Biosensors for cardiac biomarkers detection: A review Sensors and Actuators B: Chemical

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

_{, Yasar}

_Gurbuz

_,

_Javed

_H.

_Niazi

a_{SabanciUniversity,NanotechnologyResearchandApplicationCenter,OrtaMahalle34956,Tuzla,Istanbul,Turkey} b_{FacultyofEngineeringandNaturalSciences,SabanciUniversity,Orhanli34956,Tuzla,Istanbul,Turkey}

a

r

t

i

c

l

e

i

n

f

o

Received10February2012

Receivedinrevisedform22May2012 Accepted25May2012

Available online 2 June 2012 Keywords: Cardiovasculardisease Cardiacbiomarker C-reactiveprotein Electrochemical Opticalbiosensor

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b

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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:[email protected](A.Qureshi),[email protected] (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

<103_ngmL−1_lowrisk

1–3× 103_ngmL−1_{intermediaterisk} >3–15×103_ngmL−1_{highrisk(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−1_{stratificationrisk}

Medium 150 ND ND ND Yes

Heartfattyacidbinding protein(H-FABP)

Myocardialnecrosis PatientswithelevatedH-FABPlevelselevated ≥6ngmL−1_{stratificationrisk} Low 15 2–3h 8–10h 18–30h Yes TNF-␣ Inflammation/cardiacrisk factor <0.0036ngmL−1_lowrisk ≥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<3.58×106_ngmL−1 Mid3.58–4.20×106_ngmL−1 High>4.20×106_ngmL−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 10–105_ngmL−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× 103_mL−1 CRP-LOD<103_ngmL−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× 104_to5.3×106_ngmL−1_)orhuman

troponinIprotein(4.3× 104_{to4.3× 10}6_ngmL−1₎ Singleanalyte [33]

CRPandIL-6 Fluorescence/colorimetric 1–1000ngmL−1

LOD-CRP–1ngmL−1 Microfluidic_with

multianalyte

[35]

Myoglobin,creatinekinasemb(CKmb), cTnI,andfattyacid-bindingprotein (FABP) Chemiluminescence Myoglobin– 1.2ngmL−1 CKmb–0.6ngmL−1 TnI–5.6ngmL−1_and FABP– 4ngmL−1 Multianalyte [37] CRP Chemiluminescence 50–5× 104_ngmL−1 LOD–12.5ngmL−1 Multianalyte [34] CRP Chemiluminescence 10to104_ngmL−1 _{Microfluidic} withSingle analyte [14]

cTnI Colorimetric;PDMS–goldnanoparticlecompositefilm 104_to5×103_ngmL−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– 103_ngmL−1 _{Singlemarker [43]}

B-typenatriureticpeptide(BNP) SPR 5pgmL−1_to100ngmL−1

LOD– 0.005ngmL−1 Microfluidic withsingle analyte [48] CRP SPR 2–5×103_ngmL−1

LOD–103_ngmL−1 singleanalyte [44]

CRP SPR 1ngmL−1_{to5× 10}4_ngmL−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 104_ngL−1 _{Singleanalyte [46]}

TNF-␣ SPR 1–2×103_ngmL−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–7×10−3_ngmL−1

MG–70ngmL−1

CRP–700ngmL−1

Multianalyte [16]

CRP Opticalfiber 5–12.5×103_ngmL−1 _{Singleanalyte [55]}

Nervegrowthfactor(NGF) Opticalfiber 1–200ngmL−1 _{Singleanalyte [57]}

IL-6 Opticalfiber LOD–0.12ngmL−1 _{Singleanalyte [58]}

CRP Opticalfiber/fluorescence/evanescent 4.4×10−11_to2.9×10−9_ngmL−1_and1.3×10−10_to

2.29×10−8_ngmL−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−1_to2×103_ngmL−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–4.1×10−3_ngmL−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–5.4×10−11_ngmL−1 _{Singleanalyte [67]}

cTnT Non-Faradaic,impedance/Alinterdigitatedelectrodes 0.07–6.83ngmL−1 _{Multianalyte [81]}

CRP Faradaic,impedance/goldelectrodes 1.15×10−13_to1.15ngmL−1

LOD–6×10−14_ngmL−1

Singleanalyte [68] CRP Faradaic,diamondlikecarbonelectrodes LOD–10−4_ngmL−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−1_{andLOD-MPO–0.5ngmL}−1 _{Multianalyte [71]}

cTnIandCRP Faradaic,poly(dimethylsiloxane)–goldnanoparticlecomposite microreactors

cTnI–0.01ngmL−1_{andCRP–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 enablestheverylowdetectionlimitof25␮gL−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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