Article Cite This: ACS Photonics 2019, 6, 1694−1705
pubs.acs.org/journal/apchd5
All-Silicon Spectrally Resolved Interferometric Circuit for Multiplexed Diagnostics: A Monolithic Lab-on-a-Chip Integrating All Active and Passive Components Konstantinos Misiakos,† Eleni Makarona,† Marcel Hoekman,‡ Romanos Fyrogenis,§ Kari Tukkiniemi,∥ Gerhard Jobst,⊥ Panagiota Sotirios Petrou,# Sotirios Elias Kakabakos,# Alexandros Salapatas,† Dimitrios Goustouridis,§,∇ Mikko Harjanne,∥ Paivi Heimala,∥ and Ioannis Raptis*,† Downloaded via BUFFALO STATE on July 19, 2019 at 08:53:01 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
Institute of Nanoscience and Nanotechnology, NCSR “Demokritos”, Athens 153 41, Greece Lionix BV, Enscehde, 7500 AM, The Netherlands § ThetaMetrisis S.A., 12132 Peristeri Attikis, Greece ∥ VTT, FI-02150 Espoo, Finland ⊥ Jobst Technologies GmbH, 79108 Freiburg, Germany # Institute of Nuclear & Radiological Sciences and Technology, Energy & Safety, Athens 153 41, Greece ∇ Department of Electrical & Electronics Engineering, University of West Attica, Athens, 12244, Greece ‡
S Supporting Information *
ABSTRACT: Despite the tremendous advances in micro- and nanoelectronics and the fast-pacing advances in photonic circuit designs, seamless monolithic integration of electronic and photonic components on single chips still remains elusive. In this work, a radically designed silicon-based chip that monolithically integrates in a 37 mm2 footprint 10 interferometric optical sensors along with their respective optical sources, spectral analyzers, and photodetector arrays is presented. The chip is fabricated with mainstream CMOS-compatible fabrication techniques and employs optical devices operating in the visible/infrared spectrum and waveguides with a critical dimension of 1.0 μm. In addition, it exploits the newly introduced detection principle of broad-band Mach−Zehnder interferometry that surpasses the stringent requirement for external monochromatic sources and inherent limitations of traditional interferometry and introduces alternative designs of on-chip spectral analyzers and mode-filtering components, aspiring thus to become a novel lab-on-a-chip that can address the needs of next-generation analytical systems. Apart from the conceptual design, novel photonic features, fabrication steps, and out-of-the-box system development that circumvents the need for fluidic interfacing and employs only electrical interconnects, the present work tests the potential of the fully spectroscopic chip for analytical applications through real-time monitoring of immunochemical reactions and demonstrates limits of detection for antimouse IgG antibody and CRP of 60 and 8 pM, respectively. KEYWORDS: Photonics−microelectronic integration, Broad-band interferometry, Photonic circuit, Lab-on-chip, Optical biosensing
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silicon chips by exploiting the infrastructure built around silicon-based microelectronics (especially CMOS).4 Recent paradigms have exhibited remarkable levels of integration not only in terms of the number of electronic and photonic components but also in terms of functionalities.2,5,6 The realization of passive photonic devices by microfabrication technologies gave a boost in the implementation of optical bioanalytical devices, because probing matter with light allows for high sensitivity, wide dynamic range, galvanic isolation, from the control and detection electronics, immunity to parasitic effects, andin several casesmultianalyte and/or
espite the tremendous advances in micro- and nanoelectronics and the formidable achievements of photonic technologies, these two fields keep on evolving rather in parallel and only sporadically conjoin to produce integrated platforms that combine the best of both worlds.1,2 On the one hand, electronic components that rely on silicon are inherently limited in light generation. On the other hand, active optical components require the use of compound semiconductors bonded or heteroepitaxially grown onto Si wafers.3 As a result, integration of electronic and photonic components depends on complex technologies with profound effects on yield, cost, and time of production. Silicon photonics have heralded a new era by providing the means to monolithically integrate photonic components onto © 2019 American Chemical Society
Received: February 11, 2019 Published: June 13, 2019 1694
DOI: 10.1021/acsphotonics.9b00235 ACS Photonics 2019, 6, 1694−1705
