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Integrated Angle-Insensitive Nanoplasmonic Filters for Ultraminiaturized Fluorescence Microarray in a 65 nm Digital CMOS Process Lingyu Hong,† Hao Li,‡ Haw Yang,‡ and Kaushik Sengupta*,† Departments of †Electrical Engineering and ‡Chemistry, Princeton University, Princeton, New Jersey 08544, United States
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S Supporting Information *
ABSTRACT: In this work, we demonstrate for the first time massively parallelizable nanoplasmonic structures and integration of electronics in the same substrate in CMOS. We adopt the same “fabless” approach in today’s semiconductor industry with absolutely “no change” of either fabrication or processing and show that copper interconnects in an industry standard digital CMOS process (65 nm node) can be exploited to allow subwavelength optical field processing in a massively scalable fashion. We demonstrate this in the context of eliminating all external optics and enabling the first optics-free fully integrated CMOS fluorescence-based biosensor array. The system has massively multiplexed biomolecular sensing capability for DNAs with surface sensitivity comparable to commercial fluorescence readers. The angle and scattering insensitive nature of the filter, relying on coupled surface-plasmon polariton modes, allows us to eliminate all external optics and miniaturize the entire 96-sensor array system (including a LED source) within 0.1 cc of volume. The system demonstrates detection sensitivity of less than 1 molecule/μm2 or zepto moles of quantum dot based fluorophores on the chip surface. The electronic−nanophotonic codesign approach allows us to optimally partition optical and electronic filtering, enabling us to detect fluorescence signal 77 dB lower than the excitation. Such CMOS-based nano-optical systems can lead to novel chip-scale optical sensors for in vitro and in vivo applications. KEYWORDS: CMOS, plasmonics, nanoplasmonics, nano-optics, copper, biosensor, DNA, microarray
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CMOS image sensors have caught up with classic CCD detectors in recent years, all optical passive elements are typically external, bulky and expensive. Not surprisingly, fully integrated, multiplexed, and external-optics-free fluorescence sensing has not been demonstrated in CMOS. In this work, we present the first demonstration of subwavelength optical field manipulation in a commercial CMOS foundry process. This allowed full integration of a multiplexed fluorescence sensor that reaches surface sensitivity of 1 fluorophore/μm2. While prior works have shown CMOScompatible nano-optical structures in copper or otherwise,28−32 here we adopt an absolutely “no-change” approach to CMOS fabrication. We demonstrate optical nanostructures exploiting the lowest copper interconnect layers in an industrystandard 65 nm CMOS foundry process that is typically used for digital processors and wireless integrated circuits (ICs). These integrated filters allow angle-insensitive filtering of background excitation, allowing the removal of all external optical elements and detection of immobilized fluorophores directly on the chip surface. The IC integrates the nano-optical filters, optical shielding and all the necessary electronic
he ability to sense biomolecules (DNAs, RNAs, proteins) with high sensitivity and specificity in a massively multiplexed fashion in an ultraminiaturized and ultralow power system can revolutionize personalized medicine and low-cost diagnostics.1−6 If it is small enough to fit inside a pill, it can also enable complex biomolecular sensing modalities invivo that current technologies fail to address.7−9 Currently, such biomolecular detection systems are typically based on affinity-reaction (such as specific antigen−antibody for enzyme-linked-immunoabsorbent-assay (ELISA), DNAcDNA) and signals are detected by fluorescence reporters.10−15 This typically requires complex optical instrumentation including multiple filters, lenses, discrete photodetectors and mechanical scanners arranged in collimated optics making such systems bulky and expensive. This is particularly true for systems that reach surface detection sensitivities of the level of 1 fluorophore/μm2 and below. This correspods to volume senstitivites of sub-pM levels for target biomolecules, where the background excitation can be between 106−107 times larger than the fluorescence signal. To enable this new class of miniaturized and massively multiplexed sensing systems, complementary-metal-oxide semiconductor (CMOS) can play a significant role due to its scalability, yield, and ability to integrate complex systems at low cost.16−27 At optical frequencies, while the performance of © XXXX American Chemical Society
Received: April 6, 2018 Published: September 12, 2018 A
