Detection of Lipid and Amphiphilic Biomarkers for Disease Diagnostics

25 Pages·2017·1.92 MB·English

LA-UR-17-24316 (Accepted Manuscript)
Detection of Lipid and Amphiphilic Biomarkers for Disease Diagnostics
Kubicek-Sutherland, Jessica Zofie
Vu, Dung M.
Mendez, Heather
Jakhar, Shailja
Mukundan, Harshini
Provided by the author(s) and the Los Alamos National Laboratory (2017-12-11).
To be published in: Biosensors
DOI to publisher's version: 10.3390/bios7030025
Permalink to record: http://permalink.lanl.gov/object/view?what=info:lanl-repo/lareport/LA-UR-17-24316
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biosensors
Review
Detection of Lipid and Amphiphilic Biomarkers for
Disease Diagnostics
JessicaZ.Kubicek-Sutherland1 ,DungM.Vu1,HeatherM.Mendez2,3,ShailjaJakhar1
andHarshiniMukundan1,*
1 PhysicalChemistryandAppliedSpectroscopy,ChemistryDivision,LosAlamosNationalLaboratory,
LosAlamos,NM87545,USA;[email protected](J.Z.K.-S.);[email protected](D.M.V.);[email protected](S.J.)
2 DepartmentofChemicalandBiologicalEngineering,UniversityofNewMexico,Albuquerque,
NM87131,USA;[email protected]
3 TheNewMexicoConsortium,LosAlamos,NM87544,USA
* Correspondence:[email protected];Tel.:[phone]
Received:31May2017;Accepted:30June2017;Published:4July2017
Abstract:Rapiddiagnosisiscrucialtoeffectivelytreatinganydisease.Biologicalmarkers,orbiomarkers,
havebeenwidelyusedtodiagnoseavarietyofinfectiousandnon-infectiousdiseases. Thedetection
of biomarkers in patient samples can also provide valuable information regarding progression
andprognosis. Interestingly,manysuchbiomarkersarecomposedoflipids,andareamphiphilic
in biochemistry, which leads them to be often sequestered by host carriers. Such sequestration
enhancesthedifficultyofdevelopingsensitiveandaccuratesensorsforthesetargets. Manyofthe
physiologicallyrelevantmoleculesinvolvedinpathogenesisanddiseaseareindeedamphiphilic.This
chemicalpropertyislikelyessentialfortheirbiologicalfunction,butalsomakesthemchallenging
to detect and quantify invitro. In order to understand pathogenesis and disease progression
whiledevelopingeffectivediagnostics,itisimportanttoaccountforthebiochemistryoflipidand
amphiphilicbiomarkerswhencreatingnoveltechniquesforthequantitativemeasurementofthese
targets. Here,wereviewtechniquesandmethodsusedtodetectlipidandamphiphilicbiomarkers
associated with disease, as well as their feasibility for use as diagnostic targets, highlighting the
significanceoftheirbiochemicalpropertiesinthedesignandexecutionoflaboratoryanddiagnostic
strategies. Thebiochemistryofbiologicalmoleculesisclearlyrelevanttotheirphysiologicalfunction,
andcallingouttheneedforconsiderationofthisfeatureintheirstudy,anduseasvaccine,diagnostic
andtherapeutictargetsistheoverarchingmotivationforthisreview.
Keywords: biomarkers;biosensors;amphiphile;lipid;diagnostics
1. Introduction
Theutilityofadiagnosticismeasuredbyitsabilitytoproviderapidandreliableinformationto
guidetreatment. Thepastcenturyhasseenariseinmoleculardiagnosticstrategiesformeasuring
disease-specificsignaturesinpatientsamples(e.g.,blood,urine,tissueandothers)usingavariety
ofbiosensortechnologies. Abiosensorutilizesabiologicalcomponentorbiocatalysttodetectthe
presenceofananalyteandatransducertocreateaquantifiablesignalfromthisinteraction. Fora
biosensor to be used feasibly in a clinical setting, it must be highly specific for the target analyte,
accurateinpatientsamples,rapidandreliable,andresistanttonon-specificinteractionsinclinical
samples. Inaddition,forsomeapplications,especiallyinresource-poorconditions,itisalsodesirable
forthesensortobecost-effectiveandeasy-to-use[1]. Inordertoavoidfalsepositive(signalinthe
absenceofanalyte)andfalsenegative(analytepresentwithoutsignal)signals,thebiocatalystand
transducermustbecarefullyconsideredforeachtargetanalyte.
