Micromachined sensors to detect surface stress changes associated with interactions between an immobilized chemically selective receptor and a target analyte are presented. The top isolated sensing surface of a free-standing silicon plate is prepared with a thin Au layer, followed by a covalent attachment of chemical or biomolecule forming a chemically-selective surface. Surface stress changes in air are measured capacitively due to the formation of an alkanethiol self-assembled monolayer (SAM). Detection of biomolecular binding in liquid samples is measured optically using the streptavidin-biotin complex and AM. tuberculosis antigen-antibody system used for clinical tuberculosis (TB) diagnosis

Carlen, E.T.

Hsiao, J.

Kim, E.S.

Traviglia, D.

Weinberg, M.S.

Zapata, A.M.

English

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Biomolecular Sensing using Surface Micromachined Silicon Plates

Carlen, Edwin

University of Twente Research Information

BIOMOLECULAR SENSING USING SURFACE MICROMACHINED SILICON PLATESA.M. Zapata , E.T. Carlen2, E. S. Kim1, J. Hsiao1 , D. Traviglia1 , M.S. WeinbergThe Charles Stark Draper Laboratory, Cambridge, MA, USA(Tel: 617-258-1114; E-mail: azapata@draper.com)2The University of Twente and MESA+ Institute for Nanotechnology, Enschede, The Netherlands(Tel: +31-53-489-2661; E-mail: e.t.carlen@ewi.utwente.nl)Abstract: Micromachined sensors to detect surface stress changes associated with interactions betweenan immobilized chemically selective receptor and a target analyte are presented. The top isolated sensingsurface of a free-standing silicon plate is prepared with a thin Au layer, followed by covalent attachmentof chemical or biomolecule forming a chemically-selective surface. Surface stress changes in air aremeasured capacitively due to the formation of an alkanethiol self-assembled monolayer (SAM). Detectionof biomolecular binding in liquid samples is measured optically using the streptavidin-biotin complex anda M. tuberculosis antigen-antibody system used for clinical tuberculosis (TB) diagnosis.Keywords: Surface stress sensors, micromechanical biosensors1. INTRODUCTIONAdvances in MEMS have facilitated the-t >>development of novel transducers for biochemical; sensing that rely heavily on mechanical energyD [1]. These types of sensors, including resonantand static platforms such as quartz crystalmicrobalances and microcantilever beams enablelabel-free assays that are faster, cheaper and lessB t cumbersome than conventional labeled methods[2]. The surface stress sensors presented here aremicrofabricated from thin layers of single crystalCD silicon. The thin layer is suspended with all edgesclamped to a silicon substrate; therefore,physically isolating the two plate surfaces. Onesurface is used for sensing and interfaces directlywith the sample solution and the other surface isused for displacement detection, transducing thesensing surface's response to chemical stimuli.This sensor offers a number of advantagescompared to other label-free, mechanicalplatforms: 1) static detection resolution is notaffected by quality (Q) factor damping in liquidsamples, 2) plate structures are more rigid thancantilever beams and can be easily functionalizedand probed using commercially available printingtechniques, and 3) unlike cantilever beams, thedetection surface is physically isolated from thesensing surface and can be easily integrated withdifferential capacitance read-out electronics andmicrofluidic handling systems for liquid sensingapplications [3]. The surface stress sensingmechanism is different than resonant masssensing, where the resolution of the resonant masssensors is typically reduced in a liquid mediumdue to the reduction of the resonator Q-factorcaused by the liquid. Techniques have beendeveloped to improve this problem [4], however,with increased sensor complexity. Surface stresssensors detect low frequency deflection changesof mechanical structures due to differential surfacestress changes of a sensing surface. Therefore, theresolution of the surface stress sensors isminimally affected by viscous damping. Surfacestress sensors are affected in aqueousenvironments include by