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50 dB of extinction ratio between the excitation and fluorescence emission, the massive multiplexing ability allows us to sense the background to achieve another 27 dB of electronic filtering at the signal processing level at the backend, allowing an end-to-end detection capability of fluorescence power (Pf) even 77 dB below the excitation light (Pl; see Supporting Information for calculation). In the past two decades, exploiting nanoscale metallic structures to manipulate and guide light has made tremendous progress in enabling subdiffraction waveguiding,42−44 nanofocusing,45,48 improved photovoltaic devices,46,47 plasmon modulation,49 flat lenses with meta-surfaces,50,51 and plasmon resonance-based enhancements for Raman spectroscopy and biosensing.52−54 Leveraging existing CMOS technology to manipulate light at the visible and NIR at nanoscale with integrated photon detection and complex signal processing circuitry can enable a new class of multifunctional optical system on chips (SoCs) with extreme miniaturization, low-power to operate, and extremely low cost upon mass manufacturing.55,56
circuitry including photodetection, low-noise readout, and signal processing for the 96-multiplexed sensor array chip. To further leverage the angle insensitivity of the on-chip filter, we employed a low-cost, mm-sized UV LED for fluorescence excitation and a disposable biointerface, thereby miniaturizing the complete 96-sensor fluorescence system into a total volume of ∼0.1 cc. In the absence of any external reader, the systems constitutes the sensor platform, sensor and reader all integrated into the chip.33 The key enabler of the presented ultraminiaturized 96sensor fluorescence reader chip is the on-chip copper-based subwavelength nanoplasmonic filter. Unlike prior plasmonic filters that are based on resonant plasmon coupling, such as the structures proposed for RGB color filtering,34−36 the presented filter exploits the differential propagation loss of coupled surface plasmon modes in the subwavelength waveguide array structure. This unique design achieves a very high extinction ratio (40−50 dB) required for high-sensitivity fluorescence detection, and it simultaneously enables the filter to be insensitive to angles of incidence or complex fluorescence dipole excitation condition. This is key to on-chip miniaturization of a benchtop fluorescence reader where stray and scattered incident light is inevitable and fluorophores emit for a wide range of angles of incidence. Such integrated angleinsensitive design eliminates all external collimating optics37,38 and achieves detection sensitivity orders of a magnitude higher than filterless fluorescence detection systems based on fluorescence lifetime detection with complex picosecond-level laser synchronization.39,40 In this work, we present the complete design, theory and measurement results of the on-chip copper-based nanoplasmonic filter. We also demonstrate detection and multiplexing ability with DNA assays with sub-pM levels of concetration. The disposable bio-interface can be functionalized with capture antigens/antibodies for high sensitivity immunoassays as well. Furthermore, we show the scalability of fabrication of optical nanostructures in massively parallelized fashion which can be an enabling technology for future complex nano-optical systems. Miniaturized fluorescence sensing systems in a CMOS process can enable new sensing modalities for a wide range of biomolecular sensing applications, both in vitro and in vivo.
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INTEGRATED COPPER-BASED ANGLE-INSENSITIVE NANOPLASMONIC FILTER Design. In a classical fluorescence setup, both fluorescence signal and laser excitation are collimated to allow the usage of a high-performance multilayer fluorescence emission filter, which typically works within a small range of angles.37,38 In this miniaturized sensor platform without optical collimation (Figure 1), the radiation from the fluorescent dipoles on the chip surface interacts with the integrated filters in a complex fashion for a wide range of incident angles.57−59 In addition, in order to retain the ultraminiaturized form factor for point-ofcare in vitro and even in vivo diagnostics, it is preferable to use LED as opposed to a bulky laser system for fluorescence excitation. In this work, the filter needs to handle the neargrazing excitation light as well as the scattered light from the assay and other necessary structures of the CMOS chip (e.g., the bonding pads). Therefore, the angle insensitive characteristic of the filter with extinction ratios (≥45 dB) becomes a critical and differentiating factor for chip-scale fluorescence sensing. This precludes any resonant filter structures (interference-based or resonant plasmonic coupling34,35). In this work, we exploit the differential loss of the coupled subwavelength waveguiding through the copper nanostructures with the dominant coupled surface plasmon polariton (SPP) modes to achieve angle-insensitive filter characteristics. In the 65 nm industry standard CMOS process, the lowest metal layers in close proximity to the transistors have the smallest feature sizes (≈100 nm width, ≈130 nm spacing). The nanoplasmonic filter is designed to be an array of vertical subwavelength slab waveguides realized with the 4th−7th copper interconnect layers and the via layers in between, in total, measuring 1.41 μm in vertical length in the direction of the optical mode propagation (Figure 2a). Electronic signals are extracted from the photon-detection circuitry and transferred to the edge of the chip using the 1st−3rd interconnect layers and are transmitted externally. The entire optical path, filters and the electronic routing are codesigned to ensure optimal performance by maximizing light collection efficiency (minimizing circuits routing metal layers on top of the active photon-detection area) and minimizing light leakage from the sides of the chip. The metallic nanostructures fabricated in standard CMOS are examined under scanning electron microscope (SEM) after
