Point-of-Care Solution for Osteoporosis Management: Design, Fabrication, and Validation of New Technology PDF
Preview Point-of-Care Solution for Osteoporosis Management: Design, Fabrication, and Validation of New Technology
Patricia Khashayar Point-of-Care Solution for Osteoporosis Management Design, Fabrication, and Validation of New Technology Point-of-Care Solution for Osteoporosis Management Patricia Khashayar Point-of-Care Solution for Osteoporosis Management Design, Fabrication, and Validation of New Technology 123 PatriciaKhashayar CentreforMicrosystemsTechnology (CMST) FacultyofEngineeringandArchitecture GhentUniversity Ghent,Belgium ISBN978-3-319-55052-7 ISBN978-3-319-55053-4 (eBook) DOI10.1007/978-3-319-55053-4 LibraryofCongressControlNumber:2017933838 ©SpringerInternationalPublishingAG2017 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartof thematerialisconcerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation, broadcasting,reproductiononmicrofilmsorinanyotherphysicalway,andtransmissionorinformation storageandretrieval,electronicadaptation,computersoftware,orbysimilarordissimilarmethodology nowknownorhereafterdeveloped. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublication doesnotimply,evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevant protectivelawsandregulationsandthereforefreeforgeneraluse. Thepublisher,theauthorsandtheeditorsaresafetoassumethattheadviceandinformationinthisbook arebelievedtobetrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsor theeditorsgiveawarranty,expressorimplied,withrespecttothematerialcontainedhereinorforany errorsoromissionsthatmayhavebeenmade.Thepublisherremainsneutralwithregardtojurisdictional claimsinpublishedmapsandinstitutionalaffiliations. Printedonacid-freepaper ThisSpringerimprintispublishedbySpringerNature TheregisteredcompanyisSpringerInternationalPublishingAG Theregisteredcompanyaddressis:Gewerbestrasse11,6330Cham,Switzerland Idedicatethisbooktomyfamily;aspecial feelingofgratitudeisduetomyloving parents,MohsenandShahla,whosewordsof encouragementandpushfortenacitystill ringinmyears,andmysisterPariaand brotherPouria,whohaveneverleftmyside andareveryspecialtome. Preface Osteoporosisdiagnosis,whichisnowadaysgenerallymadebasedonbonemineral density(BMD)measurements,suffersfromcertainlimitations.Thus,itisbelieved thatboneturnovermarkers(BTMs)canhelpimproveosteoporosisdetection.While the use of BTMs in osteoporosis diagnosis has certain advantages over BMD and clinical risk assessment tools, not only in monitoring treatment but also in identifying those at risk, the diagnostic value of BTMs in predicting osteoporosis islow,andthusseveralBTMsareusedatthesametimewithhigheraccuracyand lowervariabilitytoovercomethislimitation. Despiterecentadvancementsinproteomicsandpersonalizedmedicine,available technologiesforBTMmeasurement,enzyme-linkedimmunosorbentassay(ELISA) and electrochemiluminescence immunoassay (ECLIA), are time-consuming and laboriousandrequirespeciallabelsandexpensiveinstruments.Withtheseapparent limitations, the focus of health-related studies has shifted toward the development ofportable,reusable,andatthesametimemultiplex,label-freepoint-of-care(PoC) biosensors particularly for diagnosis and monitoring of different diseases. Also known as on-the-spot diagnostic devices, these PoC biosensors should be simple, sensitive,rapid,specific,cheap,andeasytointerpret. Thus, there is a growing demand to fabricate different types of biosensors to provide low-cost miniaturized platforms to assess bone remodeling process more accurately.Toourknowledge,noneoftheexistingbonebiosensorshavesucceeded inthisregard. Inthisbook,amicrofluidicproteomicplatformthatcaneasilybetranslatedinto abiomarkerdiagnosticisreported.Thisplatformintegratesmicrofluidictechnology withelectrochemicalsensingandembodiesareaction/detectionchambertomeasure serum levels of different biomarkers. The unique design of the platform offers the potential for greater sensitivity as the microfluidic and electrochemical structures canbeindependentlyoptimized. This book is the result of a joint Ph.D. in biomedical engineering awarded by GhentUniversity,Belgium,andtheUniversityofTehran,Iran. vii viii Preface Thegoalofthisworkwastodevelopareliable,cost-effective,powerfuldetection andmonitoringtoolforfractureriskindicationandtreatmentmonitoringsothatthe patientcanreceivethemostappropriatetherapyandthatphysicianscanmonitorthe diseaseprogression. In this regard, a microfluidic platform capable of measuring serum levels of several biomarkers with high accuracy was developed. The plan was to develop a unique architecture which enabled low sample volume requirements, short assay time, simplicity