Point-of-Care Solution for Osteoporosis Management: Design, Fabrication, and Validation of New Technology

212 Pages·2017·6.482 MB·English

by  Patricia Khashayar (auth.)

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
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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