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label-free detection schemes.7−14 Coupled with miniaturized measurement set-ups, optical biosensors have been coming out as a practical route for viable and marketable analytical systems with a special emphasis on point-of-need (PoN) applications.15−17 Despite the ingenious designs of optical sensors (such as photonic crystals,18 microring resonators,19 and interferometers,20 just to name a few) and resourceful conceptions of on-chip elements for advanced photonic circuits (e.g., wavelength (de)multiplexers,21 on-chip spectral analyzers,22 polarization-handling components,23 etc.), light incoupling and/or out-coupling to the integrated chips has been relying exclusively on external light sources and associated optical components as well as the design of efficient grating couplers.24,25 Recently, Mach−Zehnder interefometers were coupled with array waveguides (AWGs) on the same Si chip and were successfully employed in label-free determinations,26−28 albeit with the use of external sources and detectors. Relying on external components29 though adds to the size, cost, and complexity of the final system, while it decreases its robustness against environmental conditions with serious compromises in performance and ease-of-operability. Alternative routes to fabricate on-chip light sources are still being explored, but the majority are not entirely CMOS-compatible, they may be tedious, or they are still restricted to the telecom wavelengths and fail to extend to the visible or near-infrared part of the spectrum (VIS−NIR), which is more pertinent to current analytical approaches.30,31 Thus, the integration of photonic and electronic components has become the Holy Grail for the field of analytics, which is in search of comprehensive, cost-efficient solutions, able to be massively produced via mainstream fabrication approaches. This work presents a radical approach to photonic− electronics integration that encompasses all active and passive elements of a photonic circuit and results into a unique, monolithically integrated, interferometric, and fully spectroscopic silicon chip that can become a versatile label-free analytical platform for a wide range of applications. This novel chip, manufactured with mainstream CMOS-compatible microfabrication processes, monolithically integrates in 37 mm2 a complete spectroscopic circuit consisting of an array of 10 optical transducers in the form of broad-band Mach− Zehnder interferometers (BB-MZIs) each in-coupled to its own broad-band light source and out-coupled to a broad-band spectrum analyzer in the form of an arrayed waveguide grating (AWG) and sequentially to an array of photodiodes that records on-chip the BB-MZI output spectra. Each sensor on the chip can be individually bio/chemically functionalized, offering the ability for synchronous highly sensitive label-free detection of up to 10 analytes. This way, the bio/chemical reaction progress is monitored through a transmission spectrum recorded by the on-chip array of photodiodes using only standard electrical connections, similar to any other conventional integrated circuit. Thus, the fabricated photonic circuit advances the current state-of-the-art in integrated photonic biosensors by integrating the MZI and AWG structures, arrays of broad-band light sources, and photodetector arrays for the on-chip transformation of the transmission spectrum to electrical current and also operation in a very wide spectral range that has been proven to offer significant advantages in label-free determinations restricted so far by the limitations of monochromatic or narrow-band implementations. The functionalized chip is complemented with an A5-footprint reader that takes care of the LED
multiplexed operation and photodiode array readout as well as all fluidic and electrical operations. The reader may be controlled either by a computer or smartphone/tablet, depending on the desired application. The fact that the fabricated biochip needs only electrical interconnects to turnon the integrated light sources and to record spectrally analyzed optical signals as electrical signals by the PD arrays allows for the design and implementation of a truly miniaturized reader with clear advances in terms of size, weight, and power requirements compared to the current stateof-the-art.26−28 The analytical potential of this radical fully spectroscopic Si chip and accompanying instrument is demonstrated through the real-time monitoring of the binding reaction of mouse γ-globulins immobilized onto the chip with a goat antimouse IgG antibody and the immunochemical detection of C-reactive protein, a biomarker extensively employed in daily clinical practice for the diagnosis of inflammatory conditions.