Biosensors2017,7,25;doi:10.3390/bios7030025 www.mdpi.com/journal/biosensors
Biosensors2017,7,25 2of24
Identifyingtargetanalytesandtheirpresentationinahostduringdiseaseisofcrucialimportance
for the success of such molecular diagnostic technologies. Biomarkers are indicators of biological
processesanddisease-specificbiomarkersareofgreatclinicalvaluefordiagnostics. Understanding
the interactions between the biomarker and its host is key to developing assays for the detection
ofsuchanalytesinclinicalsamples. Becauseoftheeaseofpurificationanddetection,proteinsand
nucleicacidshavebeenextensivelyusedasdiagnostictargets. Traditionally,nucleicacidsandproteins
havebeentargetedasbiomarkersforthediagnosisanddetectionofdisease, primarilybecauseof
theavailabilityofavarietyofsensitiveandtailoredmethodsforthedetectionofthesebiochemical
signatures(reference).However,anothercategoryofbiomoleculesthatplayimportantrolesinavariety
ofcellularprocesses,andcanthereforeserveasindicatorsofdiseaseandanalytesforbiosensorsis
lipidsandamphiphiles[2]. Besidesmembraneformation,lipidsalsoplayacrucialroleincellsignaling
(steroids) and energy storage. The brain is composed of 40%–81% lipid, with the myelin sheath
being78%–81%lipidicinbiochemistry[3]. Manyofthebacterialvirulencefactorsrecognizedbythe
humaninnateimmuneresponse,suchaslipopolysaccharide(LPS)andlipoteichoicacid(LTA),are
amphiphiliclipoglycans[4–10]. Thestructureoflipidsisfarmorediversethanthatofproteins,nucleic
acidsorcarbohydrates,allowingforhighlyspecificbiomarkersforavarietyofdiseases. However,
alllipidscontainatleastonehydrocarbonchainthatisinsolubleinwatermakingthemdifficultto
detectinaqueoussolutionssuchasblood,whichhaslargelylimitedtheirapplicationasdiagnostic
targets. Indeed,currently,rapiddetectionoflipidatedtargetsislargelyachievedviamethodsthat
wereoriginallydesignedforproteins. Here,wereviewadvancementsinmethodsfordetectionoflipid
andamphiphilicbiomarkersassociatedwithbothinfectiousandnon-infectiousdiseasesdescribing
theadvantagesandlimitationsofeachapproachinclinicaldiagnostics(Table1).
Table1. Advantagesandlimitationsofsensorsasclinicaldiagnostictoolsfordetectinglipidand
amphiphilicbiomarkers.
Method Advantages Limitations
Massspectrometry Sensitive Expensive
Specific Samplepreparationcanbeextensive
Worksinpatientsamples Requireshighlytrainedpersonnel
Requireslaboratoryinfrastructure
NMR-basedsensors Rapid Lowsensitivity
Reproducible Lowspecificity
Worksinpatientsamples Samplepreparationcanbeextensive
Requireshighlytrainedpersonnel
Requireslaboratoryinfrastructure
Opticalbiosensors
SPR-basedsensors Rapid Lowsensitivityinpatientsamples
Specific
Interferometry-basedsensors Low-cost Lowspecificityinpatientsamples
Sensitive
Waveguide-basedsensors Rapid Shortshelf-lifeoflabeledreagents
Reproducible
Sensitive
Specific
Worksinpatientsamples
Electrochemicalbiosensors Rapid Lowsensitivityinpatientsamples
Reproducible Lowspecificityinpatientsamples
Mechanicalbiosensors Rapid Lowsensitivityinpatientsamples
Lowspecificityinpatientsamples
Reproducibility
Biosensors2017,7,25 3of24
2. MethodsforDetectingLipidandAmphiphilicBiomarkers
2.1. StorageandProcessingofPatientSamples
Samplecollectionandpreparationisacriticalconsiderationforthedetectionanybiomarker[11],
but lipidic targets warrant further attention [12]. Common lab methods such as freeze/thaw and
chemicalextractionscanhaveaprofoundeffectontheviabilityofamphiphilicbiomarkers[13,14].
Further,dependingonthesample,lipidandamphiphilicbiomarkersareoftensequesteredbyhost
factors,furtherreducingtheiravailabilityfordirectdetection[15]. Therefore,anybiosensordesigned
todetectamphiphilicbiomarkersmustaccountfortheiruniquebiochemicalproperties.
2.2. MassSpectrometry
Lipidomicsrequiresathoroughcharacterizationofthestructureandthefunctionoflipidswithin
alivingsystem. Thestudyofamphiphilicbiomarkers,asdescribedhere,isacompositeoflipidomics,
but involves the consideration of the hydrophilic components of the biomarker in addition to the
hydrophobiclipids. Sinceitsinceptionintheearly20thcentury,massspectrometry(MS)hasplayed
asignificantroleincharacterizationoflipids[16–18],andthishasbeenextensivelyreviewed. Here,
wewillfocusontheadaptiveapplicationofMStoamphiphilicbiomarkers.