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CMOS-NANO-OPTICS INTEGRATION AND SYSTEM-LEVEL OPPORTUNITIES The embedded multilayer electronic interconnects (∼10−12 layers) realized with copper in modern day CMOS are used here to serve the dual role of interconnects and optical signal processing. With semiconductor device scaling, the feature size of the copper-based interconnect layers has also entered the deep-subwavelength regime at or below 100 nm for nodes smaller than 65 nm transistor feature size.41 Design of these nanostructures in a CMOS process must be compliant with design rule checks (DRCs) of the foundry to ensure high yield and tight process control of fabrication. This includes minimum feature sizes, minimum spacings, minimum areas as well as metal density requirements for stability of the planarization process. However, a close cointegration and codesign approach of the on-chip nano-optical structures in close proximity to the integrated electronics in the same substrate enables new system level capabilities that remain obfuscated in the partitioned approach. As an example, while the nanoplasmonic filters presented in this work achieve nearly B
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Figure 2. (a) Structure and dimension of the integrated nanoplasmonic filter, implemented in 65 nm CMOS process with minimum metal line width of 100 nm and spacing of 130 nm. (b− d) SEM images of the interconnect layers serving the dual functionalities of integrated optical filtering and electronic routing for the photon-detection circuitry underneath the filter.
Figure 1. (a) Multiplexed fluorescence sensor array chip fabricated in standard 65 nm CMOS with integrated nanoplasmonic filters, photon detection, readout, and signal processing circuitry. (b) Array architecture showing the 12 × 8 sensors with nanoplasmonic filters on top and the optical shield surrounding the filters to prevent light leakage into chip. (c) Structure of the photodiode with the optically shielded reference diode and the in-pixel readout circuitry codesigned with the integrated nanoplasmonic filter.
the top passivation layers are removed by focused ion beam (FIB), as shown in Figure 2b−d. The fabricated metallic nanostructures show exceptional consistency with the original design. The high yield of the CMOS fabrication process for both optical and electronic functionalities is exploited to enable a continuous global filter sheet across the entire array, allowing seamless scaling in the number of sensing sites for multiplexed fluorescence detection. Operating Principle. Figure 3a shows the simulated normal incidence transmission spectrum for the y-polarization (perpendicular to the slabs) and x-polarization (parallel to the slabs), respectively. The parameters of the filter structure are shown in Figure 2, the dielectric constant of copper across the wavelengths of interest is obtained from ref 60 and the dielectric material surrounding the filter structure is assumed to be glass. The simulation is performed for one unit cell of the structure applying periodic boundary conditions using Finite Difference Time Domain (FDTD) method. To comply with the design rule checks of the CMOS fabrication process, the filter is designed to allow light to pass for one polarization
Figure 3. (a) Simulated transmission spectra for the nanoplasmonic filter at normal incidence for polarizations perpendicular and parallel to the copper waveguide slabs. The spectra show ∼60 dB extinction ratio between 800 and 405 nm wavelengths for perpendicular polarization and almost complete blockage for parallel polarization across the wavelengths of interest. The absorption and emission spectrum of the chosen quantum dot fluorophore (Qdot 800) are shown inside the figure and they match the implemented filter performance. (b) Simulated filter extinction ratio with 10% metal line width process variation showing robustness of the filtering process.
along y for minimum area requirement of each copper nanostructure. As can be seen, the filter shows nearly 60 dB extinction ratio between 405 and 800 nm. Due to the subwavelength spacing between the slabs, light with parallel polarization (along x) are largely blocked across the wavelength of interest. We choose quantum dots as the fluorescence tag for their photostability, stronger emission and higher Stokes shift61,62 and they have become standardized for assay chemistry. Figure 3a also shows the fluorescence excitation and C
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Figure 4. (a) Simplified structure of the 2D waveguide array model. (b−e) Simulated mode profiles of the TM and TE modes of the lowest three orders including the SPP mode. (f) Summary of the effective mode index (propagation constant normalized by the wave-vector k0 in vacuum).