the pressure head of thesample solution, surface tension of the samplesolution and ionic strength of the sample solution.2. THEORY AND DESIGNSurface stress has been previously describedmathematically as cij= bijy + by/bvjj [5], where thetensors can be represented as scalars for surfaceswith lattice symmetry of 3-fold or larger, as(Nm-1) is the surface stress, (Jm-2) is the surfacefree energy, and E the strain. The concept ofsurface stress implies that the surface stressperforms work when straining a solid structure. Inthin samples, surface stress can producemeasurable elastic bending and deflections.Small plate deflections due to a uniform axialsurface stress are used: (a) the plate material ishomogeneous with uniform thickness t, (b) t <b/I 0, where b is the smallest plate dimension, and(c) the maximum deflection wm<t/2 [6], and (d)831-4244-0842-3/07/$20.00c2007 IEEElarge deflection shearing forces and body forcesare not considered. Fig. 1 shows plate details.() (b).... 12. x_Assuminguniform axial stress on t e40_20 0Xw 4D/....-2', *''Lateral di fnce (ren) s A stres-1jFig. I (a) Plate dimensions and forceso wridth bthickness t, and Ms(shown compressive) (b) Bendingvdueto ts(c) Rectangular plate bending profiles fornea=2b and wp=305 nm (d) Center deflection Aw.Assuming uniform axial stress on the plate surface+ossso+w trs n emdu oa ntaas r a differential surface stress(as =pt so(z-th2)-a-s m(z+tl2), where 8 is the Dirac-deltafunction, generates a stress couple of radialpflexure moment M, shown in Fig.ps(b). This isCDt equivalent to a force F at the neutral surface nsuch that the resultant force and moment on theiedgeare equal to zero. This approximation is validnear the plate center (x=O) where all deflectionsare measured. The total plate deflection wmconsists of two terms: one term due to an initial0 °deflection w,., and an additional deflection Aw dueC | to a radial surface force induced by the adsorptionof the target molecule hebndi sensing surface. Inpractice, it is rare that suspended silicon plates areperfectly flat. All suspended plates presented herehave initial plate bending due primarily to theresidual stress in the nucleation layer [3]. Since w.is much larger than Aw, then wr must beconsidered when calculating Asa The total platebending can be determined by the deflectionproduced by the combination of a uniformlydistributed lateral force q (NM-2), which is relatedto w., and a uniform in-plane force F (Nm 1),which is related as. The bending of a rectangularplate with clamped edges is estimated as w(x, y) =w'5 /Fo (I + yus) F(x, y) [3], where y is an estimatedconstant, F(x, y) is a shape function, Fo F(0, 0).Fig. l(c) shows an example of the rectangularplate bending. The differential surface stresschange can be estimated as AuszAw/w6[3]. Fig.1(d) shows the dependence of AQs and Aw on w.Although, the nucleation layer covers the entireplate surface in this article, surface stress induceddeflections can be increased by partially coveringthe plate surface, therefore, the bending momentdue to the edge of the nucleation layer adds to thetotal bending moment of the plate.3. MICROFABRICATIONThe silicon plates were fabricated usingconventional surface micromachining processeswith silicon-on-insulator (SOI) substrates anddeep reactive ion etching (DRIE). Capacitivesensors were fabricated using seven lithographysteps. Fig. 2(a) highlights the essential aspects ofthe microfabrication process. A 1 [pm-thick oxide(LTO1) etch mask film is deposited on the SOIsubstrates in a low-pressure chemical vapordeposition (LPCVD) system, followed bylithographical patterning and reactive-ion-etching(RIE) to define the sense plate. A 1.0 Ftm thicklow-stress LPCVD nitride anchor layer isdeposited, patterned, and RIE, thus defining theupper electrode anchor layer, shown in Fig. 2(a,ii).C* 9 vx 3.- - waif 4 Irrl | <~-<S'46 3N0>iNa2M,dafedyad& ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~,, <QXXX 9Q<X& .