MSionizeslipidsandsortsionsbasedontheirmass-to-chargeratio. Ithasbeenwidelyused
tocharacterizelipids[19–23],especiallywiththedevelopmentofsoftionizationtechniquessuchas
electrosprayionization(ESI)andmatrix-assistedlaserdesorptionionization(MALDI).Lipidextraction
isusuallythefirststepforlipidanalysis,andseparatesthelipidiccomponents(organicphase)from
othercomponentssuchasproteinsandnucleicacids(aqueousphase). Mostwidelyusedextraction
methodshavebeenadaptedfromFolch[24]orBlighandDyer[25],inwhichamixtureofmethanol,
chloroformandwaterareappliedforphaseseparation.However,shotgunlipidomicmethodshavealso
beendevelopedwhichomitthechromatographicseparationandsampleprocessingdescribedabove,
andanalyzesalllipidclassestogether,insteadusingionizationadditivestoprovidediscriminative
identification[16]. Thismethodmightbemoresuitableespeciallyforbiomarker-discovery,wherein
thereisnopriorartregardingthebiomarkersbeingidentified/measured.
Chromatographicmethodssuchasgaschromatography,thin-layerchromatography,high-performance
liquidchromatography(HPLC),orultra-performanceliquidchromatography(UPLC)[21,26]areused
forseparationoflipidmixtures.HPLCandUPLCarehavebroaderapplicationsinlipidanalysis[20,26]
andcanbeperformedinnormalphase(lipidclassseparationbasedontheirdifferentpolaritiesand
dipolemoments)orreversephase(separateslipidaccordingto their hydrophobicityandisbased
onfatty-acylcompositions). CouplingofseparationwithLCmethodsallowslipidstoberesolved
sequentiallywithgreaterionizationyields,decreasedionsuppressionfrommajorlipidclasses,and
increasedsensitivity[20,27]. However,thisLCstepcanbebypassed,withdirectinfusion(shotgun
lipidomics) of lipid samples, which simultaneously analyzes the lipid species in the crude lipid
extract[28,29]. Thismethoddoesnotrequireaprioridecisionsonwhichlipidspeciestomeasure,
isrelativelysimple,high-throughput,andfastwithshortdataacquisitiontimes. However,amajor
limitationofthismethodisthathighlyabundantlipidscancompeteforionizationwiththeminor
species,andmaskdetectionofthelatter.
MALDI-MSisbasedontheutilizationofanultraviolet–absorbingmatrixthatinitiallyabsorbsthe
energyofthelaserandmediatesthegenerationofions[30,31]. ESI-MSusesahighvoltageelectrospray
to aerosolize the lipid sample and generate ions to charge the lipid molecule [19,32]. MALDI or
ESI ionization can be combined with several types of mass analyzers such as triple quadrupole,
time-of-flight(TOF),iontrap,andorbitraptofurthercharacterizedifferentlipidclassesandspecies[32,33].
Both quadrupole and TOF mass analyzers are commonly used and their configuration together
(e.g., triple quadrupule (QqQ), quadrupole time-of-flight (QTOF)) as tandem mass spectrometric
instruments to break down the precursor ions into fragmented product ions, can provide further
chemical and structural resolution on the lipid species [34,35]. As an example, ESI-MS has been
Biosensors2017,7,25 4of24
successfully applied to characterize cardiolipin [35]. Cardiolipin, a biomarker implicated in heart
disease and cancer, is a phospholipid present in mitochrondria and is a component of bacterial
membrane[36,37]. Thediversityofcardiolipinmolecularspeciesisfoundinboththeidentityand
positionofitsfourfattyacylmoieties. MinklerandHoppelshowedthatreverse-phaseionpairHPLC
coupled with a triple quadrupole MS/MS linear ion trap mass spectrometer to generate MS/MS
washighlyeffectiveincharacterizingcardiolipinfromdifferentspecies(e.g.,ratliver,mouseheart,
bovineheart,dogheart)[35],withthespectrafacilitatingdeductionofthestructureofcardiolipin: the
diacylglycerolphosphateregion,themonoacylglycerolregion,andthefattyacidregion[35].
Mass spectrometry has been used by clinical laboratories to rapidly diagnose both infectious
and non-infectious diseases, detect drug toxicity, monitor treatment as well as to discover new
biomarkers[38]. However,clinicalmassspectrometrysystemssuchasVITEK® MSbyBioMerieux
areexpensivetosetupandmaintainandrequiretrainedpersonneltoperformandanalyzethetests
limitingitsutilityasapoint-of-carediagnostictool.
2.3. NuclearMagneticResonance(NMR)
NMR spectroscopy is one of the most powerful analytical techniques for lipid analysis of
biological matrices (e.g., cells, tissue, and biological fluids), owing to the natural abundance of
hydrogen in such samples [39–43]. This method is nondestructive, nonselective, and provides
detailedmolecularinformation,whichcanbeofextensivevalueinanalyzingamphiphilicbiomarkers.
