Advances in Nanophotonic Sensing TechnologiesDuring Three International Label-Free Lab-On-ChipProjects

Daniel Hill

Published online: 29 October 2011# Springer Science+Business Media, LLC 2011

Abstract We review the results from the use of variousintegrated nanophotonic sensors for label-free biosensingdeveloped in three recent European biosensor collabora-tions: SABIO, INTOPSENS, and POSITIVE. Nanopho-tonic transducers are attractive for label-free biosensing dueto their small footprint, high Q-factors, and compatibilitywith on-chip optics and microfluidics. This enables inte-grated sensor arrays for compact labs-on-chip. One appli-cation of label-free sensor arrays is for point-of-caremedical diagnostics. Bringing such powerful tools to thesingle medical practitioner is an important step towardspersonalized medicine, but requires addressing a number ofissues: improving limit of detection, managing the influ-ence of temperature, parallelization of the measurement forhigher throughput and on-chip referencing, efficient light-coupling strategies to simplify alignment, and packaging ofthe nanophotonics chip and integration with microfluidics.From SABIO, we report a volume sensing sensitivity of240 nm/RIU and detection limit of 5×10−6 RIU, and asurface sensing limit of detection (LOD) of 0.9 pg/mm2 forat 1.3 μm for an eight-channel slot-waveguide ringresonator sensor array, within a microfluidics integratedcompact cartridge. In INTOPSENS, ongoing efforts have sofar resulted in various nanophotonic transducer designswith volume sensing sensitivities as great as 2,169 nm/RIUand LODs down to 8.3×10−6 RIU at 1.5 μm. Earlyexperiments from the POSITIVE project have demonstratedvolumetric sensitivities as high as 1,247 nm/RIU at 1.5 μm.

Keywords Nanophotonics . Slot-waveguides . Ringresonators . Porous silicon . Label-free . Biosensing .

Lab-on-chip

1 Lab-on-a-Chip Devices and Nanophotonic Sensors

Personalized medicine is a key target for improved qualityof life and Lab-on-Chip (LoC) technologies for use at pointof care (PoC) have been proposed for providing suitablesolutions and therefore will have a massive socioeconomicimpact. They provide this by scaling analytical chemicaland biological instruments down to a single chip [1]enabling: automation of the analysis, increased mobility ofthe instrument, shorter response times, reduced manualsample handling, and a low cost per test. To date, mostestablished biosensing technologies that might be used inLoC devices use special reporter molecules or particles,known as labels, to read out the measured quantity. Mostoften, the labels are fluorescent or radioactive, but gold ormagnetic nanoparticles are also used. Label-based sensorscan measure down to the single-molecule level, but theyhave a number of drawbacks. One limitation, often cited, isthe time and cost of the labeling step [2]. Another is thecomplexity that the label adds to the reaction under study,and thus an increased risk of misinterpretation [3]. A morefundamental limitation is however that, since the labels aretypically added in a secondary step, most labeled methodscan only measure the static endpoint of a reaction and donot provide real-time information. This information can,however, be gained by label-free (LF) methods, which cangive quantitative real-time information in physical units.

Refractive index (RI) sensing is widely used for real-time monitoring of chemical processes and, when used withseparation techniques such as liquid chromatography or

D. Hill (*)UMDO, Institut de Ciència dels Materials,Universitat de Valencia,Catedrático José Beltrán, 2,46980 Paterna, Valencia, Spaine-mail: daniel.hill@uv.es

BioNanoSci. (2011) 1:162–172DOI 10.1007/s12668-011-0026-1

capillary electrophoresis, universal solute detection systemscan be created [4]. As affinity sensors within LoC devices,silicon nanophotonics have found much use recently [5] forvarious reasons. Since the refractive index of aqueousmacromolecular solutions is linear with macromoleculedensity [6], sensors measuring the RI close to a surface byusing the evanescent field from a guided wave can be usedto measure the mass of macromolecules, such as proteins,binding on the surface. RI-based nanophotonic sensingmethods can thus be used for LF monitoring of biomolec-ular interactions on surfaces, as for example is done in thecommercially successful surface plasmon resonance (SPR)-based sensors [7]. Furthermore, they can be made with thesame microfabrication technologies as electronic micro-chips allowing efficient production of highly integratednanophotonics devices at wafer scale. Thus, to bring thepowerful tool of label-free sensing into the hands of a wideruser base, there is a strong interest in integrating nano-photonic sensors into LOC technologies that can be used atthe PoC, e.g., to the single medical practitioner or in aremote field situation. In this paper, we mainly discuss theapplication of nanophotonic transducers that due to theirsmall footprints and ease of integration with other on-chipoptical and fluidic functions are particularly interesting assensors for LoC devices. In doing so, we will review theirdevelopment within three European collaborative projects:SABIO, INTOPSENS, and POSITIVE.