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MATERIALS AND METHODS Fully Spectroscopic Chip Design. The complete electrophotonic circuit uses as its main building block a unique silicon-based monolithic optical transducer (Si-MOT),32,33 fabricated by mainstream silicon technologies with a critical dimension on the 1 μm scale and based on stoichiometric LPCVD-deposited Si3N4 waveguides. Employing Si3N4 enables low-loss optical transmission, wafer-scale processing, and costefficient fabrication.34 Si-MOT manages to monolithically integrate on a single chip a light source self-aligned to a planar waveguide (WG), the other end of which is coupled to a photodiode (PD) (Figure 1a). The light source is an avalanche diode, which when reverse-biased beyond its breakdown point, emits in a wide VIS−NIR spectrum (530−950 nm)35 and through a carefully designed processing flow is self-aligned to the waveguide. To achieve this, the LED P2+ emitter is
Figure 1. (a) 3-D schematic representation of one couple of BB-MZIs with their respective light sources, spectral analyzers, and a common array of 10 PDs. (b) Layout of the complete electrophotonic chip. The numbers denote: (1) LED contact pads; (2) LED junctions; (3) on-chip mode converters; (4) BB-MZI sensing arms; (5) on-chip mode converters; (6) on-chip spectral analyzers (AWGs); (7) array of PDs; (8) PD bus lines; (9) PD pads. 1695
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implanted through the silicon nitride layer; the boron atoms compensate a pre-existing base N+ implant (phosphorus) so that the metallurgical junction of the LED is precisely placed under the up-going segment of the waveguide. The SiO2 spacers next to the LED and the detector provide for the smooth waveguide bending from the vertical to the horizontal direction and from the horizontal back to the vertical, respectively, offering an effective optical link, which minimizes the bending losses (more details can be found in ref 36). The Si-MOT concept has been augmented to actualize for the first time true on-chip multiplexed spectroscopic interferometric analysis. In particular, the planar waveguide is engineered to become a BB-MZI, the sensing arm of which can be tuned for the detection of a particular biomolecule through selective biofunctionalization. The output waveguide of the BB-MZI is then coupled to an on-chip spectral analyzer in the form of an AWG that divides the BB-MZI output spectrum into 10 spectral bands (right part of Figure 1a). The spectral response is measured through an array of 10 PDs integrated on the same electrophotonic Si chip, because each output WG of the AWG is self-aligned and coupled to one PD. In the present work, each Si chip accommodates 10 optoelectronic sensors, the layout of which is illustrated in Figure 1b. Each sensorbased on the Si-MOT design consists of its optical source and the BB-MZI transducer biofunctionalized for a specific analyte but is expanded further from the basic Si-MOT and acquires its own spectral analyzer fed to a 10 PD detection array, which is shared by one more neighboring sensor (Figure 1a). Hence, each chip contains 10 BB-MZI sensors divided into 5 couples connected to 5 arrays of PD detectors, as depicted in Figure 1b. Principle of Operation: Broad-Band Mach−Zehnder Interferometry. The principle of detection of the photonic chip is the recently introduced broad-band Mach−Zehnder interferometry36 that employs broad-band light at its input. The BB-MZI has been shown to circumvent the inherent limitations of single-wavelength MZ interferometry (SWMZI), known as signal fading and phase ambiguity, and offers a great versatility in terms of design and applications, because changes of the geometrical characteristics of the interferometric arms (mainly their core thickness) results in drastic changes of the transmission spectrum. Very briefly, in SWMZI, the interferometer affects the intensity of the in-coupled light in the well-known sinusoidal eq 1: T (λ) =
ΔK rs =
2π ΔNr,s(λ) λ
= 2π (αλ + β)
(2)
then any event that changes the effective index of the sensing arm by δNs will result in a spectral shift δλ given by36 δλ =
δ Νs ∂( Νs λ ) δnc = · = (λm − λm − 1)L( δNs λ ) αλ ∂nc α
(3)