Specifically, high-field proton nuclear magnetic resonance (1H-NMR) spectroscopy is used for
molecular profiling [44,45], allowing for identification of biomarkers associated with diseases
such as tuberculosis [46], cancer [47,48], heart disease [49]; and others, identifying underlying
causes of disease [50], and identifying diagnostic biomarkers and new therapeutic strategies [51].
High-resolution,one-dimensional(1D)1HNMRisreproducibleandsimple[39,49].Ithasbeenapplied
for metabolomics since the 1980s [39,42], specifically, for plasma [48], serum [52], urine [43], and
feces[53]. Tissuesarenormallyexaminedintact[54]andspectracanbeobtainedrapidly(<5min)[39]
withdetectionlimitsaround1to10µMat>500MHz[42,50]MSandNMRareoftenusedinconjunction
withoneanother[55,56]. AlthoughNMRhasdecreasedsensitivityincomparisontoMS[50,57],ithas
otheradvantagessuchasbeinganon-destructivetechniquewithminimalsamplepreparation,and
providingquantitationofmolecularstructures[18,57]. Couplingthetwomethodyieldssignificantly
greatersensitivity(attomoletofemtomole)[58].
LipidomicNMRisassociatedwithmanychallenges. Asubstantialnumberofbiomoleculesin
complexmatriceshavesimilarresonances,duetosimilarprotonchemicalshiftranges[42,59]resulting
inconsiderablepeakoverlap[46,60]andalowsignal-to-noiseratio[61]. Minimizingpeakoverlapand
increasingsensitivitycanberesolvedbyusinghighresolutionspectrometers(800to950MHz)[62].
Resonancelinebroadening[63]attributedtotheformationofmicellesinanaqueousenvironmentor
constrainedmolecularmovementasaresultoflipidaggregationintobilayers[42]isanotherchallenge
thatisovercomebychemicalorphysicaltreatmentofthesample[42].
The use of two-dimensional (2D) NMR [64,65] can also overcome the above challenges of 1D
1HNMR.2Dtechniquessuchastheheteronuclearsinglequantumcoherence(HSQC)methodare
bettersuitedforlipidprofilingbecauseofitsabilitytodetectresonancesforboth1Hnucleiand13C
nuclei[61],thusallowingforcorrelationbetween1Hand13Cchemicalshiftsandprovidingelucidation
ofC-Hbondswithinastructure[50]. Althoughdetectionof13CispossiblewithHSQC,thenatural
abundanceof13C[66]isrelativelylow,requiringpriorenrichmentofcellswith13C[46]. 2DNMRis
moretimeconsuming(>1h/spectrum)incomparisonto1D1HNMR(<10min)[43,50,67]. Absolute
quantitationoflipidsusingNMRremainsachallenge[68].
HighresolutionNMRhasbeenshowntodetectavarietyofdiseasesdirectlyinhumantissue
andfluidsamples[69]. PortableNMR-basedbiosensorshavealsobeendevelopedforuseasmedical
diagnostics[70]. However,portableNMRsensorssufferfromlowsensitivityandspecificitywhile
high-resolutionNMRtoolsrequireexpensiveequipmentandtrainedpersonneltoanalyzeresults.
Biosensors2017,7,25 5of24
Biosensors 2017, 7, 25 5 of 25
2.4. Biosensors
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Figure 1. Examples of biosensor techniques incorporating lipids for the detection of analytes include
Figure1.Examplesofbiosensortechniquesincorporatinglipidsforthedetectionofanalytesinclude
(a) optical (b) electrical and (c) mechanical.
(a)optical(b)electricaland(c)mechanical.
2.4.1. Optical Biosensors Detecting Lipids
2.4.1. OpticalBiosensorsDetectingLipids
Optical biosensors measure binding-induced changes in light from the sensor surface. Surface
Opticalbiosensorsmeasurebinding-inducedchangesinlightfromthesensorsurface. Surface
plasmon resonance (SPR) is a label-free technology that has been used to detect the interaction of
plasmon resonance (SPR) is a label-free technology that has been used to detect the interaction of
amphiphilic molecules with lipid bilayers, for ligand screening and biomarker discovery [71]. In SPR
amphiphilicmoleculeswithlipidbilayers,forligandscreeningandbiomarkerdiscovery[71]. InSPR
sensing, a ligand is immobilized on a gold-coated surface while an interacting molecule is injected in
sensing,aligandisimmobilizedonagold-coatedsurfacewhileaninteractingmoleculeisinjectedin
aqueous solution, and the change in resonance associated with binding is measured optically using a
aqueoussolution,andthechangeinresonanceassociatedwithbindingismeasuredopticallyusinga
spectrometer. SPR has been optimized for measuring protein-protein affinities using the BIAcore™
spectrometer. SPRhasbeenoptimizedformeasuringprotein-proteinaffinitiesusingtheBIAcore™
(GE Healthcare) system. In original iteration, SPR systems lack resolution, cannot multiplex, and are
(GEHealthcare)system. Inoriginaliteration,SPRsystemslackresolution,cannotmultiplex,andare
difficult to miniaturize. However, several researchers are working on overcoming these limitations,
difficulttominiaturize. However,severalresearchersareworkingonovercomingtheselimitations,
which may provide for more robust sensors in the future [72]. SPR is not conducive for detection of
whichmayprovideformorerobustsensorsinthefuture[72]. SPRisnotconducivefordetectionof
amphiphiles in biologically relevant conditions, as required for diagnostic applications.