2 The SABIO Approach

2.1 The Socioeconomic Problem

Although technically feasible, the routine screening of viraldiseases such as hepatitis B and C at whole-populationlevel is not currently implemented largely due to the highcost of current assays and the burden on diagnosticlaboratories. However, many common viral infections inEurope have a huge negative socioeconomic impact, whichwould be ameliorated with improved screening programs.SABIO proposed to construct a technology platform thatwould overcome the current objections for routine popula-tion screening of some common diseases, including viralinfections, namely by making the diagnosis inexpensive tocarry out and practical at a “point of care” setting, such asin the surgery of general practitioners. With the platform toserve both as proof of concept, and as a first step to thepractical implementation of point-of-care multiarray diag-nostic for socioeconomically important diseases, the pro-posed chip was to detect markers in the blood for thefollowing medical conditions: hepatitis B, cytomegalovirus,and liver cancer. The proposed chip to be developed in thisnine partner EC-financed project was to have consisted of

nanophotonic biosensors for label-free biomolecular recog-nition based on novel photonic structures named slot-waveguide ring resonators with immobilized biomolecularreceptors on their surface.

2.2 SABIO Cartridge

To demonstrate the possibility of using a slot ring resonatordevice in a point-of-care setting, the SABIO measurementcartridge, Fig. 1, was designed for non-critical alignment ina reader instrument. An on-chip fluidic channel networklayer was included to transport the samples to the sensors.Alignment pins and precision dicing of the chip minimizethe position inaccuracy when mounting the cartridge in theinstrument. Light was coupled into the cartridge fromabove, using an alignment-tolerant grating coupler [8]. Thelight was then distributed to the different sensors on thechip and coupled out through the edge of the chip in a waythat allows automatic alignment to a photo-diode array [9].The fluidic layer is on top of the optics. A full descriptionof the cartridge design can be seen here [10].

2.3 SABIO Nanophotonic Sensors

Researchers have been working on ring resonators since the1980s [11–13] and inspired by Almeida et al’s demonstra-tion [14, 15] of slot-waveguides in 2004; the SABIOproject targeted the implementation of these in a ringresonator format for biosensing. The theory of the ringresonator, side-coupled to a straight bus waveguide hasbeen extensively studied [16]. Here, we just conclude that ifwe start from a model according to Fig. 2 and a makenumber of assumptions,

Fig. 1 The SABIO cartridge

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& all waveguides are unimodal per polarization orienta-tion and do not couple between polarizations,

& all losses are incorporated in the attenuation constant ofthe individual modes,

& back reflections are negligible,& transitions between modes are adiabatic,& interaction between modes is negligible outside the

coupler regions,& only symmetric devices are considered,& the coupling is lossless,

we can arrive at an expression for the power transmission inthe forward direction through the bus waveguide.

PT ¼ Dþj j2 ¼ a2 þ tj j2 � 2a tj j cos q þ yð Þa2 þ a2 tj j2 � 2a tj j cos q þ yð Þ

In this expression, a and θ are the loss and phase shift fora round trip in the ring, τ is the straight-throughtransmission of the coupler and ψ is phase shift in thecoupler. In Fig. 3, PT is plotted as a function of the round-trip phase shift θ when a ≈ τ. In this case, the transmissiongoes to zero in the dips. With this condition fulfilled, thedips become sharper when a and τ are close to 1, i.e., whenthe losses are small. θ is proportional to the effective indexof the ring guide and inversely proportional to thewavelength, so changing any of these would mean movingalong this graph.

To achieve a good resolution in terms of refractive indexof the sample, we must strive for

& a large change in the effective index of the guide for agiven change in sample index or, expressed in another

way, a large sensitivity in terms of resonance wave-length shift per refractive index unit (RIU),

& low losses in the resonator ring to achieve sharp dips inthe ring transmission characteristics

& an efficient method to determine the changes in the dipposition,

& suppression of changes in the effective index frominterferences, such as the temperature.