where the subscript m denotes the m-th order peak with two successive peaks λm, λm−1 corresponding to a phase difference of 2π i.e., φ(λm) − φ(λm−1) = 2π, whereas nc is the refractive index of the cover medium over the MZI’s sensing arm. For the special case of designing a BB-MZI with δΝr,s/λ independent of the wavelength over the spectral range of the in-coupled light, the sinusoidal output spectrum will undergo a solid shift δλ, as given by eq 3. Discrete fourier transform (DFT) of such an output spectrum yields a dominant peak in the wavenumber domain with the phase identified by the argument of the complex DFT value at the main peak. Such a capability has already been demonstrated through label-free multiplexed model binding assays (biotin−streptavidin and mouse IgG−antimouse IgG)36,37 and through the label-free detection of bovine milk in goat milk with an LOD of 0.04% (v/v) bovine milk in goat milk and a dynamic range 0.1−1.0% (v/v).38 In all cases, an external commercial spectrometer was employed coupled to the chip via an optical fiber. In this work, on-chip spectral analysis of BB-MZI with the entire photonic circuit monolithically integrated and contained in a single silicon chip of a less than 40 mm2 footprint is demonstrated for the first time. Chip Surface Activation/Biofunctionalization. The chips were chemically activated through silanization after cleaning and hydrophilization by oxygen plasma treatment (30 s, 10 mTorr) in a reactive ion etcher. For the silanization, the cleaned chips are immersed in a 0.5% (v/v) aqueous solution of 3-(aminopropyl)triethoxysilane (APTES; Sigma-Aldrich, Munich, Germany) for 2 min. After that, they are gently washed with distilled water and cured at 120 °C for 20 min. The biofunctionalization of the chips is accomplished by spotting the biomolecule solution (100 μg/mL mouse γglobulins or 200 μg/mL anti-CRP antibody in 0.1 M carbonate buffer, pH 9.2) at sensing window areas using the BioOdyssey Calligrapher Mini Arrayer (Bio-Rad Laboratories Inc., Hercules, CA). In order to homogeneously cover the sensing window areas with the biomolecule solution, a spotting protocol involving deposition of multiple overlapping spots was applied. In each chip, four out of the five waveguide pairs are spotted with the biomolecule solution, whereas the last pair is used for the determination of the nonspecific binding signal. The spotted chips were incubated overnight at 4 °C in a 75% humidity environment and then, they are washed with 10 mM Tris-HCl buffer, pH 8.25 (washing buffer), and blocked using a 10 g/L BSA in 0.1 M NaHCO3, pH 8.5, for 1 h. The blocked chips are washed with washing buffer and distilled water, dried under a nitrogen stream, and kept in a desiccator until use. CRP Assay. The chips that have been functionalized with the anti-CRP antibody (affinity purified goat antihuman CRP antibody from Scripps Laboratories; San Diego, CA) and blocked as described above were placed onto the docking station of the portable reader. Once the fluidic and electrical connections were secured, assay buffer (Tris-HCl 50 mM, pH 7.8, 5 g/L BSA, 0.5 g/L bovine IgG, 9 g/L NaCl) was run over
Å Ñ i 2π ΔΝr,sL yzÑÑÑÑ Iout 1 1 ÅÅÅ zzÑÑ = [1 + cos(Δφ)] = ÅÅÅÅ1 + cosjjjj zÑÑ Iin 2 2 ÅÅÇ λ k {ÑÖ
(1)
where Ιin and Ιout are input and output optical power at a specific wavelength λ, respectively, Δφ is the phase difference between the sensing and the reference arms induced by a change ΔNr,s of the effective RI difference between the two arms ΔNr,s(λ) = Nr(λ) − Ns(λ), and L is the length of the MZI arms. In a ΒΒ-MZI, the spectral characteristics of the transfer function depend on λ in a unique way through the term ΔNr,s(λ), rendering the dependence of the transfer function on the wavelength more intricate but also amenable to elaborate tailoring by appropriate photonic design of the waveguides. In particular, it has been shown36 that when the propagation constant difference between the two arms is made linear for the spectral range of interest 1696
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Figure 2. (a) Photograph of the fully spectroscopic chip, where the sensing areas of the MZIs (blue rectangle), the AWGs (purple arrow), and the contact pads of the light sources and photodiodes are discernible (green rectangles); optical microscope images of the fully spectroscopic chip showing (b) its front end (top) with 6 out of the 10 LED junctions and their contact pads as well as the first mode-converters, (c) 6 out of the 10 sensing areas of the BB-MZIs with the windows opened through the top cladding layer, (d) 5 BB-MZI output WGs with the second modeconverters along with 2 pairs of AWGs (the third AWG pair is partly visible in the right-hand side of the photograph), and (e) 3 out of the 5 PD arrays where the AWGs converge in pairs. The bus lines connecting all PDs can also be seen.