amphiphilesinbiologicallyrelevantconditions,asrequiredfordiagnosticapplications.
Interferometry, particularly Backscattering interferometry (BSI), allows for label-free detection
Interferometry,particularlyBackscatteringinterferometry(BSI),allowsforlabel-freedetection
of both surface-bound and free-solution molecules [73]. In BSI, excitation of a microfluidic channel
ofbothsurface-boundandfree-solutionmolecules[73]. InBSI,excitationofamicrofluidicchannel
containing the sample, as well as the channel surface, usually made of polydimethylsulfoxide or
containingthesample,aswellasthechannelsurface,usuallymadeofpolydimethylsulfoxideorglass,
glass, yields interference fringes. Changes in fringes associated with binding of two molecules are
yieldsinterferencefringes. Changesinfringesassociatedwithbindingoftwomoleculesaremeasured
measured in the backscatter region. BSI detects interactions of amphiphiles with lipid bilayers [74]
inthebackscatterregion. BSIdetectsinteractionsofamphiphileswithlipidbilayers[74]directlyin
directly in human serum [75]. However, in complex samples, non-specific interactions and
humanserum[75]. However,incomplexsamples,non-specificinteractionsandassociatedchanges
associated changes in refractive index can prove challenging, but can be limited using suitable
inrefractiveindexcanprovechallenging,butcanbelimitedusingsuitablesurfacefunctionalization
surface functionalization chemistry [76]. BSI is inexpensive, sensitive and very versatile, making it a
chemistry[76]. BSIisinexpensive,sensitiveandveryversatile,makingitapromisingcandidatefor
promising candidate for detection of amphiphilic biomarkers in patient samples.
detectionofamphiphilicbiomarkersinpatientsamples.
Ellipsometry measures the refractive index of a thin film, and has been utilized in label-free
Ellipsometry measures the refractive index of a thin film, and has been utilized in label-free
assays where changes in refracted light are measured upon interaction of giant lipid vesicles with a
assayswherechangesinrefractedlightaremeasureduponinteractionofgiantlipidvesicleswitha
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ellipsometry (TIRIE) [77]. This sensor can detect µm size particles such as cells, capsules and
(TIRIE)[77]. Thissensorcandetectµmsizeparticlessuchascells,capsulesandliposomes. However,
liposomes. However, the ability to detect such particles in tissues, as well as the overall clinical
utility of this system is yet to be demonstrated.
An optical assay based on UV absorption of lipid-functionalized gold nanorods has been used
to detect the lipopeptide myristoyl-Lys-Arg-Thr-Leu-Arg, and a variety of other lipid biomarkers, in
Biosensors2017,7,25 6of24
theabilitytodetectsuchparticlesintissues,aswellastheoverallclinicalutilityofthissystemisyetto
bedemonstrated.
Biosensors 2017, 7, 25 6 of 25
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2.4.2. Electrochemical Biosensors Detecting Lipids
2.4.2. ElectrochemicalBiosensorsDetectingLipids
Electrochemical sensing is a label-free method where an electrode is used to directly detect
Electrochemical sensing is a label-free method where an electrode is used to directly detect
associated reactions [91]. Electrochemical biosensors have had the greatest commercial success
associatedreactions[91].Electrochemicalbiosensorshavehadthegreatestcommercialsuccess(glucose
(glucose monitors) due to their low-cost, ease of use and miniaturization properties [92].