Readers are recommended to see another review paper[10] for further information on ring resonators, theirdevelopment over the years and how they, including slot-waveguide types, compare to other sensor types. Briefly, Siplanar waveguide ring resonators, and even more so, slot-waveguides ring resonators are very attractive for label-freebiosensing due to their small footprint, high Q-factors, andcompatibility with on-chip optics and microfluidics. Theirdesign permits parallel sensor operation which not onlyyields higher throughput by multiple analyses of onesample, or simultaneous analyses of multiple samples, butit can also provide reference channels for drift compensa-tion and control experiments. Such reference measurementsare particularly important for automated labs-on-chipswithout temperature stabilization. In our case, the opticalchip (Fig. 4) was designed with six measurement channelsand two reference channels, and channel to slot-modeconverters were used for conversion between the twowaveguide types before and after the ring resonatorcoupling regions, where the bus slot-waveguides have railwidths of 400 nm and a slot width of 200 nm, Fig. 4. Thecoupling gap was 350 nm, and in the sensing ring,asymmetric slot-waveguides with the inner rail widened to550 nm were used for high optical confinement and lowbending loss.

The optical chip consisted of a silicon substrate, with theintegrated nanophotonic components etched with nanome-ter precision into a silicon nitride thin film, embedded in asilicon dioxide cladding on its surface. Silicon nitride waschosen over silicon as it is nearly impervious to diffusion of

Fig. 3 The transmitted power PT of a side coupled ring resonator, fora=0.87 and τ=0.85, as functions of the round trip phase shift θ andignoring dispersion. Figure adapted from [17]

Fig. 2 A waveguide ring resonator side coupled to a bus waveguide.B, b, D, and d are the complex mode amplitudes. Ring modes arelower case and bus modes uppers case. κ is the complex couplingcoefficient and τ the complex transmission coefficient. Figure adaptedfrom [17]

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moisture and sodium ions, maintaining a stable refractiveindex when operating in biological liquids. Additionally,silicon oxide and nitride have a relatively high-refractiveindex contrast, thus permitting close spacing of integratedoptical components, by allowing small bend radii withoutexcessive bending losses. Moreover, in comparison to theeven higher RI contrast in the silicon/silicon oxide system,it eases the problems from loss due to surface roughness.Stitching issues from electron beam lithography and surfaceroughness from subsequent dry etching limited devicereproducibility, further design and fabrication details canbe found elsewhere [9]. General design rules were basedaround achieving a good resolution in terms of the RI of thesample, and so the following were striven for:

& a large change in the effective RI of the guide for agiven change in sample RI or, expressed in another way,a large sensitivity in terms of resonance wavelengthshift per refractive index unit (RIU),

& low losses in the resonator ring to achieve sharp dips inthe ring transmission characteristics

& an efficient method to determine the changes in the dipposition,

& suppression of changes in the effective index frominterferences, such as the temperature.

Furthermore, in relation to the design, the choice of laserwavelength was a trade-off between a narrow slot-width at

short wavelengths, that may limit analyte access, and lightabsorption in water at longer wavelengths, that limits theresonator Q factor. The choice of the standard telecommu-nication wavelength of 1,310 nm was a compromiseensuring that a wide selection of sources is available.

To present a useful alternative to current technology,novel sensors need to achieve an LOD of the order of 10−6

RIUs or better. Considering the fact that commonly usedwaveguide materials, and the liquid solvents of interesthave thermo-optic coefficients of magnitudes 10−5–10−4

RIU/K, it is clear that minimizing temperature interferenceis essential. An uncompensated sensor requires temperaturestabilization to of 10–100 mK to reach the requiredperformance. In the SABIO device, the design freedomachieved with the slot-waveguide was used to achievesensors with reduced temperature sensitivity and then on-chip referencing was used to overcome the residualtemperature sensitivity. The measurements described inthe next subsection were made over 7 K operating window,without external temperature control and individual sensorcalibration. This is further described elsewhere [18].

2.4 SABIO Measurements and Discussion

Sensing experiments were performed using a dilution seriesof ethanol and methanol plugs in a running buffer ofdeionized (DI) water to determine the volumetric RI

Fig. 4 A top view of the layout of the nanofabricated SABIO opticalchip (occupying a 3×7 mm2 area): Light is injected at the surfacegrating coupler (c) and split, by the multi-mode interference splitter(b), to the six sensing channels M1–M6 and the two reference

channels REF1 and REF2. The sensing channels consist of slot-waveguide ring resonators which can be seen in inset (a) alongside anenlargement of the coupling region

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sensitivity and limit of detection of the SABIO sensorchips. Full details of the sensing experiments including thatof the fitting algorithm used to push the wavelength noisesignificantly below the laser tuning step can be foundelsewhere [19]. At 1,300 nm, an index sensitivity of Sn=246 nm/RIU was determined, sensor resolution wasdetermined to be 1.2 pm following the convention of usingthree standard deviations σ of the total system noise as ameasure of the sensor resolution, with the volumetric RILOD given by Ln=R/Sn and thus equal to 5×10−6 RIU.