for the TM and TE modes for 405 nm fluorescence excitation and 800 nm fluorescence emission wavelengths. Figure 4f summarizes the effective mode indices and the propagation losses of the modes. As can be seen, the fundamental TM mode (TM0) for both 405 and 800 nm wavelengths have an effective index larger than the refractive index of the dielectric media SiO2. This mode is known as the surface plasmon polariton (SPP) mode, which does not “cutoff” as the dimensions of the waveguide system scale down. The loss of the SPP modes, therefore, mainly depends on the dielectric behavior of copper at these frequencies, which experiences significant propagation loss at 405 nm (∼35.24 dB/μm) compared to wavelengths near 800 nm (∼0.95 dB/μm). This nonresonant characteristic is critical for sensitive detection in the presence of uncollimated and scattered excitation and fluorescence. It is critical to understand the effect of the cavity modes represented by various types of TE and higher order TM modes, when extinction ratios as high as 50 dB are being considered (Figure 4). In contract to the SPP modes whose mode loss is primarily determined by the material loss for different wavelengths (Figure 5a), the loss of these cavity modes is affected strongly by the geometry and dimensions of the waveguide with respect to the wavelength (Figure 5b). As can be seen, while the TM0 mode loss for 800 nm is orders of magnitude lower than that at 405 nm, the reverse is true for the TE0 mode. For optimal filter performance, the cavity modes must be suppressed to the largest extent possible. In this work, the spacing among the waveguide slabs is chosen to be 130 nm, the minimum allowable dimension in the implemented 65 nm CMOS technology, thereby keeping the loss of both the cavity and SPP modes at the excitation wavelength of 405 nm high.
emission spectrum of the chosen Qdot 800 fluorescent tag which can be efficiently excited at around 400 nm and emits at around 800 nm, compatible with the filter performance.63 In principle, the filter can be designed to process both polarizations with different geometric shapes (such as subwavelength nanopillar arrays as an example) and therefore double the photon collection efficiency. In this work, since the filter blocks the parallel polarization at 405 nm excitation wavelength, it removes the need for any external polarizer as well. Additionally, although theoretically process variation can have some effect on the optical performance of the filter. The nonresonant absorptive nature of the filter design enable the optical performance to preserve. As shown in Figure 3b, the simulated extinction ratio keeps its value at 55−65 dB for 10% metal line-width variation. A modal analysis of the periodic, subwavelength copper waveguide array is carried out to explain the filtering principle. A 2-dimensional (2D) waveguide array which shares similar principle of operation is first considered. In this 2D system as shown in Figure 4a, the structure is periodic along the y dimension with unit cell pitch of 230 nm, the structure is assumed to be infinite along x and the modes propagates along z. Due to the periodicity of the system, coupled waveguide modes are excited which is characterized by the Bloch wave vector along y dimension (k∥), depending on the excitation condition (incident angles for plane-wave excitations or different dipole excitations). To study the quality of the modes, we first consider the case when k∥ = 0, corresponding to plane wave excitation normal to the surface of the filter along z dimension. Expectedly, the waveguide supports different orders of TE and TM modes. Figure 4b−e show the simulated absolute values of Hx and Ex field distributions D
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ACS Photonics TM mode: cos(k Λ) − cos(k1a)cos(k 2b) +
ϵ k yz 1 ijj ϵ1k 2 jj + 2 1 zzzz ϵ1k 2 { 2 jk ϵ2k1
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(1)
× sin(k1a)sin(k 2b) = 0
TE mode: cos(k Λ) − cos(k1a)cos(k 2b) +
Figure 5. Coupled SPP (TM0) modes and cavity modes (TE0) have distinct losses as wavelength and waveguide spacing vary for kx = ky = 0 (normal incidence). The implemented minimum spacing of 130 nm in 65 nm CMOS ensures both SPP and cavity modes experience high propagation losses at 405 nm, critical for angle-insensitive filtering.
