5 páginas, 10 figuras.-- PACS: 42.25.-p; 42.82.-m; 85.85.+[EN] We show the design, fabrication and testing of micro/nanobiosensor devices based on highly sensitive optoelectronic and nanomechanical transducers. Most of the devices are fabricated by standard Silicon CMOS microelectronics technology according to a precise design for achieving a high sensitivity for biosensing applications. Three biosensors have been developed: (a) a Surface Plasmon Resonance biosensor, (b) an integrated, Mach-Zehnder micro/nano interferometer device based on optical waveguides, and (c) nanomechanical biosensors based on microcantilevers.[ES] En este artículo se muestra el diseño, la fabricación y la caracterización de dispositivos micro/nanobiosensores basados en transductores optoelectrónicos y nanomecánicos. La mayoría de los dispositivos se fabrican con tecnología estándar de Si compatible CMOS, después de un cuidadoso diseño de los transductores para conseguir biosensores de alta sensibilidad. Se muestran tres tipos de biosensors: (a) un biosensor de Resonancia de Plasmón Superficial (b) un micro/nano Ínterferómetro integrado Mach-Zehnder basado en guías de ondas ópticas y (c) biosensores nanomecánicos basados en micropalancas.Peer reviewe

Calle Martín, Ana

Domínguez, Carlos

Lechuga, Laura M.