The surface mass sensing performance was also studiedby measuring the binding of anti-BSA to a surfaceselectively activated by a layer of the molecular linkerglutaraldehyde. Increasing concentrations of anti-BSA werethen injected in a running buffer of phosphate-bufferedsaline and the resonance shift monitored until saturation. Ashift in resonance wavelength at saturation was estimated atΔλ=2.55 nm. The sensitivity for surface mass detection isgiven by Sm=Δλ/σp, where σp is the surface density of amolecular monolayer. The surface density of a monolayerof anti-BSA measured using dual polarization interferom-etry with the Farfield AnaLight 4D system was σp=2.0 ng/mm2. Using the value of the resonance shift from theexperiment, a mass sensitivity of Sm=1.3 nm/(nanogramsper square millilmeter) was determined. The surface massdetection limit given by Lm=R/Sm=0.9 pg/mm2.

The detection limits achieved in SABIO of 5×10−6 RIUand 0.9 pg/mm2 were due to the use of multiple transducerson the chip to compensate for external disturbances, thehigh sensitivity of the slot-waveguide ring resonators andthe low system noise 1.2 pm, achieved by fitting ananalytical model to the spectrum, thus effectively utilizingall the information available [18] In terms of surfacesensing, the LoD presents a significant improvement on apreviously published [20] value of 28 pg/mm2. As in thecase of volume sensing, the improvement is mainly due tothe reduced system noise. The LODs achieved in SABIOcompare favorably to other published ring resonator results(see Fig. 5). In the top right hand corner, we find the high-sensitivity, low-quality factor (Q) devices such as SPR.Since they have a very large index contrast at the metal/dielectric interface, high E-field strengths (therefore highsensitivity) are obtainable in the dielectric sample. Howev-er, the resonance is not sharp as plasmon propagation islossy. On the opposite end of the scale, in the bottom lefthand corner, we find non-planar toroid ring and sphericalresonators, made by reflowing glass. These have most ofthe electric field in the core, and in general, a lowsensitivity. However, losses in the dielectric resonators areextremely small, and due to the reflow, surface roughness isvery small and scattering low. In the center, we find acluster of planar waveguide ring resonators. These deviceshave lower sensitivities than the plasmon-based devices,

because of the lower index contrast, but as they are madeonly of dielectrics, they do not suffer the electron scatteringlosses. Theoretically, they are lossless except for bendinglosses in reality; however, photon scattering from theroughness of the lithographically defined sidewalls limitthe resolution and Q. The slot WGs allow higher sensitivityand thus come higher up in Fig. 5. The guide is still singlemode and the modes are in theory lossless except forpossible bending losses. But there is a catch, if the extraside surfaces in the slot guide are created by lithographyand etching, the number of rough surfaces has alsoincreased. The penalty is increased losses, a lower Q, andin turn increased noise, i.e., a drift to the right in the figure.This was observed for guides of multi-slots [21]. Theoret-ically, they have even higher refractive index sensitivity, butthis comes at the cost of an increased loss due to an overallgreater side surface area contributing to the light scattering.

3 The Continuation in INTOPSENS

3.1 Socioeconomic Problem

Sepsis is a serious medical condition characterized by awhole-body inflammatory state caused by microbial infec-tion. Sepsis can lead to septic shock, multiple organdysfunction syndrome, and death, and claims ∼146,000lives/year in the EU and an associated EUR 7.6 billion/yearhealth care costs. Although the patients suffer from a high

Fig. 5 Detection limit analysis, from [17]. The x-axis is the log of thewavelength resolution and the y-axis is the log of the device sensitivityin terms of wavelength shift per RIU. The gray scale then representsthe minimum detectable refractive index change. Examples includedare Hu et al. [22] Haneumegowda et al. [23] Fan et al. [24] Carlborg etal. [21] (the SABIO device), Claes et al. [25] (an INTOPSENSprototype), Yalcin et al. [26] De Vos et al. [27] Pfeifer [28], andKabashin et al. [29]