monitors)duetotheirlow-cost,easeofuseandminiaturizationproperties[92]. Amperometricsensors
Amperometric sensors measure current typically as a result of electron transfer during the binding
measurecurrenttypicallyasaresultofelectrontransferduringthebindingbetweenamolecule,and
between a molecule, and chemically functionalized surface, as exemplified in the detection of LPS
chemicallyfunctionalizedsurface, asexemplifiedinthedetectionofLPSusingredoxdiacetylenic
using redox diacetylenic vesicles on a sol-gel thin-film electrode [93]. Another example is the
vesicles on a sol-gel thin-film electrode [93]. Another example is the binding of cholera toxin to a
binding of cholera toxin to a glycosphingolipid ganglioside GM1 coated gold surface, which has
glycosphingolipidgangliosideGM1coatedgoldsurface,whichhasbeendevelopedintoasensitive,
been developed into a sensitive, low-cost sensor [94]. Potentiometric sensors measure potential or
low-costsensor[94]. Potentiometricsensorsmeasurepotentialorchargeaccumulationandhavebeen
charge accumulation and have been used to detect lipid antigens [95] such as amphiphilic
usedtodetectlipidantigens[95]suchasamphiphiliccholesterolusinglipidfilms[96,97],without
cholesterol using lipid films [96,97], without interference from ascorbic acid, glucose, urea or other
interference from ascorbic acid, glucose, urea or other proteins and lipids. In addition, label-free
proteins and lipids. In addition, label-free electrochemical biosensors have been developed for the
electrochemical biosensors have been developed for the detection of lipids such as low-density
detection of lipids such as low-density lipoprotein (LDL) with high sensitivity and specificity [98–
lipoprotein(LDL)withhighsensitivityandspecificity[98–100]. Thesesensorshavealsodisplayed
100]. These sensors have also displayed long-term stability and high reproducibility with small
sample volumes, which make for a promising clinical tool. However, a problem with
electrochemical detection of biomarkers is specificity of detection in complex biological samples, as
these platforms are more sensitive to even small perturbations in the background, which is always a
possibility in clinical measurements.
Biosensors2017,7,25 7of24
long-termstabilityandhighreproducibilitywithsmallsamplevolumes,whichmakeforapromising
clinicaltool.However,aproblemwithelectrochemicaldetectionofbiomarkersisspecificityofdetection
incomplexbiologicalsamples,astheseplatformsaremoresensitivetoevensmallperturbationsinthe
background,whichisalwaysapossibilityinclinicalmeasurements.
2.4.3. MechanicalBiosensorsDetectingLipids
Mechanical biosensors provide rapid and sensitive measurements without extensive sample
processing, ideal for clinical application. The two main mechanical sensing techniques are based
oncantileverandquartzcrystalmicrobalances(QCM),bothofwhicharelabel-free. QCMdetects
changesinresonancefrequencyonthesensorsurfacefromincreasedmassduetoanalyteminding[92].
QCM has been used to observe the formation of supported lipid bilayers in real-time [101] and
lipid exchange between a bilayer and vesicle [102]. The latter measures mechanical bending of a
receptor-functionalized microcantilever upon binding of the target molecule, as demonstrated for
LPS[103]. Anotherexampleisthemeasurementoftheinteractionbetweenamyloid-βandthecell
membraneasanearlyindicatorofAlzheimer'sdisease[104]. Amajordrawbacktocantileversensors
isthattheyoftenoperateinair,ratherthanliquidsamples,limitingtheirclinicalutility.
3. LipidBiomarkersforInfectiousDiseases
Rapiddiagnosisisimportanttohaltthespreadofbothendemicandemergingpathogens, as
wellasmulti-drugresistantorganisms[105]. Infectiousdiseasesarecausedbybacterial,viral,fungal
andparasiticorganismsandareroutinelydiagnosedusingculture,microscopy,serologyandgenetic
tests[106],allofwhicharetime-consumingandperformedbytrainedlaboratorypersonnel. Several
biosensors have been developed for diagnosis of infectious diseases [11], however all come with
challenges that limit their application in the clinic. Many biomarkers associated with infectious
diseasesareamphiphiles,whicharedifficulttodetect,especiallyinaqueousblood(Table2).
Table2.Selectlipidandamphiphilicbiomarkersusedforthediagnosisofinfectiousdiseases.
Biomarker Disease Location InteractingMolecules Reference
Sepsis Blood LBP,HDL,LDL [107,108]
Lipopolysaccharide holotransferrin [109]
(LPS) Urinarytractinfection Urine n.d. [110]
Antimicrobialresistance Anysample n.d. [111]
Lipoteichoicacid HDL,LDL,VLDL [108,112]
Sepsis Blood
(LTA) LBP,holotransferrin [109]
Lipoarabinomannan Urine n.d. [113]
Tuberculosis
(LAM) Blood HDL [15]
Lipomannan(LM) Bovinetuberculosis Blood HDL [84]
OmpK36porin Antimicrobialresistance Anysample n.d. [111]
LBP,HDL,LDL,VLDL,
Hemozoin(HZ) Malaria Blood apolipoproteinE, [114]
α-1-antitrypin
LBP:LPS-bindingprotein;HDL:high-densitylipoprotein;LDL:low-densitylipoprotein;VLDL:verylowdensity
lipoprotein;n.d.:notdetermined.