Tamayo de Miguel, Francisco Javier

Revista Mexicana de Física

Domínguez Horna, Carlos

Digital.CSIC

PLENARIA REVISTA MEXICANA DE F´ISICA S 52 (2) 94–98 FEBRERO 2006Nanobiosensors based on optoelectronic and nanomechanical transducers forgenomic and proteomic applicationsL.M. Lechuga, J. Tamayo, A. Calle, and C. Domı´nguezGrupo de Biosensores, Centro Nacional de Microele´ctronica (CNM), CSIC,Isaac Newton, 8 – 28760 Tres Cantos, Madrid (Spain),e-mail:laura@imm.cnm.csic.esArticulo invitadoWe show the design, fabrication and testing of micro/nanobiosensor devices based on highly sensitive optoelectronic and nanomechanicaltransducers. Most of the devices are fabricated by standard Silicon CMOS microelectronics technology according to a precise design forachieving a high sensitivity for biosensing applications. Three biosensors have been developed: (a) a Surface Plasmon Resonance biosensor,(b) an integrated, Mach-Zehnder micro/nano interferometer device based on optical waveguides, and (c) nanomechanical biosensors basedon microcantilevers.Keywords: Biosensor; nanotechnology; optoelectronics.En este artı´culo se muestra el disen˜o, la fabricacio´n y la caracterizacio´n de dispositivos micro/nanobiosensores basados en transductoresoptoelectro´nicos y nanomeca´nicos. La mayorı´a de los dispositivos se fabrican con tecnologı´a esta´ndar de Si compatible CMOS, despue´sde un cuidadoso disen˜o de los transductores para conseguir biosensores de alta sensibilidad. Se muestran tres tipos de biosensors: (a) unbiosensor de Resonancia de Plasmo´n Superficial (b) un micro/nano interfero´metro integrado Mach-Zehnder basado en guı´as de ondas o´pticasy (c) biosensores nanomeca´nicos basados en micropalancas.Descriptores: Biosensores; nanotenologia; optica integrada.PACS: 42.25.-p; 42.82.-m; 85.85.+j1. IntroductionThere is an increasing interest in systems based on mi-cro/nanotechnologies for ultrasensitive and miniaturizedbiosensors [1]. Genomic and proteomic sensing are fieldswhere new laboratory analysis (faster, direct, more accu-rate, selective, with high throughput, and cheaper than con-ventional methods) are required. On the other hand, newmethods allowing label-free and real time measurement ofsimultaneous interactions (such as antigen/antibody or DNAhybridization) must be developed. A highly multidisci-plinary approach including microelectronics, MEMS, mi-cro/nanotechnologies, molecular biology, nanobiotechnol-ogy and chemistry are needed for the implementation of suchnew analytical devices [2]. Biosensing devices fabricatedwith optoelectronics and MEMS micro/nanotechnologies arepowerful devices which can fulfill these requirements. In thisline, our group is working on two different approaches [3]:(A) A platform based on optoelectronic biosensors(evanescent wave detection). Two optical biosensorshave already been developed: a portable Surface Plas-mon Resonance Sensor (presently being marketed)and an integrated Mach-Zehnder interferometer de-vice. For the latter, the use of standard Si microelec-tronics technology makes possible the integration ofoptical, fluidic and electrical functions on one opticalsensing circuit in order to obtain a complete lab-on-a-chip. A limit of detection close to femtomolar isachievable with this sensor in a direct format [4].(B) A platform based on nanomechanical biosensors. Mi-crocantilever biosensors are a new class of high sen-sitivity biosensors capable of performing local, highresolution and label-free molecular recognition mea-surements [5]. Moreover, nanomechanical biosen-sors based on microcantilevers have recently been re-ported as a promising alternative to current DNA-chips, promising permiting real-time monitoring ofDNA without a need for labeling. For that reason, weare working on the development of a portable multi-biosensor microsystem based on an array of micro-cantilevers [6] able to detect analytes with femtomolarsensitivity, and capable of discerning single base vari-ations in DNA strands.2. Receptor immobilization at the nanometerscaleFor biosensing purposes, a layer of receptor molecules (pro-teins, DNA,. . . ) that are capable of binding the analytemolecules in a selective way must be previously immobilizedon the transducer surface. The immobilisation of the receptormolecule on the sensor surface is a key point for the final per-formance of the sensor. The immobilization procedure mustbe stable and reproducible, and must retain the stability andactivity of the receptor. One of the most promising strate-gies is the covalent immobilization on gold-coated surfacesusing thiol self-assembled monolayers (SAM) [7]. For exam-ple, a widespread method is the functionalization of ss-DNAwith an alkane chain termed in a thiol (-S-H). Sulphurs formNANOBIOSENSORS BASED ON OPTOELECTRONIC AND NANOMECHANICAL TRANSDUCERS FOR. . . 