166 BioNanoSci. (2011) 1:162–172

related mortality: 20–30%, this can be significantly reducedwhen a suitable anti-biotherapy is administered within 24–48 h. The current lack of documentation (negative bloodculture), however, has led to both an empirical therapy withmultiple drug treatments which has resulted in growingantibiotic resistance, and an excess of high-cost investiga-tion. The EC project consortium of INTOPSENS istherefore developing a state-of-the-art diagnostics platformvia an integrated microfluidic sample preparation techniquecapable of rapid bacteria isolation from whole blood. Thisintegrated Lab-on-a-Chip platform involves capturing ofintact bacteria from relatively large blood volumes ∼5 mL,pre-concentration, purification, and cell lysis to extract thegenomic material for on-chip PCR amplification andsensing.

3.2 INTOPSENS Cartridges

The INTOPSENS platform is also intended for use in a PoCsetting, and its packaged disposable part has been designedfor non-critical alignment in a reader instrument with afluidic channel network layer to transport the samples to thesensors. The integration of sample handling on-chip for theINTOPSENS device faces all of the same complexity asSABIO with regards to anti-clogging, removal/preventionof air bubbles, and transport of assay fluids throughmicrofluidics channels, pumps, and valves. But while theSABIO design allowed six immunoassays, aiming fordetection of biomarkers for liver cancer, hepatitis (B/C) orcytomegalovirus, INTOPSENS aims to test for some 64bacterial DNA-strands in a fluidic blood sample for sepsisand to determine to which antibiotics these bacteria areresistant to. Here, it is should also be noted that theINTOPSENS device includes on-chip amplification ofDNA with polymerase chain reaction (PCR), so there is atrade-off between assay time (number of PCR cycles) andminimum LOD required of the photonic sensors. Thefluidic layer is on top of the optics (Fig. 6) whose light iscoupled both in and out from above through alignment-tolerant grating couplers in a design that permits thesimultaneous interrogation of multiple sensors.

Input is based on flood illumination, from the distance ofseveral centimeters, of hundreds of grating couplers by acollimated broad laser beam with a width of some 2 mm.This design permits the alignment of a wide parallel freespace on the order of tens of microns and scales with thewidth of the collimated beam. The light from multipleoutput gratings are detected in parallel by an infra-red CCDcamera mounted on a microscope. The distance betweenthe microscope objective and the output grating couplers isseveral centimeters. In order to minimize temperatureinfluences, both active system control and on-chip refer-ences are used [10] The INTOPSENS temperature control

has the added task of having to maintain a constanttemperature during measurements, which occur in betweenPCR temperature ramping cycles.

3.3 INTOPSENS Nanophotonic Sensors

3.3.1 Ring Resonator-Based Sensing

With a minimal footprint required per nanophotonictransducer, one starting point for sensor development wasthat of a silicon-on-insulator (SOI) ring resonator. This hadpreviously [30] demonstrated a volumetric RI sensitivity of70 nm/RIU and LOD=1.3×10−5 RIU as well as a LOD of7 ng/ml for biotin–avidin recognition. In an aim to improveon LOD with the same high degree of integration as seenelsewhere [32], slot-waveguide racetrack resonators with100 nm wide slots in the high-index-contrast SOI materialsystem were nanofabricated [25]. In contrast to the electronbeam lithography patterned SABIO devices, massfabrication-compatible optical lithography was used toproduce the devices, and footprints of just 13×10 μm,opening the way toward cheap, disposable chips.

Experiments with aqueous salt solutions [25] demon-strated a volumetric refractive index sensitivity of 298 nm/RIU and a LOD=4.2×10−5 RIU for these SOI slot ringresonators (see Fig. 7). The sensitivity value lay within arange of theoretical values from an empty to a liquid-filledslot demonstrating that liquid had penetrated the narrowslot region. After silanizing the sensor surface a biotin–avidin recognition experiment was then performed [25]where a limit of detection of 10 ng/ml was recorded,corresponding to a poorer surface mass detection limit thanthat of the SABIO device at 5 pg/mm2. The saturation shift,which was 3.5 times that of the SABIO device, lay betweenthe theoretical values for avidin only binding outside of the

