BE/EE189DesignandConstructionofBiodevices
Project2:“On-chip”Real-timePolymeraseChainReaction(RT-PCR)
Week3–OpticalDetectionandSystemTest
Systemreadyforin-classtestingon:1Mar2016
Reportdue:10Mar2016
Instructions
1. Tobecompletedingroupsof2-3.
2. Yourthermocyclingshouldbeworkingbynow.
3. Handle all optics very carefully. Do NOT touch the surfaces of the lenses, filters,
objectivelensesandcamerasensors.Whenindoubt,plsask.
4. Pleasefeelfreetoaskfordemonstrationsofhowtheopticsshouldbemounted.When
indoubt(andifthere’snoonetoconsult),visitwww.thorlabs.comforsomeideas.We
got all our opto-mechanic components there. It should get more intuitive as you start
playingaroundwiththecomponents.
5. Onlyonelabreportisrequiredofeachgroup.
6. YourlabreportshoulddocumentclearlybutconciselyhowyourdeviceandVIwork.The
labreportwillbegradedpartiallyonfunctionalityofthesystem.However,ifyourdevice
doesn’t function perfectly, do not fret. A large emphasis will placed on understanding
theshortcomingsandpointsoffailure(ifany)ofyoursystem.
Opticaldetection
Epifluorescencedetectionwillbeused.Theopticaldetectionsystem(essentiallyahomemade
microscope) consists of an LED light source (see spec sheet attached), a filter set (excitation,
dichroicandemission),anobjectivelens(infiniteconjugate,specsheetattached),atubelens
andacamera.
Figure1.Suggestedsetup.
Q1.Whatisthefocallengthoftheplano-convexlens(50mm,75mmor100mm)?Howdidyou
finditout(withoutreferringtoitspartnumber)?
Q2.Howwouldyouorienttheplano-convexlens?Shouldtheflatsurfacefacetheobjectiveor
thecamera?Hint:Oneorientationworksbetteratfocusingacollimatedlightintoapoint.
Q3.Whatisthemagnificationofthemicroscopesysteminthesetup?Howdidyoufindout?
ImportationConsiderations–
1. Sincefluorophorebleachingcanoccur,itisprudenttoturnontheLEDonlyduringsignal
acquisition.
2. Atwhichpartofthecycleshouldthesignalacquisitionbedone?
3. How would you determine the background signal and eliminate/consider that in your
data?
4. Since background light contributes to noise, how would you maximize the signal-tonoiseratio?
Your LabView program should be able to acquire and plot a replication curve. A replication
curveisrequired.Ameltcurveanalysisisnotrequired.
Systemtest
Atubeofcompletereactionmix(containingtemplateDNA,DNAprimers,polymerase,dNTPs,
SYBRetc)willbegiventoyouatthebeginningofclass.We’retotestforthepresenceofaGMO
gene.Here’sanexcerptfromtheBioRadGMOinvestigatorkitwe’reusing,justsowehavean
ideaofwhatwe’retryingtoamplify:
“TheGMOInvestigatorkitisdesignedtotestforthepresenceoftwodifferentGMO-associated
DNA sequences: the 35S promoter of the cauliflower mosaic virus, and the terminator of the
nopaline synthase gene of Agrobacterium tumefaciens. These DNA sequences are present in
mostoftheGMcropsthatareapprovedfordistributionworldwide.”
Testthefunctionalityofyoursystemduringtheclass.
Atypicalamplificationplotlookslikethegraphinfigure2.
Figure2.Typicalamplificationplot.
Q4.Whywouldthecurvelooklikethis?
Q5. Did you get a typical amplification curve? What are the sources of noise and design
inadequacies?Howcanthesystembeimproved?
PostlabQuestions
Post-labQn1:Duetothetimeandresourcelimitations,weareonlyabletodoaveryrough
simulationofwhatan“on-chip”RT-PCRdeviceshouldbeableto.(Weprobablyshouldn’teven
callitanon-chipsystem.)Whatarethepotentialadvantagesofatrueon-chipplatform?Think
alongthelinesofsamplepreparation,reagentmixing,reagentvolume,portability,etc…
Post-labQn2:Withoutconsultingcurrentliterature,designafullyon-chipPCRdevice.(A
conciseblockdiagramofdesignfeatureswilldo.Acompleteblueprintisnotrequired.)Thispart
oftheexerciseisnotgradedbasedonfeasibility(butitcannotbecompletelyoutofthisworld).
Justbeimaginativeandhavefun!Startfromanintacttissuesample.Theendpointinmind
wouldbethedeterminationoftheabsenceorpresenceofaspecificDNAsequence.
Post-labQn3:Readsomerecentpapersonon-chipPCR.Whichpartofyourimaginarydesign
hasalreadybeentackled?(Citethepapersyoureferredto.)Whataretheremaining
engineeringchallenges?(Ifthereisn’tanyremaining,thinkaboutanydesignopportunities.
Includeassumptionsmade(e.g.availabilityoftechnologiesand/orprocesses).