95a strong bond with gold, and thus thiol-derivatized ssDNAspontaneously forms a single self-assembled monolayer uponimmersion on clean gold surfaces. This can be applied also tosilicon surfaces, using silane monolayers covalently attachedthe SiO2 or Si3N4 sensor surfaces [7]. Several aspects mustbe taken into account in the development of the immobiliza-tion procedures such as the non-specific interactions, an opti-mized surface density of the receptor in order to prevent sterichindrance phenomenon, or the regeneration of the receptor.We have developed immobilisation procedures at thenanometer-scale which seek to fulfill all the requirements de-scribed above, based on thiol-chemistry, silanization or es-terification, depending on the type of sensor surface and theapplication [7]. Different examples will be shown in the fol-lowing, in the description of each biosensor platform.3. Optoelectronic biosensors platformIntegrated optical (IO) devices are increasingly being usedas transducers for optochemical sensing applications [7]. IOdevices combine high sensitivity, mechanical stability, minia-turization and the possibility of mass-production. Most of theintegrated optical sensors make use of the evanescent field de-tection principle for sensing[8]. In an optical waveguide, thelight travels inside the waveguide, confined within the struc-ture by Total Internal Reflection (TIR). A detailed study ofhow light travels inside the waveguide shows that the lightis transmitted through a model of the electromagnetic fieldcalled “guided modes” (as shown in Fig. 1). Although light isconfined inside those modes, part of it (evanescent field, EW)travels through a region that extends outward, around a hun-dred nanometers, into the medium surrounding the waveg-uide (see Fig. 1). This EW field can be used for sensingpurposes. When a receptor layer is immobilized onto thewaveguide, as shown in Fig. 1, exposure of this surface tothe partner analyte molecules produces a (bio)chemical inter-action, which takes place on the surface of the waveguide andinduces a change in its optical properties that is detected bythe evanescent wave. Thus, evanescent wave sensors are se-lective, sensitive devices for the detection of very low levelsof chemicals and biological substances, and for the measure-ment of molecular interactions in-situ and in real time [7].FIGURE 1. Biomolecular interaction sensing by the evanescentwave detection principle in an optical waveguide sensor.3.1. Surface plasmon resonance biosensorOne of the best known and most developed EW biosensorsis the Surface Plasmon Resonance (SPR) sensor, because ofits sensibility and simplicity [9,10]. Surface plasmons areelementary excitations which result from a collective oscil-lation of the free-electron plasma at a metal-dielectric filminterface. In a SPR sensor, a thin metal film (usually Au)is evaporated on the dielectric material surface. The sensingmechanism is based on variations of the refractive index ofthe medium adjacent to the metal sensor surface during theinteraction of an analyte to its corresponding receptor, pre-viously immobilized at the sensor surface in the region ofthe evanescent field. The recognition of the complementarymolecule by the receptor causes a change in the refractiveindex, and the SPR sensor monitors that change. After themolecular interaction, the surface can be regenerated using asuitable reagent to remove the bound analyte without dena-turing the immobilized receptor.We have developed a home-made portable SPR sensorprototype (see Fig. 2) as a highly sensitive field analyticalmethod for environmental monitoring. As a proof of its util-ity in the detection of pathogens, we have determined sev-eral pesticides, such as the chlorinated compound DDT andthe neurotoxins of the carbamate (carbaryl) and organophos-phorus types (chlorpyrifos). For the determination of thesecompounds, a binding inhibition immunoassay was applied.For DDT, the assay sensitivity was evaluated in the 0.004-3545g µg/l range of pesticide concentration by the determi-nation of the limit of detection (0.3 g µg/l). For carbaryl,the dynamic range of the sensor is 0.12-2 g µg/l, a detectionlimit of 0.06 µg/l. Similarly the immunoassay for chlorpyri-fos determination afforded a high sensitivity working in the0.02-1.3 g µg/l range. Figure 3 shows