× sin(k 2b) = 0
k yz 1 jij k 2 jj + 1 zzzsin(k1a) 2 jk k1 k 2 z{
(2)
where k1 = ϵ1k 02 − β 2 and k 2 = ϵ2k 02 − β 2 , ϵ1 and ϵ2 are the dielectric constants of copper and SiO2 respectively, a and b are the slab thickness of copper and SiO2 respectively, and π π − Λ < k < Λ . Solving the complex propagation constant β for different k∥, we obtain the band diagram. The propagation loss band diagram for the fundamental TM and TE modes for both 405 nm fluorescence excitation and 800 nm fluorescence emission wavelengths are shown in Figure 6a−d. For 800 nm wavelength, the fundamental TM modes (coupled SPP modes) exhibit low propagation loss around 1−2 dB/μm, while the loss of the same mode for 405 nm is around 35 dB/μm. On the other hand, the fundamental TE mode (coupled cavity modes) for 405 nm wavelength shows 18−23 dB loss and above 100 dB loss for 800 nm. The actual fabricated structure, as shown in Figure 2a, is periodic in both x and y dimensions (pitches are both 230 nm) due to the via layers. To capture this effect, we show the simulated minimum propagation losses (including both SPP and cavity modes) against the kx,ky in the first Brillouin zone of the periodic structure at the excitation and fluorescence wavelengths in Figure 6e,f. Considering this worst case of filtering, we see that despite of the multiple modes, there exists around 30 dB/μm differential propagation loss for the two wavelengths. This angle-insensitiveness allows us to eliminate
Subwavelength spacing is key to ensure that background excitation is rejected regardless of which modes are excited, hence the robust high-performance optical filtering. Expectedly, migrating to finer technology node (such as a 32 nm CMOS) will further enhance the filter performance due to stronger elimination of TE0 mode for 405 nm. Angle-Insensitive Filter Characteristics. As mentioned above, for chip-scale fluorescence-sensing, the filter needs to suppress the near-grazing and scattered excitation light and also allow fluorescence emission from a variety of incident angles to pass efficiently. For the same periodic structure shown in Figure 4a, the coupled modes are characterized by the modal order, complex propagation constant and the Bloch wave vector k∥ along the y dimension. The electric or magnetic field satisfys u(y + nΛ) = u(y)exp(jnΛk∥), where Λ is the pitch of the waveguide array (230 nm). For the 2D waveguide array structure, the solution of the complex propagation constant (β) can be derived analytically.
Figure 6. (a−d) Analytically calculated mode loss as a function of the Bloch-wave vector k∥ in the first Brillouin zone for the coupled-SPP TM0 and the cavity TE0 modes at the excitation and fluorescence wavelengths for the 2D slab waveguide array. (e, f) Lowest mode loss for different kx and ky in the first Brillouin zone for the CMOS integrated 3D filter structure with periodicity in both x and y, proving the angle-insensitivity of the filter upon all excitation conditions. (g, h) Scaling down from 65 to 32 nm CMOS with tighter spacings suppresses the cavity modes strongly and further improves the performance. (i, j) Fluorescence emission pattern on the chip surface for xy random oriented fluorescent dipole and z oriented dipole, respectively. E
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Figure 7. (a) Measured normalized transmission spectrum for y-polarization (perpendicular to slabs) showing ∼47 dB extinction ratio between 800 and 405 nm wavelengths. (b)Measured normalized transmission for 800 and 405 nm wavelengths with varying angles of incidence and polarizations.