Light in

Camerareadout

Sample in

Valving, vacuum,

waste

Covalently bonded layer

stack

Peltier temp. ctrl

Fig. 6 Cartridge II, where intact bacteria enter following separationand up-concentratation of bacteria from whole blood using inertialmicrofluidic techniques in combination with selective blood lysis incartridge I. In cartridge II, after bacterial lysis and integrated multiplexPCR, the PCR product hybridizes on an 8×8 photonic biosensor array

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slot and for it lying both inside and outside of the slot.Thus, it was demonstrated that surface chemistry forselective label-free sensing of proteins can be appliedinside a 100 nm wide slot region. Compared to the SABIOdevice, the LOD was poorer due to a lower resolution orquality factor principally caused by bending and mismatchlosses although sidewall inclination, roughness from sili-con’s greater sensitivity to nanofabrication limitations, thepresence of a biochemical layer, and absorption alsocontribute. Calculations did show however that the LODreached was more than sufficient for detecting typical DNAconcentrations of 104 ng/ml for 46 PCR cycles taking90 min. The initial project targets were however 1 ng/mland then 0.1 ng/ml, equivalent to 3 ng/mm2, for DNAhybridisation, and so in an aim to improve upon 104 ng/mlwhilst maintaining high integration, various alternativedesigns were implemented.

3.3.2 Further Ring Resonator and Mach–ZehnderInterferometer-Based Sensing

Several of the alternative highly integrated nanophotonicsensors were based on modifications to existing ringresonator and slot ring resonator waveguide-based sensordesigns such as:

& The use of notch ring resonator filters instead of add-drop filters

& Increasing the sensor circumference& Switching to 1,300 nm where water is less absorbent& Combining quasi-TE and quasi-TM modes

Modified ring resonators led to further aqueous saltsolution sensing experiments that demonstrated a volumet-ric limit of detection of 5×10−6 RIU and biotin/avidinexperiments demonstrated [30] a surface mass LOD of2 pg/mm2.

Other alternative sensors realized through nanofabrica-tion technologies were based on significantly differentdesigns. The first of which was based around the use ofthe Vernier effect through suitably designed cascaded ringresonators which were folded so as to permit highintegration. In a similar experiment to that reported abovefor the SOI slot-waveguide-based sensor in flowing variousconcentrations of aqueous NaCl solutions, a volumetric RIsensitivity of 2,169 nm/RIU was recorded [31] for theVernier ring resonators corresponding to a LOD of 8.3×10−6 RIU. Although its LOD is still poorer than that of theSABIO device and of other single ring resonator-basedsensors [32], this first experimental result is promising forfuture optimized designs. The second alternative sensor ofsignificantly different design was a SOI based implemen-tation of a Mach–Zehnder interferometer (MZI) with foldedwaveguides. Not only has it robustness against temperaturechanges and optical losses [33], it is compatible withadvanced waveguide technology such as slot-waveguidesand slow light, and combination of interferometers withdifferent interaction lengths enables extended dynamicrange. Through flowing various concentrations of aqueoussalt solutions over the MZI a volumetric sensitivity of465 nm/RIU and LOD=5×10−5 RIU was measured. Athird alternative sensor pursued of significantly differentdesign included that of 4-μm sized SOI-based diskresonator which was estimated to have a surface massLOD=1 pg/mm2.

3.3.3 Towards DNA Sensing

In parallel to these activities, various SOI-based photoniccrystal (PhC) waveguide-based nanophotonic sensordesigns [34, 35] were realized, whose in-depth presentationhere is also beyond the scope of this article. Flow throughof DIW-ethanol solutions in these PhC waveguides dem-onstrated volumetric sensitivities and detection limits from83 nm/RIU and 2×10−4 RIU to 175 nm/RIU and 3×10−6

RIU. The higher performance sensors demonstrated asensitivity of 12.5 ng/ml and surface mass LOD=2 pg/mm2 for BSA–AntiBSA recognition [36] and a 22-nM limitof detection for double-stranded DNA sensing [37] on anICPTS functionalized surface. Single-strand DNA hybrid-ization experiments on the same type of device andmeasurement procedure demonstrated [38] a 20-nM LODwhich is 50×min concentration that needs be detected. Thesuccessful functionalization chemistry (ICPTS) was portedacross to ring resonator-based devices where double-stranded DNA sensing was demonstrated down to a limitof detection of 37 nM. For the same devices, single-strandDNA hybridization was seen down to 40 nM for anAPTES-functionalized surface, which is 27×min concen-tration that needs to be detected.

