Microfluidics & BioMEMS Biomolecules on a chip Biomolecular detection

1. Microfluidics & BioMEMSBiomolecules on a chipBiomolecular detection 7.3.2018 Tarja Nevanen VTT Technical Research Centre of Finland

2. Definition of BioMEMS • BioMEMS: • BiologicalorBiomedicalMicro-Electro-MechanicalSystems • Alsodeviceswithoutelectro-mechanicalcomponents, (DNA and proteinarrays) areoftencategorizedunderBioMEMS • Bashir 2004: ”devicesorsystemsthatareused for processing, delivery, manipulation, analysis, orconstruction of biological and chemicalentities • lab-on-a-chip (LOC) and micro-totalanalysissystems (µTAS): bioanalyticalsystemsscaleddown and integratedonto a single chip.

3. Dimensions • MEMS deviceshavedimensionsrangingfrom 100 nm to 1 mm • Diameter of DNA alpha helix: 2 nm • Proteins: 1- tens of nanometersscale • Viruses: 30-100 nmscale • Bacteria: 0.2-10 µm • Mammalian cells: 10-30µm

4. BioMEMSapplications • Global BioMEMS market expected in 2018: $6.6 billion (forecast made byYoleDeveloppement) • MEMS sensors for thebiologicalsystems, no biocomponentincluded • Pressuresensorse.g. bloodorintraocularpressuresensors • Inertialsensorse.g. defibrillators, pacemakers • Hearing-aidtransducer • MEMS sensorshavingbiocomponent • Diagnosticdevices • Medicalapplications • Tools for research

5. DrivingforcestowardsBioMEMS 1. • Diagnostics • Demandfor higher-qualitymedicalcare: • Preventivecare • Minimallyinvasiveprocedures • Personalizedmedicine • Point-of-caretesting, home testing • Continuousmonitoring • Solutions needed for ”Medicalcare to all” • Simple, user-friendly • Low-cost • manufacturing • powerconsumption • Lesswaste Jung 2015

6. Whymicrosystems for diagnostics? A typicalmicrofluidicsystemmoves, mixes and controlssmallvolumes of fluids (nanolitres) componentsinclude: needles, channles, pumps, mixers, valves, sensors, filters, dispensers, reservoirs A typicalpoint-of carediagnosticmicrofluidicsystemusesbiofluids as a sample. Device performssampling, samplepreparation, analytedetection, signalamplification (data analysis and resultsdisplay) Microscalediagnosticsystemsenable: Miniaturization lowersreagent and sampleconsumption lowpowerconsumption fasteranalysis Automation higherthroughput controlledenvironment: decreasedriskof contamination bettercontrol of assayparameters reducesneeds for highlyskilledpersonnelas endusers Integration(themain challenge) sampling,samplepreparation,analysis, detection, data transmission multiplexedanalysis

7. DrivingforcestowardsBioMEMS 2. • Medicalapplications • A greatchallenge: integration of diagnosticdeviceswiththerapeuticdevices • Commercial products ( according to YoleDeveloppement) • Kidneydialysismonitoring (pressuresensors) • Hearing aids (siliconmicrophones, microelectrodes) • Smartpills for imaging • Microneedles for transdermaldrugdelivery • Drugdelivery: implantabledevices (passive / responsive), Transdermaldelivery ( e.g. microneedlearrays), smartpills • Drugdevelopment: • Automated, multiplexed, high-throughputscreens for drugcandidatelibraries

8. DrivingforcestowardsBioMEMS 3. • Researchtools • For omics • Genomics, peptidomics, transcriptomics, proteomic, glycomics, lipidomics etc. • Systems biology • High-throughputstudies • Lowerconsumption of valuable samplesand reagents Alberts et al.

9. DrivingforcestowardsBioMEMS4. Novelpossibilities • Single cellanalysis / function in a realtime • Measurements of mRNA, protein, chemicalprofiles, oxygen, pH • Wholegenomesequencing • Also as a function of controlled stimulus • Understanding of basiccellularfunctions ( e.g. apoptosis, differentiation,) and theirimplications on diseasestates (e.g. malignacy) • Novelbiologicalfindings: exosomes • ”Human-on-a-chip” • ”Organ-on-a-chip” Yeo (2016) Bashir (2004)

10. Specificfeaturesintroduced to MEMS bybiomolecules • BIOMOLECULES AND CELLS ARE SUBJECTED TO ANON-NATURAL ENVIRONMENT! • Fabrication /constructionsolutions • Maintain the functionality of thebiomolecules (thermo-sensitive) • Preservestabilityinbiointerface and in packaging • Bioapplications • Currentapplicationsminiaturizedortotallynovelapplications • Properties of thesamples and analytesvary • Biocompatibility(interaction of thematerialwithe.g.humanbody) • Concern for in vivodevices (experiencefrome.g. pacemakers) • Safetyissues • prevention of cross-contamination/ infection • materialchoices, costs, life cycle of thedevice