~...mSWed~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~,~~~~LT02~ ~ ~ ~ ui*Fig. 2: (a) Simplified sensor microfabricati on processflow(b) Top view SEM of released sensor structure (c)SEM ofreleased capacitor structure.The remaining LTO1 mask layer is removed in adilute hydrofluoric acid (HF) solution. A 3 rim-thick LPCVD oxide layer (LT02) is depositeddefining the separation gap. The LT02 layer isthen patterned and etched as shown in Fig. 2(a,iii).The 4 [pm-thick LPCVD low-stress polysiliconlayer is next deposited, patterned and etched (RIE)thus opening the release holes and defining theupper polysilicon plate structure. Electrical832I-4244-0842-3/07/$20.00c2007 IEEEcontacts are formed by metallization (30 nmCr/500 nm Au) and liftoff, shown in Fig. 2(a,iv).Next, the handle layer is patterned and etchedusing DRIE, shown in Fig. 2(a,v). Next, thehandle layer is patterned and etched using DRIE,shown in Fig. 2(a,v). The remaining oxide layersare then removed in a 3:1 HF:H20 solution,shown Fig. 2(a,vi). Sensor structures for opticaldetection were also fabricated from SOI substratesusing two lithography steps to define and releasethe sensing plate, shown in Figs. 2(a,i) and 2(a,v).Studies of biomolecular interactions in liquidsamples are facilitated by fabricating a 3-D silicon-PDMS (polydimethylsiloxane) hybrid structureusing simple replica molding and bondingprocesses [7]. The PDMS structure is made from amaster mold from a silicon substrate. A curingagent and PDMS prepolymer (Sylgard 184Silicone Elastomer, Dow Corning) are mixed in> t 1:10 weight ratio, and degassed in a vacuumdesiccator. The prepolymer mixture is then pouredonto the master mold and cured at 70°C. Afterr Qt curing, the PDMS mold replicas are peeled offfrom the mold and cut to fit the dimensions of themicrosensor die. The final structure is a 2-mmthick square layer containing a v-shaped channelwith dimensions of 1.75 ptm x 3.5 ptm x 200 ptm,shown in Fig. 3. Both silicon and PDMS surfacesCFig. 3: PDMS microfuidulic cell bondedc to 2-senseplate device (900 ,un-diameter), inlet/outlet tubing.are activated by oxygen plasma (100 mTorr, 100W) for 30 s. The process generates hydrophilicsurfaces that can form a permanent bond. Plasmatreated surfaces are immediately aligned andcontacted to initiate bonding, and left for 20 min.while applying pressure. Coring of the PDMS andinsertion of tygon tubing (Dow Corning, No. 2tubing) enables the off-chip fluidic connections.The volume of reagent needed to fill the v-shapedcell is 6 tL.4. EXPERIMENTS AND RESULTSSelf-assembling alkanethiol monolayers on thinAu(1 1 1) layers are used for plate bendingcharacterization in ambient air. The high affinityof thiols for gold surfaces facilitates their use togenerate well-defined organic surfaces with awide range of chemical functionalities displayedat the sensing interface. The bottom side of thesilicon plates is sputtered coated with a 30 nm Aunucleation layer (8 nm Ti adhesion layer).Capacitance measurements are done with custom-built electronics circuitry, in a low-noise common-mode rejection configuration, shown in Fig. 4(b).( (Fotsmconora c oondfsured9 eFig. 4l (a Sesr Irsmesue 1-oea ethiol1ur ~~~~~~1S "IV0 vpr f) 204mcrut (c tes fitr_ (d)8reposeto wher Aifferettedwith w6=305 nm, g=3.2 pu m and b=480,u m.Sense and reference capacitors are driven by amodulation signal, and the differential capacitanceis converted to a voltage with a charge-integratingamplifier. Prior to testing, the sensor baseline isfirst established for 60 s before exposing thesensing surface to the test vapor. The Au coatedsensing surface is then exposed to the alkanethiolvapor from a large sealed reservoir of liquidz1.3mL for a time of 300 s. Fig. 4(b) shows the sensorresponse of a test device. Prior to exposure, theoffset voltage of z500 mV is consistent withinitial plate deflection of Wdz305 nm. Theexposure of the sensing surface at t=60 s to vaporphase 1-dodecanethiol (Aldrich No. 471364)results in a surface stress change AQsz0.42 Nm 1smaller than values previously reported by ourgroup using optical interferometry [3], but similarto other reports [8].Deflections induced by binding of a biologicalreceptor immobilized at the plate's sense surface833I4244-0842-3/07$20.00c2007 IEEEto an analyte flowing in a liquid matrix, we]studied using the optical lever detection methccommonly used with atomic force microscopy. Aimmunological