all external optics and ensures robust filtering in the presence of oblique or scattered excitation and fluorescent dipole emission. As expected, the filter performance improves with technology scaling as we migrate to a finer node such as 32 nm CMOS process due to the suppression of the cavity modes, as shown in Figure 6g,h. Figure 6i,j shows the calculated dipole radiation power density in the same Brillouin zone of the filter.57−59 As expected, the majority of fluorescence emission power has large kx and ky values due to dipole being placed at the air/SiO2 interface. Therefore, maintaining low loss in the entire Brillouin zone at 800 nm is critical to collect fluorescence power as much as possible for high sensitivity detection. Filter Experimental Characterization. The transmission spectrum of the optical filters are characterized with the integrated photodetectors by measuring the responsivity (V/S) of the sensor chip divided by the responsivity of a test chip (the same photosensing circuits without the filter), under the illumination of lasers of various wavelengths. This captures the spectral responsivity of the filters. Figure 7a shows the measured normal incidence transmission spectrum (normalized at the 830 nm) for the y-polarization (perpendicular to the slabs). As can be seen, around 47 dB extinction ratio between 800 nm fluorescence emission and 405 nm excitation wavelengths is obtained. The slight discrepancy between the measurement and simulation is attributed to multiple nonidealities. First, the simulation and analysis assume a copper waveguide array, while in reality CMOS foundry processes employ barrier diffusion layers to prevent copper ions from diffusing into the active devices. Since SPP mode properties are
highly sensitive to the material property at the interface, this factor is expected to have large contribution to the discrepancy. In addition, the dielectric consists of multiple thin layers proprietary to the foundry, some of which can be considerably different from SiO2. In spite of that, measurement results show a 47 dB of filtering achievable with the CMOS integrated nanoplasmonic filters. Figure 7b show the measured normalized filter transmittance for the excitation and emission wavelengths across various angles of incidence and polarizations. The measurement demonstrates a extinction ratio varying between 45 and 60 dB. In essence, the subwavelength nonresonant nature of the nanoplasmonic structure ensures the rejection of near-grazing or scattered excitation light from all angles, which is critically important for sensitive biomolecular assays. This allows us to eliminate collimation, objective lens, and other external optical filtering elements and replace the typical excitation laser by a ultracompact low-cost LED for an overall ultraminiaturized system.
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INTEGRATED CMOS FLUORESCENCE MICROARRAY SYSTEM Electronics-NanoOptics Codesign in Commercial 65 nm CMOS. In this work, the nanoplasmonic filter and the photon detection, readout, and signal processing circuitry are all codesigned and cointegrated in a single chip in a 65 nm industry standard CMOS process without any post processing or change in fabrication (as shown in Figure 1a). The chip encompasses 8 × 12 sensors in a 2 mm2 area for detecting multiple biological analytes simultaneously and each sensor is 100 μm × 100 μm in size. The 96 pixels are addressed and F
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Figure 8. (a) CMOS fluorescence sensor chip copackaged with an UV LED for fluorescence excitation and a removable thin glass coverslip as the biointerface whose position is set by the silicon fixtures. (b) Coverslip is printed with biocapture probes with a commercial DNA arrayer, and the assay is performed in the well.
Figure 9. (a, b) Measured waveforms of CTIA and CDS outputs when the array is readout in series (pixel by pixel). (c, d) Single pixel noise voltage distribution in dark and under excitation. (e) Optimization of limit of detection defined as (laser power/fluorescence power) for the given filter performance of SNR = 1 showing nearly 70 dB of detection capability. (f) The limit of detection can be increased to 77 dB for longer averaging time (∼1 min).
leakage from the side of the chip through tiny gaps (≈1 μm) between routing traces needs to be taken into consideration. This effect is mitigated by designing optical shield (nanostructured metal layers with maximum density allowed by the fabrication process) surrounding the entire periphery of the 96 sensors (Figure 1b). Inside each sensing pixel underneath the nanoplasmonic filter are the 80 sensing photodiodes (in parallel) with 80 metal-shielded reference photodiodes with a differential capacitive transimpedence amplifier (CTIA), as shown in Figure 1c. The design details of the photodetector structure and considerations have been addressed in our prior work.55,56 The amplifier (OTA) gain of the CTIA is around 35.0 dB, the feedback capacitance of the CTIA is around 15.6 fF, and the diodes are controlled by the switches made of four
accessed by the column and row decoders in a timemultiplexed fashion, as shown in Figure 1b. Flexible readout including programming the integration time and the readout sequence is allowed to maximize the photon detection sensitivity, and the signals are further processed by the differential correlated double sampling circuits to reduce the correlated noise and offset (Figure 1b). In the optical sensing region of the chip, the routing of the circuitry is achieved with the lowest three metal layers, while the nanoplasmonic filter is realized with the 4th to 7th metal layers, as previously mentioned (Figure 2c,d). The filter is designed to be a uniform global optical structure covering the entire 96 pixels (Figure 1). However, due to the small dimension of the chip and stringent extinction requirement at the excitation wavelength, light G
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Figure 10. (a) Measured sensor response versus surface density of quantum dot-based fluorophores. (b) Fluorescence image for the lowest detectable surface concentration of quantum dots at 1 dot/μm2. (c) Multiplexed detection of three different spots at different locations of the sensor array.