3.1. Sepsis
Sepsis is an infection of the bloodstream resulting in uncontrolled activation of the immune
system[115]. Morbidityandmortalityresultsfromorganfailure,whichcanbeavoidedwithearly
andeffectivetreatment,whichinturn,requiresrapiddiagnostics[116]. Thereareseverallipidand
amphiphilicbiomarkersassociatedwithsepsis. LPS(alsoknownasendotoxin)isaclassicpathogen
associated molecular pattern (PAMP) shed by Gram-negative bacteria. LPS activates the innate
immunereceptor, Toll-likeReceptor2, resultinginacytokinecascade, whichisthemechanismof
Biosensors2017,7,25 8of24
endotoxicshock[117]. ThepresenceofLPSinpatientbloodisaclearindicatorofsepsis. However,
detectionofLPSinaqueousbloodiscomplicatedbythemolecule’samphiphilicbiochemistry,which
drivesittoassociatewithhostcarrierlipoproteins[118]andothermoleculessuchasLPS-binding
protein (LBP), high-density lipoprotein (HDL), low-density lipoprotein (LDL), very low-density
lipoprotein (VLDL) and bactericidal/permeability-increasing protein [109]. Similarly, LTA is an
amphiphiliccellwallcomponentofGram-positivebacteriawhosepresenceinbloodalsoindicates
sepsis. StaphylococcusaureusLTAinhumanbloodpreferentiallybindstoHDL(68%), thentoLDL
(28%),andminimallytoverylowdensitylipoprotein(4%)[112]. LBPalsobindstoLTA[109]. These
associationscomplicatethedetectionofamphiphilicLPSandLTAinblood.
3.2. MycobacterialInfections
Lipoarabinomannan(LAM)isabiomarkerfordiagnosisoftuberculosis(TB)[119–122]. LAMis
anamphiphilicheatstablelipoglycan,whichformsanintegralcomponentofthemycobacterialcell
wall[119,120],andissecretedduringTBinfection. LAMhasprovedtobeanexcellentdiagnostictarget
especiallyinsmearnegativepatients(HIV-positive,pediatricpopulationandothers). Somelimitations
of LAM include assay cross reactivity with common oral flora like Candida and Actinomyces
that can result in lower predictive value of detection [123]. LAM assays can be improved using
standardized sample processing methods and more sensitive sensor technology, as well as more
specificantibodies[124]. LAMisreadilydetectedinurine[88,89],butnotinblood,likelybecause
itassociateswithHDLinblood[15]. Tothisend,lipoproteincaptureassaysthatutilizeantibodies
totargetHDLhavebeenusedtopulldownboundLAMinserumsamples[15,83,84]. Thismethod
targetshostpathogeninteractions,andcanbeappliedtootheramphiphilicbiomarkersinblood[83].
Pathogen biomarker based measurements can be used in both human and animal hosts [84,113].
However,thehighcostofsuchtechnology,andbiochemicalnatureofLAMarecurrentchallengesto
thedevelopmentofsuchassays[124].
3.3. AntimicrobialResistance
Theglobalspreadofantimicrobialresistanceunderlinestheneedforrapidandaccurateassays
forthedeterminationofresistantorganismspriortoantibiotictreatment. Acommonmechanismof
antibioticsisinhibitionofbacterialcellwallcomponents,whicharelipidoramphiphilicmolecules[125].
Modificationsofmembranelipidsarecommonmechanismsofantimicrobialresistanceinbacteria[126–128]
andthedirectdetectionofsuchchangeswouldsupporteffectivetreatment. Infact,targetingPAMPs
that are highly conserved, such as membrane lipids, for molecular diagnostics has a tremendous
benefitoverapproachestargetingmorerapidlyevolvingaspectsofbacterialphysiology(i.e.,PCR).
MALDI-TOF MS has been used to detect antibiotic resistance caused by lipid alterations [111].
E.g.,detectionofLPSalterationsthatreducethemoleculesnetnegativechargeresultinginresistance
toantimicrobialpeptidessuchascolistin,andOmpK36porinlossinKlebsiellapneumoniaeresulting
inresistancetocarbapenems. However,theinitialcostofsettingupaclinicalMALDI-TOFsystemis
prohibitivetowardapplicationinroutinediagnosticsettings.
3.4. Malaria
Hemozoin is an insoluble amphiphilic crystalline byproduct formed from the degradation of
bloodbyparasitessuchasPlasmodiumfalciparum,thecausativeagentofmalaria[114]. Hemozoinis
releasedintocirculationuponerythrocytelysis,anditspresenceinpatientbloodisadirectindication
ofmalarialinfection[129]. Detectionofhemozoinhasmostlybeenachievedusingmicroscopyand
flowcytometryinblood[130]. Theamphiphilicnatureofhemozoinhampersitsdirectdetectionin
bloodduetosequestrationbyhostimmunecomponents[129]suchasapolipoproteinEandLBP[114].