diagnostic model for Atuberculosis based on the binding of tibacterium's Early Secretory Antigen Target ((ESAT-6) to its corresponding polyclonantibody (anti-ESAT-6) was used. The ESAT-antigen was immobilized at the Au coated surfacusing dithiobis- [succinimidylproprionate] (DSPa thiolated crosslinker for Au binding, andprimary amine-reactive NHS ester for amidcrosslinking to proteins. Following DSP surfacactivation, the sensor is incubated overnight wilESAT-6, and then placed on the optical lever seup. All reagents are introduced into the PDMchannels manually with 1-ml syringes and 19G;needles. Fig. 5 shows and example of the senseresponse to 120 nM anti-ESAT-6. The data has4 80 7et"-_xa0 S0,-4OO..2200?-6N .InjectionnoiseIiIILI.2% glycerol/ 7.5 pM BSA in 120 nrM anti-ESAT6PBS 2% glycerol/PBS in 2% glycerol/PBSI I I I I I I t0 20 40 60 ik10 olime (mr)Fig. 5: Sensor response to anti-ESAT6 introduction.not been normalized for pressure and temperatureperturbations, therefore the sample injection spikeand temperature relaxation can be seen. Thesensor' s baseline is first established with PBScontaining 2% glycerol, representative of the anti-ESAT-6 matrix. No deflections due to non-specific binding of 7.5 ptM BSA solution areobserved. The response to BSA was essentiallythe same as observed for the PBS solution.Introduction of 120 nM anti-ESAT-6 generated ameasurable response markedly different, and mostlikely attributed to antigen-antibody binding. Thenoise during binding is likely due to bubbles in thePDMS channels and observed in previousexperiments. Larger antibody concentrations (notshow) yielded larger response magnitudes.Surface plasmon resonance (SPRImagerlI, GWCTechnologies) studies confirm the associationbinding rate for the TB system does not follow theexpected Langmuir adsorption kinetics (k 19000M-'s-1), likely due to non-specific binding.5. CONCLUSIONSThe microfabricated surface stress plate sensorspresented here are advantageous, in our view,compared to cantilever beam structures in threeimportant ways: 1) not affected by degradation ofquality factor, 2) plate structures are less fragilemore than beams and 3) the detection surface isphysically isolated from the sensing surface andeasily adapted to other readout techniques inliquid solutions. Although the microfabricationtechnology used to manufacture the plate sensorsis more complex than that used for cantileverbeams, surface micromachining technology is wellestablished and provides a path to low-cost massproduction of sensors. The measurementspresented here indicate that the plate structureswith electronic readout are as sensitive as thecantilever beam-optical readout systems.6. ACKNOWLEDGMENTSThe authors thank Draper Laboratory for funding,Connie Cardoso, Mert Prince, and ManuelaHealey for fabrication assistance, John Lachapellefor electronics, Caroline Kondoleon forpackaging, and Fusion Antibodies for thetuberculosis immunological reagents.REFERENCES[1] N.V. Lavrik, M.J. Sepaniak, P.G. Datskos, Rev. of Sci.Instruments, vol. 75, No. 7, pp. 2229, 2004.[2] P. Skladal, J. Braz. Chem. Soc., vol.14, No.4, pp. 491,2003.[3] E.T. Carlen, M.S. Weinberg, C.E. Dube, A.M. Zapata,J.T. Borenstein, App. Phys. Let., vol.89, pp. 173123, 2006.[4] T. Burg and S. Manalis Appl. Phys. Lett., vol.83, No.13,pp.2698,2003.[5] R. Shuttleworth Proc. Phys. Soc.,vol.A63, pp. 444, 1950.[6] S. Timoshenko, Theory of Plates and Shells. McGraw-Hill Book Company, Inc., 1959.[7] B.H. Jo, L.M. Van Lerberghe, K.M. Motsegood and D. J.Beebe, J. Microelectromech. Syst.,vol.9, No.1, p. 76, 2000.[8] M. Godin, P. Williams, V. Tabard-Cossa, 0. Laroche,L. Beaulieu, R. Lennox, and P. Grtitter, Langmuir, vol. 20,p.7090,2004.834-4244-0842-3/07/$20.00c2007 IEEE-10~~4De1114,pqppô14 VfP0k0 -- I"mmwmII0 10.:.. ............................................a ..rIt............................................

Biomolecular sensing using surface micromachined silicon plates

Theory ofPlates and Shells.

https://doi.org/10.1109/SENSOR.2007.4300259

Biomolecular sensing using surface micromachined silicon plates

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University of Twente Research Information