Figure 11. (a) Functionalization and assay protocol for DNA detection using quantum dot-based fluorescence tag. (b) Measured sensor response with varying target DNA concentration from 100 fM to 100 pM with conjugated pairs and nonconjugated pairs (negative control).
light source, the sensor IC, the fixtures and the biointerface) occupies a volume of around 6 × 6 × 3 mm3, as shown in Figure 8a. The glass coverslip is prepared by a DNA printer/ arrayer to be functionalized with multiple different captures molecules at different sites to detect multiple different target analytes simultaneously, as shown in Figure 8b. Limit of Detection of the Sensor and Biological Measurements. With the on-chip row and column decoders, there are multiple ways to readout the signals of the sensor array. Figure 9a,b shows an example of the analog waveform of CTIA and CDS outputs for the in-series readout mode (pixel by pixel). For any single pixel, the diode is reset first, followed by integration, as shown in the differential output of the CTIA. When the CTIA is in integration mode, the CDS is in reset until the end of CTIA integration, where the maximum differential signal can be recorded with reduced correlated noise and offset. The measured electrical and optical performance of the chip is shown in Figure 9. The measured average noise in dark and under laser excitation is ≈0.5 mV and ≈3.5 mV respectively. Under excitation, the noise is primarily contributed by the photon shot noise. With regard to net filtering of the excitation and the background, backend electronic filtering can be added on top of the 47 dB of extinction ratio achieved optically with nanoplasmonic filters. This can be done by allowing the array to sense the average residual background and filter it electronically. The combination of optical and electronic filteringallow us to reach the detection limit of fluorescence power (Pf) 70 dB below excitation power with S.N.R. = 1. This can be further increased to 77 dB for longer acquisition time by averaging for ∼1 min
cascading PMOS transistors in order to suppress the switch leakage current during diode integration.56 Since the 65 nm digital CMOS process is for general purpose processors and nonoptimized for photon detection, such differential design converts the dark current in a well-matched distributed pixel in each sensor as common mode, which can be suppressed. This allows a longer integration time (≈1 s) to increase the sensitivity. System Design for Fluorescence Biosensing. The complete ultraminiaturized fluorescence microarray system consists of the CMOS chip with the integrated filters, detection, read-out, and signal processing circuitry, UV LED source, and carefully placed tiny silicon-wafer based fixtures that allows automatic alignment with a disposable biointerface for multiplexed detection (Figure 8a). The multiplexing ability is important for screening and also allows multiplicative readouts from a single assay, including the blank control, to drastically improve the statistics. This reduces the false-positive and false-negative rates, a critical factor in all practical medical diagnostics. As mentioned before, the angle-insensitive nanoplasmonic filter allows a vertically positioned low-cost UV LED to be used to replace traditional bulky laser sources for fluorescence excitation. The LED is placed to the side of the chip to maintain the ultracompact form factor and the light is incident from near grazing angles to the surface of the glass slip and is rejected by the filter. The NIR emission from the spatially multiplexed fluorescent tags, on the other hand, passes through the filter efficiently, gets detected by the photodetector arrays, and then electronically processed by the IC. The entire sensing part of the system (including the excitation H
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(see Supporting Information for calculation). The cointegration of the electronics and optics, thereby, allows us to partition the filtering optimally reaching an end-to-end effective filtering stronger than any one component. Additionally, as the system leverages the mature CMOS photosensing technology, the high-yield CMOS foundry process for nanoplasmonic filter fabrication, robust filter design, and the exceptional photostability of quantum dot-based fluorophore, the measurement is very stable and consistent. All measured chips have shown very similar performance. The sensitivity of the chip in detecting quantum-dot-based fluorophores is quantified as shown in Figure 10a. A tiny droplet with fixed volume of V = 0.5 μL and varying quantum dot volume concentration ρ (diluted from 1 μM stock solution) is placed on the thin glass slip, let dry, and placed on the surface of the chip, after which the sensor response is read out upon LED excitation. The tiny droplet preserves a spherical shape and occupies approximately the surface area of 2/3
( 34V )
S ≈ π 1/3
Figure 12. (a) Multiplexed detection from a single assay at positive/ negative control sites for DNA hybridization on three different spots of the sensor array.