the calibration curvesobtained from the pesticides carbaryl and chlorpyrifos, re-spectively.FIGURE 2. Portable SPR sensor prototype system including sensor,optics, electronics and flow delivery system.Rev. Mex. Fı´s. S 52 (2) (2006) 94–9896 L.M. LECHUGA, J. TAMAYO, A. CALLE, AND C. DOM´INGUEZFIGURE 3. Calibration curve for the immunoassays determination by using SPR technology of (left) Chlorpyrifos (right) Carbaryl.The reusability of the sensor was demonstrated after 250assay cycles, without significant variations in the averagemaximum signal. This immunosensing technique, togetherwith the portable surface plasmon resonance sensor devel-oped, can be applied as a fast, cost-effective field-analyticalmethod for the monitoring of any chemical and biologicalcompound if the corresponding receptor is available.FIGURE 4. Mach-Zehnder interferometer configuration.FIGURE 5. Structure of the MZI devices (left) ARROW-MZI(right) TIR-MZI.FIGURE 6. Integrated Mach-Zehnder interferometer: details of theMZI Y-junction and sensor area.FIGURE 7. Sensitivity evaluation of a TIR-MZI nanodevice by us-ing solutions of varying refractive indices.3.2. Integrated Mach-Zehnder interferometersFrom the different types of IO sensors[8], the interferometricarrangement is the most sensitive and the only one that pro-vides an internal reference for the compensation of refractive-index fluctuations and unspecific adsorption. Due to the highsensitivity of the interferometer sensor, the direct detectionof small molecules (even without inhibition immunoassays)would be possible with this device [11]. A detection limit of10−7 in the refractive index (or better) can be achieved withthese deviceswhich opens up the possibility of developinghighly sensitive devices, for example, for directly determingthe extreme protein (femtomolar) concentration [11].In an interferometer (see Fig. 4) two light beams of equalintensity are made to travel across two areas of a waveguide(one is the sensor and the other is the reference) and are fi-nally combined, creating an interference pattern. When a bio-chemical reaction takes place in the sensor area, only the lightthat travels through this arm will experience a change in itseffective refractive index. At the sensor output, the intensityof the light shows a sinusoidal variation that depends on thedifference of the effective refractive indexes of the sensor andreference arms (∆N) and on the interaction length (L), andRev. Mex. Fı´s. S 52 (2) (2006) 94–98NANOBIOSENSORS BASED ON OPTOELECTRONIC AND NANOMECHANICAL TRANSDUCERS FOR. . . 97FIGURE 8. (left) Immobilization of the receptor on the MZI surface by covalent attachment (right) Immunoreaction with the specificantibody.FIGURE 9. Working principle in nanomechanical cantilevers: (left)cantilever with a thin gold layer for covalent attachment of the re-ceptor (right) bending of the cantilever due to biomolecular inter-action.can be directly related to the concentration of the analyteto be measured. We have fabricated two integrated Mach-Zehnder interferometric devices using two technologies: (a) aMZI Microdevice based on ARROW waveguide [12] and (b)a MZI Nanodevice based on TIR waveguide [13].(a) MZI Microdevice based on ARROW waveguide:For the development of a highly sensitive integrated opti-cal sensor based on the Mach-Zehnder interferometer con-figuration, it is necessary to design optical waveguides thatsatisfy two conditions: monomode behaviour and high sur-face sensitivity. ARROW (Anti Resonant Reflecting OpticalWaveguides) structures based on Silicon technology meetthese requirements. This optimised waveguide consists ofa rib-ARROW structure with a silicon oxide core layer(ncore=1.485) and thickness greater than 2 µm; a silicon ox-ide second cladding layer with a refractive index of 1.46and a fixed thickness of 2 µm; and a silicon nitride firstcladding layer, 0.12 µm thick, with a refractive index of 2.00.The waveguide is overcoated with a thin silicon nitride layer(nov=2.00) and with a silicon oxide layer (n=1.46) with athickness of 2 µm. The rib depth is 60% of the core thick-ness and the rib width should be less than 8 µm to obtainsingle-mode behaviour. The structure of the ARROW devicecan be seen in Fig. 5.(b) a MZI Nanodevice based on TIR waveguide.In Fig. 5, the cross section of the MZI TIR waveguide isshown. The basis of the TIR structure is: (i) a Si wafer,(ii) a 2 µm thick thermal Silicon-Oxide layer (n=1.46), (iii) aLPCVD Silicon Nitride layer of 100 nm thickness (n=2.00),which is used as a guiding layer. To achieve monomodebehavior, a rib structure must be defined with a depth ofonly 4 nm by a lithographic RIE step. Finally, a Silicon-Oxide protective layer is deposited by LPCVD over the struc-ture with a 2 µm thickness (n=1.46), which is patterned andetched by RIE to define the sensing arm of the interferome-ter. Photographs of one integrated MZI device can be seen inFig. 