of fabrication and use, and low-cost instrumentation and at the same time portability and high sensitivity and specificity for the biomarkers. At the end, the measurement results were compared with the current gold standard, electrochemiluminescenceimmunoassay(ECLIA). Thestepstakentoachievethefinalgoalconsistedof: • The development of a functional conjugated AuNP-antibody nanoprobe to be usedtomodifythesurfaceoftheelectrodeforsensingpurposes:Thisnanoprobe was directly reacted with antigen following their preincubation in solution, or the probe was assembled onto the electrode to bind with antigen, by so-called “in-solution”or“on-chip”methods,respectively. • Stable immobilization of the nanoprobe on the electrode, while keeping the antibodies’ bioactivity in microreactor environment, and thus supporting high- quality and low-background noise in electrochemical sensing: In this work, gold electrodes were coated with AuNPs by electrodeposition using cyclic voltammetry.Thisgoalisnotlimitedtothedevelopmentofthemodifiedsurface but also includes characterization of the modified surfaces using advanced chemicalsurfaceanalysistechniquesincludingXPS,STEM,andAFM. • Validationofthebiosensor:Oneofthemostcrucialstepsinbiosensorfabrication isassessingthesensitivityandselectivityofthesystem.Inthiswork,acalibration curve was plotted for each of the measured markers, and the final results were comparedwiththatofthecurrentstate-of-the-artECLIA. • Development of microfluidic chips: The application of micro-manufacturing technologyanddevelopmentofmicrofluidicchipsallowedconsiderablethrough- put portability and the capacity for a high level of integration and thus fulfilled the requirements of fluidic sample-based point-of-care diagnostics. This also lowered the consumed reagent and sample volume as well as the analysis time without compromising the detection limit of the device, which are required for PoCdevices. • Validationofthefinaldevice:Inthisstage,theparameterswhichweretakeninto accounttoimprovethesensitivityandspecificityofthemicrofluidicplatformare studied.Hence,thereproducibilityofthemeasurementswasinvestigatedthrough comparingtheresultswithECLIA. Ghent,Belgium PatriciaKhashayar January24,2017 Acknowledgments “TobeadoctoristobeanintermediarybetweenmanandGod,”FelixMarti-Ibanez said famously in his To Be a Doctor book. This realization struck a chord with meandmademestudymedicine.Thankstomyfamily,Ilearnedthat“Oneofthe essentialqualitiesoftheclinicianisinterestinhumanity,forthesecretofthecare ofthepatientisincaringforthepatient.”SoIdedicatedmylifetohelppatients. As time passed, I struggled with the idea that I could not help all my patients no matter how hard I tried. I imagined that in months and years to come, I would stand by their bedside as patients experienced complications due to late diagnosis ordeferredtreatment.Icouldn’thelpbutstrivetoseekasolutionthatwouldcome totheiraid. Adopting the motto “Science is knowing and Technology is doing. Science focuses on understanding the natural phenomena, while technology focuses on understandingthemadeenvironment,”Irealizedthatbioengineeringwouldprovide theperfectblendandpresentmewiththeremedytomyinternalstruggle.Myfinal goal was to incorporate my medical knowledge into developing new devices that wouldfillthegapsinmodernmedicineandthushelppeoplearoundtheworld. And thus I started this project, which later become a new beginning and a life changer. I am heartily thankful to Prof. Bagher Larijani, Prof. Jan Vanfleteren and Prof. Ghassem Amoabediny as well as my colleagues Dr. Rik Verplancke and Dr.FrederikLeysfortheirguidanceandpersistenthelp. Andfinally,aspecialthankstomyfamily.WordscannotexpresshowgratefulI amtomymom,mydad,mysisterParia,andmybrotherPouria,forallthesacrifices theyhavemadeonmybehalf.Theirprayerformewaswhatsustainedmethusfar.I thankthemforalwaystakingcareofme,fortheirloveandpatience,forsupporting me all through my life, and for encouraging me to strive toward my goals. They alwaysencouragemetobeabetterperson. Ghent,Belgium PatriciaKhashayar January24,2017 ix Contents 1 Introduction .................................................................. 1 1.1 ImportanceofPoint-of-CareTestinginOsteoporosisManagement.. 1 1.1.1 TheConceptofBoneRemodeling............................. 1 1.1.2 WhatIsOsteoporosis?.......................................... 1 1.1.3 OsteoporosisDetectionTechniques............................ 6 1.1.4 EarlyDiagnosisinOsteoporosisDetection.................... 8 1.2 TheAimofCurrentBook .............................................. 9 1.2.1 PoCandOsteoporosis .......................................... 10 1.3 OrganizationoftheBook............................................... 