MethodslikeLipoproteincapturecanpotentiallybeadaptedforthedetectionofamphiphilichemozoin
inblood.
Biosensors2017,7,25 9of24
4. LipidBiomarkersforNon-infectiousDiseases
Non-infectiousdiseasesincludingcancerandcardiovasculardisordersarearesultofamalfunction
in normal physiological processes. Therefore, identifying specific biomarkers for diagnosing
non-infectiousdiseasespresentsamajorchallenge. Ratherthantargetingaforeignmoleculethatis
absentinahealthypatient,thediagnosisofnon-infectiousdiseasesoftenmeasuresirregularitiesin
expressionofhost-derivedmolecules[131,132]. Manylipidandamphiphilicmoleculesassociated
withhumanmetabolismcanbeusedasbiomarkerswhendifferentiallydetectedindiseasedversus
healthy individuals (Table 3). Detection of such biomarkers can potentially be used to determine
prognosis,monitorrecurrence,evaluatediseaseprogression,andpredictapatient’sriskofdeveloping
aparticulardisease[133].
Table3.Selectlipidandamphiphilicbiomarkersusedfordiagnosingnon-infectiousdiseases.
Interacting
Biomarker Disease Location Reference
Lipoproteins
Cholesterol Cardiovasculardisease Blood HDL,LDL [134,135]
Cancer Blood HDL,LDL [136–143]
Preeclampsia Blood HDL,LDL [144–147]
Lipotoxicity Blood HDL,LDL [148]
Triglycerides(TG) Cardiovasculardisease Blood LDL,VLDL [132,149,150]
Cancer Blood LDL,VLDL [136–143]
Preeclampsia Blood LDL,VLDL [144–147]
Lipotoxicity Blood LDL,VLDL [148,151,152]
Cardiolipin(CL) Cardiovasculardisease Blood LDL,HDL,VLDL [153]
Braintissue,
Cancer n.d. [37,154]
Prostatetissue
HDL: high-density lipoprotein; LDL: low-density lipoprotein; VLDL: very low density lipoprotein;
n.d.:notdetermined.
4.1. CardiovascularDiseasesandDisorders
Cardiovascular diseases (CVD) [155] account for 17.5 million deaths, accounting for 31% of
alldeathsworldwide[155]. Heartattacksandstrokes, mainlycausedbyablockagethatprevents
bloodfromflowingtotheheartorbrainaccountforabout80%ofallCVDdeaths. Inmanycases,
dyslipidemia, or abnormal levels of lipids in the blood, is a major mediator of CVD [156,157].
Itistypicallycharacterizedbyincreasedlevelsoftotalcholesterol, increasedlevelsoflowdensity
lipoproteincholesterol(LDL-C),increasedlevelsofTG,decreasedlevelsofhighdensitylipoprotein
cholesterol(HDL-C),modifiedfunctionoflipidmolecules,oracombinationofsomeorallofthese
factors [156–158]. Cholesterol is a major component of cell membranes, and contributes to their
structural integrity and fluidity [131]. Cholesterol is also a precursor of vitamin D and important
steroid hormones such as progesterones, glucocorticoids (cortisol), androgens (testosterone) and
estrogens [131,159]. Cholesterol homeostasis is critical to cardiovascular health. High cholesterol
can raise CVD risk [135,160], by creation of sticky plaque deposits along arterial walls, eventually
blockingbloodflow[125,160],causingheartattacksandstrokes. LDLandHDLareimportantcarriers
ofcholesterol. IncreasedlevelsofLDL-Ccancausecholesterolbuildupinarteries,resultingintheir
narrowing,raisingcardiovascularrisk[161]. HDLisascavenger,carryingcholesterolawayfromthe
arteriesandbacktotheliverforbreakdown[134]. AhighTGlevelcombinedwithhighLDL-Cand
lowHDL-Cislinkedwithfattybuildupsinarterywalls[132,149,150].
AssessingCVDrisklargelyinvolvesserumlipidprofiling,focusedonmeasuringtotalcholesterol,
HDL-C, LDL-C, and TG levels [162], largely by use of labeled optical immunoassay platforms.
Forinstance,theAlereCholestechLDX®Systemcombinesenzymaticopticaldetectionwithsolid-phase
technologytorapidlyandsensitivelymeasuretotalcholesterol,HDLcholesterol,triglyceridesand
glucoseinafingerprickofblood[163]. Cholesterolismeasuredenzymaticallyinseruminaseries
of coupled reactions that hydrolyze cholesteryl esters and oxidize the 3-OH group of cholesterol,