6. Several devices, using both technologies, have beenfabricated in our Clean Room facilities. The devices are im-plemented with a microfluidic unit, electronics, data acquisi-tion and software for optical and biochemical testing.For evaluating the sensor sensitivity, a calibrating curvewas recorded using solutions with different refractive in-dexes, as depicted in Fig. 7 for a TIR-MZI device. Forthe TIR device, the lower detection limit measured was∆no,min = 7.10−6, which means an effective refractive indexof ∆N = 4·10−7. For the ARROW-MZI devices, a minimumdetectable refractive index variation of ∆no,min = 2·10−5was obtained.We have applied the MZI biosensors for the detection ofthe insecticide carbaryl [2]. The introduction of different con-centrations of the specific monoclonal antibodies porducesan immunoreaction proportional to this concentration, as ob-served in Fig. 8 (right).Rev. Mex. Fı´s. S 52 (2) (2006) 94–9898 L.M. LECHUGA, J. TAMAYO, A. CALLE, AND C. DOM´INGUEZFIGURE 10. Real time simultaneously monitoring of five microcantilevers. (left) Immobilization and blocking treatment, and (right) hy-bridisation bending signal.Nanomechanical biosensor platformMicrocantilevers, such as those used in Atomic Force Mi-croscopes, have been recently employed as this new class ofbiosensors [14]. The so-called nanomechanical biosensorshave demonstrated that they are capable of detecting single-base mismatches in oligonucleotide hybridization without la-beling [5], as well as performing protein recognition [15]with extreme sensitivity. The working principle for nanome-chanical biosensors relies on the induced surface stress pro-duced when molecules bind to a surface (see Fig. 9). Surfacestress mainly arises from electrostatic, van der Waals, stericinteractions, etc., between the adsorbed molecules.Cantilever bending (deflection) measurements are carriedout by using the well-known optical beam deflection method.A laser beam is focused on the free end of the cantileverand the deflection of the reflected beam is measured with afour-segment photodetector. For example, for the DNA hy-bridization detection, nucleic acids are immobilized on oneside of the micromachined lever. Exposure of the cantileverto a sample containing complementary nucleic acid gives riseto a cantilever bending (deflection) of a few nanometers.To test the reliability of such approach, we have usedarrays of Si cantilevers fabricated in our Clean Room forthe real-time detection of (a) immobilization of DNA strandsand (b) hybridization with the corresponding complementaryDNA strands, as shown in Fig. 10.1. C. Domı´nguez, L.M. Lechuga, and J.A. Rodrı´guez, Integratedoptical chemo- and Biosensors. In Integrated Analytical Sys-tems (Ed. S. Alegret, 2003) Elsevier Science BV. Amsterdam(The Netherlands).2. L.M. Lechuga et al., Proceedings of Photonics West (SPIE), In-tegrated Optoelectronics Devices, 5357 (2004).3. www.imm.cnm.csic.es/biosensores/home.html4. F.S. Liegler and C.R. Taitt (eds). Optical Biosensor: Presentand future. Elsevier, Amsterdam (NL), (2002)5. Fritz et al., Science 288 (2000) 316.6. www.optonanogen.com7. L.M. Lechuga. Optical Biosensors. In Biosensors and ModernBiospecific Analytical Techniques. Ed. L. Gorton (2005). Else-vier Science BV. Amsterdam (The Netherlands). (In press).8. R.P.H. Kooyman and L.M. Lechuga. In “Handbook of Biosen-sors: Medicine, Food and the Environment”, ed. E. Kress-Rogers, CRC Press, Florida (USA), (1997) 169.9. J. Homola, S.S. Yee, and G. Gauglitz. Review. Sens. Act. B 54(1999) 3.10. L.M. Lechuga, A. Calle, and F. Prieto, Quı´mica Analı´tica 19(2000).11. L.M. Lechuga, F. Prieto, and B. Sepu´lveda. InterferometricBiosensors for environmental pollution detection. In OpticalSensors for Industrial, E (2003). Springer.12. F. Prieto, A. Llobera, A. Calle, C Domı´ngue,z and L.M.Lechuga, J. Lightwave Tech. 18 (2000) 966.13. F. Prieto et al., Nanotechnology 14 (2003) 907.14. H. Wu et al., Proc. Natl. Acad. Sci. USA 98 (2001) 1560.15. M. ´Alvarez et al., Biosensors & Bioelectronics 18 (2003) 649.Rev. Mex. Fı´s. S 52 (2) (2006) 94–98

Nanobiosensors based on optoelectronic and nanomechanical transducers for genomic and proteomic applications

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