chip to serve for 10 sensors, while the planar waveguides were engineered to ΒΒ-MZIs, the sensing arms of which can be “personalized” through selective biofunctionalization for specific analytes allowing for multianalyte determinations. The 10 MZIs were divided into 5 couples (Figure 1b) with the scope to have duplicates for every analyte. Particular care was devoted to the design of the MZIs with extensive simulations of the effective refractive indices (Neff) of the reference and sensing arms. Neff and appropriate ΔNr,s values were calculated for the entire range of the avalanche LED emission spectrum as a function of: (i) the WG core thickness for both the sensing and reference arms; (ii) the cover medium index of refraction; and (iii) the adlayer refractive index that builds up over the sensing arm during a binding reaction event. The magnitude of ΔΝr,s is the outlining factor that in conjunction with the length
the biochip for 2 min. Then, the CRP calibrators prepared in assay buffer, or CRP-free human serum were introduced and run for 6 min. After that, a solution containing 10 mg/L of anti-CRP antibody in assay buffer was run for 4 min. The biochip surface was regenerated by running 0.1 M glycine/HCl buffer, pH 2.5, for 3 min, followed by washing and equilibration with assay buffer prior to the next run.
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RESULTS AND DISCUSSION Monolithic Spectroscopic Si Chip: Design, Simulation, and Principle of Operation Verification. The monolithic spectroscopic chip was designed for multianalyte determinations at a minimum area through the implementation of radical photonic concepts (Figures 1b and 2a). First of all, an array of 10 sources has been incorporated on a single 1697
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Figure 3. (a) Layouts of AWGs: (1) symmetric AWG; (2) antisymmetric AWG; (3) design of the antisymmetric 1 × 10 AWG employed in the photonic chips. Inset: SEM image of the AWG at the output where 5 out of the 10 1.2 μm wide strip-WGs with a gap of 0.8 μm can be seen. Simulation results of the antisymmetric AWG employed in the fully spectroscopic chip showing (b) the generation of 10 spectral bands as would be recorded at an imaging screen right at the output of the AWG and (c) the spectral content at the 10 output channels. (d) Comparison of the individual recording of each output AWG channel by an external spectrometer to the normalized TE spectrum when multiplied with the simulated AWG performance. Inset in (d): normalized TE content of the LED emission spectrum.
of the MZI can provide the expected spectral shifts of the MZI transfer function (as was analyzed in the previous subsection). The output waveguide of each MZI is then fed into an onchip spectral analyzer in the form of an AWG with M = 1 input WG and N = 10 output WGs that divide the MZI output spectrum into 10 spectral bands. Each band is detected by 1 of the 10 PDs, because each output WG of the AWG is selfaligned and coupled to 1 PD. Each couple of MZIs/AWGs share 1 array of 10 PDs, so in total, the chip contains 50 PDs (5 arrays of 10 PDs each). Such a scheme is possible, because the LEDs are turned on sequentially in a multiplexed format, and the PDs can independently record the signal from the transducers, all controlled by the reader. A total of 10 bus lines are used to connect 5 parallel PDs to a single contact pad, and thus, the total number of contact pads for the PDs is 12, equal to the corresponding number of contact pads required for the LEDs (10 plus 2 for the ground connections). A guard ring has been placed around the photodiode array in order to maximize the signal-to-noise ratio of the detector current, by shielding it from the LEDs.
The AWGs employed in this work are based on a novel design that has stark differences with respect to conventional AWGs most commonly employed for telecom applications, where AWGs are designed to allow multiple wavelengths to combine and then spatially separate into a distributed array of waveguides (Figure 3a1). The operation of the conventional AWG scheme is presented in S-1. In general, the order m of the AWG determining how many wavelengths fit in the required path length difference ΔL is given by ΔL = m(λi /Neff (λ)), i = 1, 2, ..., N
(4)
where λi is the free space wavelength entering the AWG through the i-th input WG out of the total N input WGs, and Neff is the effective refractive index (RI) of the WGs, which depends on λi. In our case, the AWG concept was adapted to accommodate the stringent requirements imposed by the operation in a very broad band spectrum. In the spectroscopic chip, the required path length differences are relatively small so that the order of the AWG is only m = 1, a very challenging condition to achieve 1698
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bands but their consistent separation both spectrally and spatially as well as their finesse, which is comparable to the simulated one. This is evident in the recorded bands, which are well-separated on average by 25 nm and have the same finesse, as predicted by the simulations. The photonic circuit contains two additional but equally important components: two on-chip mode converters, one placed just before the MZI input and one at the MZI output. For the interferometers to operate properly (see next subsection) the WGs must be single-mode (SM) for the entire emission spectrum of the LEDs. The most suitable type of WG for the VIS−NIR part of the spectrum is a rib-WG with a rib width
