.Oneofthemoststrikingadvantagesofsuch nanoma-terialsistousesurfaceactivatedmagneticnanoparticles(SAMN).
These SAMNenable functionalizationand concentrationof
tar-getproteinmoleculesdirectlyfromthecomplexmixtures,such asblood/serum.Concentrationoftargetproteinfromthecomplex serumcanbeachievedbymagneticnanoparticlesfunctionalized withspecificaffinityligands(antibodies/aptamers).Themagnetic propertiesofnanoparticlesprovidemagneticseparationsimplyby
placinga magnet in closeproximityof thesuspension, making
themconcentratewithcapturedtargetproteins.This methodol-ogyholdsgreatpromiseinelectrochemicalchipbasedbiosensing, mainlyfordrawingthetargetcapturedmagneticnanoparticleson thesensorsurfaceimmobilizedwithspecificaffinityligands.This methodenableswashingawaythemagneticnanoparticleswithno
targetwhilewithdrawingthemagnetaway,andleavingbehind
only thosethat boundtothetargetproteinsfor specific detec-tionandquantification.Theconceptofusingnanomaterialsasone ofthecandidatestofurtherimprovethesensitivity in develop-inghighlysensitivedevicesforearlydiagnosisandpoint-of-care applications.Earlydiagnosiswillaidinincreaseinthehuman sur-vivalratethatrequiresprecisediagnosis,whichisonlypossible withmultiplebiomarkerdetectionforCVDrisk.However, appro-priateinvestmentandfundingsupportmechanismsareneededto facilitatemovingthistechnologyfromresearchtothecommercial applications.
Acknowledgements
ThisworkwassupportedbyDPTProgramofSabanci
Univer-sity,NanotechnologyResearchandApplicationCenterandpartially
by the Science and Technological Research Council of Turkey
(TUBITAK)undertheGrantno.110E287andtheauthorsthankfor thesupport.
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