11. Nucleicacids in microsystemsas componentsortargetanalytes

12. Nucleicacids • DNA versus RNA • DNA in procaryotes and eucaryotes • RNA in viruses (also DNA) • Genes and non-codingsequences • Notice the differencies in stability, single/doublestranded,sensitivity to contaminationin BioMEMSapplications • Location • Eukaryotes (humans, animals, plants): DNA is in nucleus (membrane-boundedintracellularcompartment), tightlypacked in chromosomes • Prokaryotes (e.g.bacteria): no compartment for DNA, additionalsmallgeneticelements: e.g. plasmids • Viruses: DNA/RNA packed inside the virus coat • Notice the location and packingmode of the geneticelementwhendesigningBioMEMSapplications for nucleicaciddetection, payattentionespecially to thesamplepreparationrequirements

13. DNA in BioMEMS • Convenient to handle (stable) • Manyprotocols and deviceconstructions • Isolation, digestion, amplification, hybridization, detection • Compatible with manysurfacematerials (proteinsmaydenaturateand losetheirfunctionality) • Single stranded DNA/RNA mayhaveunspecificattachment to surfaces • Self-complementarity is a unique feature ! • Utilized in identification, immobilisation, construction, isolation, amplification

14. The double-helixstructure of DNA Doublestrand (ds), single strand (ss) Long and narrowmolecule Linear, circular Single DNA strandrecognizes a complementarysequence on another single strand of DNA= Hybridization Important in amplification (PCR) and constructs (targetting, aptamers, Origamis) Veryspecific Alberts et alMolecularbiology of the cell

15. Semiconservative = both DNA strands act as a template for new DNA Aim: identicalcopies Amount of DNA: 2n , n= cycles of replication Replication (important in PCR applications) In vivo: naturalconditions, manyassisting proteinsareincluded In vitro (PCR): non-naturalconditions (temperature, buffer, thermostablepolymerases primers) Parentalstrands Primer: a small sequence of a single-stranded DNA. Attaches to the template DNA and initiates replication Alberts et al

16. Typicalgeneticanalysis in macroscale Lagally and Mathies (2004) J.Phys.D:Appl.Phys 37: R245-

17. BioMEMSapplication 1: DNA extraction - fromwholeblood • Wholebloodcontainsredbloodcells, white bloodcells (leucocytes, manydifferenttypes, sizesvary), platelets, plasma (proteins, lipids and smallmolecules) • DNA is in the nucleus of white bloodcells. • One way to extract DNA fromwholeblood is to trap the white bloodcellsby a microfilter (whileeverythingelseflowsthrough), thenlyze (destroy) the white cells and extract DNA bysilica. Circulating human blood. Photo by Bruce Wetzel and Harry Schaefer. Figuresfrom Li et al. Sensors and Actuators A, 2007, 139: 139-144.

18. An example: Genomeanalysis of a single cell(Marcyet al2007, 450 citations (2018)) • Amplification of a genome of a single cell • Notallinteresting and relevantmicro-organismscanbecultivated • Need for analysis of the genome of individualcellsisolatedfrome.g. soil, geysirs • MultipleDisplacementAmplification (MDA) • 60 nlreactions, 107copies of the genome • Compared to standard 50µl reaction • Benefits: • Non–specificsynthesislowered, amplificationbiasreduced (thesedrawbacks of MDA in standardscale) • Highqualitymaterial to sequencing

19. Marcy et al 2007

20. BioMEMSapplications2.DNA analysis, sizing, separation Determinationof the size of DNA or DNA fragmentscutbyrestrictionenzymes Electrophoresisseparates DNA fragmentsbased on theirsize. The nucleic acids are transported in a capillary or microfluidic channel by an electric field Calibrationladderas a reference. Commerciallyavailable(e.g. Agilent, CaliperLabChip) Benefits: lowerconsumption(1 µl sample and reagents) highsensitivity (0.1 ngfragments) reducedassaytime(12 samples in 30 min) highdegree of automation and data handling Application potential: -Researchtool search for positiveclones, qualitychecking -Forensicstudies Geneticfingerprinting: highlyvariablerepeatingsequences, differentnumbersbetweenindividuals identification of humanremains, paternitytestingetc

21. BioMEMSapplications 3: DNA as a constructionmaterial • Used in nanoscalestudiesalready • DNA hybridizationmakespossible to design 2-3D structuresfrom DNA and also • to functionalize the constructs • DNA origames • Aptamers Pilo-Pais et al 2011