unshielded chips exposed to scattering, the sensor achieves competitive sensitivity with state of the art sensors. The nanoplasmonic-electronic codesign approach in CMOS chips, demonstrated for the first time, can enable a new class of complex, ultraminiaturized, chip-scale optical biosensors for in vitro and in vivo applications. Conclusions and Outlook. We demonstrate for the first time that nanoscale copper-based electronic interconnects in an industry standard CMOS process can allow subwavelength optical field processing in the visible/near-IR. We utilize these interactions to enable on-chip angle-insensitive nanoplasmonic filters for an external-optics-free massively multiplexed fluorescence sensing chip. The codesign and cointegration of the nano-optics and multiplexed sensor array allow us to combine optical filtering of around 47 dB with electronic filtering of 30 dB for an end-to-end fluorescence detection sensitivity reaching down to 77 dB below excitation. We present the theory and design of the subwavelength nanoplasmonic waveguide array including characterization. The angle-insensitive and robust filtering characteristics allow the extreme miniaturization of a complete fluorescence sensing microarray into a chip-scale format, eliminating all traditional external optical components including lenses, filters, and photodetectors arranged in collimated optics. The system demonstrates measured detection limits down to zeptomoles of fluorophores on the surface (∼1 dot/μm2 of surface density). This work also demonstrates scalability of realizing these complex nanoplasmonic structures in the CMOS fabrication process. Given the integration capability of both electronics and now optics, the technology can be potentially scaled into tens of thousands, if not hundreds of thousands of sensing sites, in a cost-effective manner enabling a new class of ultraminiaturized sample-to-answer biomedical devices for both in vitro and in vivo applications.
; therefore, the surface density can be
estimated σ = 0.827ρV1/3. The sensor shows a linear response as a function of surface concentration and the lowest level of detection is measured to be around 1 dot/μm2. This extremely low level of surface density is considered to be at the singlemolecule level. This is verified in the fluorescence image measured using a benchtop fluorescence microscope (Figure 10b), where the slides are prepared using the same procedure in Figure 10a. Note that the active area of each pixel is around 55 μm × 55 μm and, therefore, the total number of quantum dots is only around 5 zepto moles. This exceeds the surface sensitivity levels of modern fluorescence scanners and readers that are typically in the attomole range. It is important to note the low standard error at each measurement point in Figure 10a and it demonstrates the repeatability of the experiment and the low noise of the sensor. DNA detection is tested using the miniaturized fluorescence sensor. The glass slips are placed and temporarily fixed in separate glass wells (Figure 8). Subsequently, the coverslips are functionalized for the nucleic acid detection. The functionalization and assay procedures for DNA is shown in Figure 11a. Finally, detection is achieved using streptavidin conjugated Qdot 800 fluorescent tag. Figure 11b shows the measured sensor response and standard error with DNA hybridization assays with 33 base pairs. For conjugate and nonconjugate pairs (negative control). Here, we define the standard error as the standard deviation of the measured signals from multiple samples of the same assay. Even though the sensor noise itself is low (which reflects in the low standard error in detecting Qdots on the sensor surface in Figure 10a), the preparation of the multiple slides and their functionalization with the spotting instrument have variations from slide to slide. This shows up in the slightly larger error bars in the response measurement (Figure 11b). The error bars can be reduced with better control and repeatability of the surface chemistry. In presence of these variations, the chip demonstrates a linear response with limit of detection (LOD) of 100 fM at SNR ∼ 2. Figure 12 shows an example of measurement of a multiplexed DNA hybridization experiment with one positive and two negative controls illustrating the expected high detector response at the conjugated site and almost no signal at the nonspecific sites. In spite of eliminating all external optical elements and measurements done in
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsphotonics.8b00440. Detailed sensitivity analysis (PDF).
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Kaushik Sengupta: 0000-0001-7074-0248 I
DOI: 10.1021/acsphotonics.8b00440 ACS Photonics XXXX, XXX, XXX−XXX
ACS Photonics
Article
Author Contributions
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L.H. and K.S. designed the system. L.H. performed the theoretical analysis and experiments on the optics, circuits, and the sensitivity of the entire integrated sensing system. L.H. and H.L. performed the biological (fluorescence assay) experiments. K.S. and H.Y. supervised the work. L.H. and K.S. wrote the manuscript. Funding
This work was funded by National Science Foundation (NSF; ECCS 1610761, ECCS 1711067), Qualcomm Innovation Fellowship, Princeton Project X, Princeton Intellectual Property Fund. Notes
The authors declare no competing financial interest.
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DOI: 10.1021/acsphotonics.8b00440 ACS Photonics XXXX, XXX, XXX−XXX