11 References..................................................................... 14 2 LiteratureOverview......................................................... 17 2.1 BoneTurnoverMarkersandOsteoporosis............................. 17 2.1.1 Introduction..................................................... 17 2.1.2 WhatAreBiomarkers .......................................... 17 2.1.3 BoneTurnoverMarkers(BTMs)............................... 19 2.1.4 BTMClassification............................................. 22 2.1.5 BiomarkerSetbacks(withFocusonBTMs) .................. 27 2.1.6 Conclusion ...................................................... 29 2.2 BoneBiosensors:KnowingthePresentandPredictingtheFuture... 32 2.2.1 CurrentApproachestoAssessBoneRemodeling............. 32 2.2.2 ConclusionandFutureDirections ............................. 37 2.3 ProteinImmobilizationStrategiesforBiosensingPurposes.......... 39 2.3.1 Physisorption.................................................... 39 2.3.2 ElectrostaticInteraction ........................................ 39 2.3.3 CovalentBinding ............................................... 41 2.3.4 BioaffinityImmobilization..................................... 44 2.3.5 Conclusion ...................................................... 45 References..................................................................... 45 xi xii Contents 3 ScientificBackground ....................................................... 53 3.1 OverviewonImmunosensors........................................... 53 3.1.1 Introduction..................................................... 53 3.1.2 WhatIsaBiosensor ............................................ 54 3.1.3 MainTypesofImmunoassays.................................. 56 3.1.4 ImmunosensorComponents.................................... 57 3.1.5 DetectionTechniques........................................... 58 3.2 Electrochemistry:Principles............................................ 61 3.2.1 CyclicVoltammetry ............................................ 64 3.2.2 DifferentialPulseVoltammetry................................ 67 3.2.3 Chronoamperometry............................................ 67 3.2.4 Chronocoulometry.............................................. 68 3.2.5 ElectrochemicalImpedanceSpectroscopy .................... 69 3.3 BasicConceptsinMicrofluidicDevices............................... 70 3.3.1 MaterialsfortheMicrofluidicDevices ........................ 73 3.3.2 SurfaceModification ........................................... 75 3.3.3 Introducing Sample/Reagent and Moving Fluid WithintheMicrofluidic......................................... 76 3.3.4 Detection........................................................ 77 3.3.5 Design........................................................... 78 References..................................................................... 78 4 ConjugatedAuNP-AntibodyNanoprobeFabrication andValidation................................................................ 83 4.1 Introduction ............................................................. 83 4.2 SynthesisofaConjugatedAuNP-AntibodyNanoprobe.............. 84 4.2.1 SynthesisofAuNPs ............................................ 84 4.2.2 AuNP-AntibodyConjugation.................................. 86 4.3 CharacterizationofConjugatedAuNP-AntibodyNanoprobe ........ 86 4.3.1 CharacterizationofAuNPs..................................... 86 4.3.2 CharacterizationofAuNP/AbNanocomplex.................. 88 4.4 OptimizationofAuNP/AbNanoconjugatePreparation............... 91 4.4.1 EnvironmentalFactors.......................................... 91 4.5 Conclusions ............................................................. 92 References..................................................................... 93 5 ElectrochemicalChipPreparation......................................... 95 5.1 Introduction ............................................................. 95 5.2 ElectrodeSelection...................................................... 96 5.2.1 CarbonElectrodes .............................................. 96 5.2.2 GoldElectrodes................................................. 100 5.3 SurfaceModificationofGoldElectrodeswithAuNPs................ 106 5.3.1 GoldDepositionTechniques................................... 106 5.3.2 PerformanceAnalysis .......................................... 112 5.3.3 Reproducibility,Reusability,andStabilityTests.............. 114 5.4 CharacteristicsofAuNP-CoatedGoldElectrodesUsingECV....... 115 5.4.1 ElectrochemicalBehavior...................................... 115
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