Fig. 7 A comparison [25] between the experimental resonancewavelength shift of a normal-waveguide based ring resonator andthe theoretical and experimental resonance wavelength shift of ourslot-waveguide-based ring resonator for top refractive index

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It should be noted that the many of the values quoted aboveare poorer than theoretically calculated ones. This washypothesized halfway into the project as to be due to masstransport problems to the sensors. These values are stillhowever 3–4 orders of magnitude lower than the DNAconcentrations from realistic numbers of PCR cycles allowingthe INTOPSENS partners to run faster hybridization sensingassays by reducing the number of PCR cycles run.

4 The POSITIVE Approach

4.1 Socioeconomic Problem

Food allergies are common in 1–2% of adults and up to 8%of children—a serious public health problem that affectsover 15 million Europeans from infants to the elderly andtheir prevalence are increasing. In some cases, they canprovoke clinical reactions whose most severe is anaphylax-is, with respiratory and/or cardiovascular problems thatmight result in death. To date, routine GP allergy testing ofchildren under two consists of repeated and uncomfortableskin tests that are difficult to administer. Consequently forimprovement in quality of life, there is a need for aconvenient diagnostic platform that can quickly and safelymonitor for specific IgE to common food allergens,particularly for young children, so that a suitable diet canbe recommended or certain foods prohibited. Alternativetests in place are blood based mostly using the FEIA,RAST, and ELISA techniques, and are usually lab-based.PoC devices do exist, but are either limited to testing only afew allergens at a time and give at best semi-quantitativedetermination. The EC project POSITIVE is thereforedeveloping a state-of-the-art diagnostics technology todetermine the sensitization down to 0.1 kU/L to hundredsof food allergies within 15 min from a 100-μL whole bloodsample at a cost of 10 Eurocents per allergy.

4.2 POSITIVE Cartridge Development

The POSITIVE platform is also destined for PoC use andso has its packaged disposable part designed as well fornon-critical alignment, although in difference to theprevious projects the analyte flow is not lateral to thephotonics chip but from front to back. The integration ofsample handling on chip is somewhat similar to thatundertaken in both previous projects with regards to anti-clogging, removal/prevention of air bubbles, and transportof assay fluids through microfluidics channels, pumps, andvalves. Moreover, similar to the INTOPSENS project, thePOSITIVE design allows a large number of assays, 48, butsimilarities end there as it does not require additionalsample preparation complexities of lysing nor PCR and so

everything is contained within a single cartridge. Tominimize temperature influences, both active system con-trol and on-chip references are also used.

4.3 POSITIVE Nanophotonic Sensors

The very high surface to volume ratio of porous siliconpermits [39] very high surface densities of bound antibody–antigen complexes in a reduced volume that through certainnovel optical interactions can leads to scores of sensingareas on a 1-cm2 chip with LODs down to below 0.1 pg/mm2, or 5×10−6 RIU, significantly beyond the state of artfor highly integrated label-free sensors at point of care.Overall, a successful sensor in such a system willprincipally depend on the controllable and reproduciblefabrication of porous silicon layers with nanometer precisepores size so as to maximize sensor optical properties,while at the same time, allow the flux of an analyte throughthe membrane itself avoiding pore clogging. Currentfabrication technologies permit stable pore growth withpore sizes of 100–400 nm for membrane thicknesses up tosome 10 s of microns. These permit two interestingapproaches for sensing: birefringent membranes for polar-imetry [40] or pore size modulation microcavity-basedstructures [41] for interferometry. For brevity as we onlyreport on sensing results from the former approach, we onlyintroduce the concept of sensing by that.

Bulk silicon is an isotropic material due to its diamondcubic crystal structure; however, porous silicon (PSi) preparedfrom (110) Si surface-oriented substrates presents a highanisotropy due to pores grow preferentially along the [100]and [010] crystallographic directions [42] as seen in Fig. 8.The orientation of the pores along those directions results ina difference in the refractive indices along the [001] and the

[001]

[110][100]

[010]

IncidentBeam [110]

Fig. 8 Scheme of an anisotropic PSi layer produced from a (110)surface-oriented Si wafer

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½110� directions [43]. This difference or birefringence,therefore, depends on the refractive index of any materialthat enters the pores [44] and forms the basis for sensing.

4.4 POSITIVE Measurements and Discussion

At this current early stage of the project polarimetry-basedmeasurements of the birefringence for porous silicon mem-branes filled with air, and thereafter with ethanol andisopropanol have only been performed. The PSi birefringentproperties were characterized for a set of samples consisting ofnanoporous silicon etched into p-type (110) Si with resistivityof 0.01–0.001Ω/cm. Pores sizes were in the range of few tensof nanometers, as shown in Fig. 9a. Samples were preparedby electrochemical etching using a solution composed of HF/ethanol=3:7 by volume, considering an initial HF concen-tration of 48%. A current density of 25 mA/cm2 was usedduring the etching. For subsequent optical characterization,porous membranes were detached from the bulk silicon layerfollowing a procedure outlined here [45].

Several (110) PSi membranes of various thicknesseswere fabricated and transferred to a glass substrate in whicha 3- to 4-mm circular hole was made in order tocharacterize the birefringence as a function of optical path.Before measuring the birefringence of the fabricatedsamples, a thermal oxidation at 200°C for 12 h was carriedout in order to stabilize pore surfaces during the measure-ments [46]. The optical anisotropy of fabricated sampleswas then determined by analyzing the state of polarizationof the light transmitted through them [47].

The bulk refractive index sensitivity of the PSi membraneswas determined by filling the samples with the differentliquids: ethanol (nEtha≈1.36) and isopropanol (nIsop≈1.377).A few seconds after filling the cube with the liquids, thetransmission spectra stabilized, indicating that the liquids hadcompletely filled the pores. From this data, experimentalbirefringence values were derived (Fig. 9b) and thebirefringence change between the samples filled withisopropanol and filled with ethanol obtained from curvesfitted to that data. These values are 1.8×10−3, 1.1×10−3, and

0.98×10−3 for the wavelengths of 810, 1,300, and 1,500 nm,respectively. The values are in good agreement withtheoretically predicted values from a model developed withinthe consortium [47]: 1.3×10−3 at 810 nm, and 1.2×10−3 at1,300 and 1,500 nm, thereby validating that model. In orderto compare PSi membranes with other sensing platforms,optical sensitivity was obtained in terms of nanometers/RIU,being 626 nm/RIU at 810 nm, 1,135 nm/RIU at 1,300 nm,and 1,247 nm/RIU at 1,500 nm.

5 Conclusions

The bulk refractive index sensitivity of 1,247 nm/RIU at1,500 nm for initial anisotropic PSi membranes in POSI-TIVE is of the same order of the magnitude as the2,169 nm/RIU of the Vernier ring resonators in InTopSensand one order greater than the 246 nm/RIU of the Si3N4

ring resonators in SABIO. This, combined with its intrinsichigh degree of sensor integration indicates a high potentialfor sensing applications. Currently, higher optical qualityPSi membranes are being prepared for both chemical andbiomolecular sensing experiments in an optimized experi-mental setup where we expect to see better sensitivity stilland low limits of detection.

Acknowledgments As project manager of all three projects, there aremany contributors with whom I have worked directly and am mostgrateful for their efforts. I thank Jesús Álvarez Álvarez, Hans Sohlström,and Kristinn Gylfason for their photonics contributions in both SABIOand POSITIVE. Other direct contributors to the SABIO work summa-rized here are Andrzej Kaźmierczak, Fabien Dortou, Laurent Vivien, JonPopplewell, Gerry Ronan, and Carlos A. Barrios. Other direct contrib-utors to the INTOPSENS work summarized here include Tom Claes,Peter Bienstman, Asger Krüger, Martin Kristensen, Jaime GarciaRupérez, Veronica Toccafondo, and Javier Garcia. Further directcontributors from the POSITIVE project for the article include VladimirChirvony, Isaac Suárez, Paolo Bettotti, and Juan Martínez Pastor. As Ireview the collaborative projects SABIO, INTOPSENS, and POSITIVE,many others have contributed. The work reported here was financed bythe European Commission through the sixth framework project FP6-IST-SABIO, and the seventh framework projects FP7-ICT-INTOPSENS andFP7-ICT-POSITIVE, respectively.

(a)

500 nm 500 nm600 800 1000 1200 1400 1600

0.06

0.07

0.08

0.09

0.1

0.11

0.12

0.13

0.14

Δn

λ [nm]

AirIsoEtha

(b)Fig. 9 Surface SEM images oftwo fabricated and experimen-tally characterized samples: aPSi sample with pore size in theorder of a 10 nm, b birefrin-gence data obtained from themeasured spectra (dots) andsimulated curves using theBruggeman model (dashedlines) for air (green), isopropa-nol (blue) and ethanol (purple)

170 BioNanoSci. (2011) 1:162–172

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