High coupling efficiency contact imaging system having micro light pipe array for a digital enzymelinked immunosorbent assay Hironari Takehara1,3, Mizuki Nagasaki1, Kiyotaka Sasagawa1,3, Hiroaki Takehara1,3 Toshihiko Noda1,3, Takashi Tokuda1,3, Hiroyuki Noji2,3, and Jun Ohta1,3 1
Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan 2 Graduate School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan 3 CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan
[email protected]
Abstract—The enzyme-linked immunosorbent assay (ELISA) is a diagnostic technique used for detecting the presence of viruses or tumor markers. To detect target biomarkers, antigenantibody reactions followed by fluorescent reactions are carried out in an ELISA. Recently, digital ELISAs have been proposed to achieve higher sensitivity. In the digital ELISA, fluorescent reactions are carried out in an array of femtoliter-scale microchambers. The concentration of the target biomarkers is determined by counting the number of microchambers with and without fluorescence using a fluorescence microscope. We have been developing compact digital ELISA systems by replacing the fluorescence microscope with a dedicated stacked photodiode CMOS image sensor as a fluorescence detection tool. High coupling efficiency for fluorescence and low coupling efficiency for excitation light are key problems that need to be overcome to achieve a practical contact fluorescence imaging system. Here, we present an array of light pipe absorption filters directly connected with microchambers. The manufacturing processes are also described. Our structure makes it possible to achieve a high sensitivity that is comparable to that achieved by employing the digital ELISA with a fluorescence microscope and provides a miniaturized digital ELISA system. Keywords—ELISA, Digital ELISA, CMOS image sensor, Fluorescence detection, Light pipe array
I.
INTRODUCTION
Compact medical diagnostic systems that perform highsensitivity detection of biomarkers with low power consumption can be widely deployed in clinical practice. That is because these systems can be powerful tools in the battle to suppress epidemics of infections in resource-limited countries. In addition, home diagnosis leads to early detection of diseases and effective medical treatment. The enzyme-linked immunosorbent assay (ELISA) is a diagnostic technique used for detecting any antigens, for example viruses or tumor related ones, in serum, plasma, and urine. There are various types of ELISA. The typical protocol of a sandwich ELISA is as follows. First, target biomarkers
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(e.g., antigens) are specifically captured by capture antibodies (which are covalently attached on the inner surface of the reaction chambers) by an antigen-antibody reaction. After the unbound antibodies are washed away, biotinylated detection antibodies are added that bind with the antigen. Next, streptavidin enzyme conjugates bind with the detection antibodies, which are then subjected to a fluorescent reaction. The enzymes enhance the fluorescent reaction, producing fluorescent products in an aqueous solution of fluorescent substrate. The concentration of the target biomarkers is determined by the fluorescence intensity. Recently, digital ELISAs have been proposed to achieve higher sensitivity [1], [2]. Kim et al. showed a one-millionfold higher sensitivity compared to that of a conventional ELISA for the detection of prostate specific antigen (PSA) when they applied microbeads for capture antibody immobilization and a femtoliter-scale microchamber array for the fluorescent reaction [2]. The diameter of the microbead is slightly smaller than that of the microchamber, so that each microchamber can contain only one microbead in it. The microchambers are small enough to detect fluorescent products in a single chamber containing only a single molecule of the enzyme. The concentration of the target biomarkers is determined by counting the number of microchambers with and without fluorescence (Fig.1). Digital 20 nm
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Fig. 1. Digital ELISA. Concentration of the target biomarker is determined by counting the number of microchambers with and without fluorescence.
We have been developing compact digital ELISA systems in which the fluorescence microscope is replaced with a dedicated CMOS image sensor as a fluorescence detection tool. In these systems, an excitation attenuation filter should be placed between the chamber array and the CMOS image sensor. In this study, we clarify the critical issue and describe a structure, and a manufacturing process for a compact system that can achieve a high fluorescence coupling efficiency.
pipe structure can provide a high CE by collecting the fluorescence from the microchambers using reflection on its inner surface. 1 Point light source
d a Photo detector
II.
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LENSLESS DIGITAL ELISA SYSTEMS
A. Lensless Contact Imaging We have demonstrated two types of lensless digital ELISA systems. However, the sensitivities were not high enough for practical use. The systems can be characterized by their methods for attenuating excitation light. If the excitation light is not attenuated enough, the CMOS image sensors are saturated by excitation photocarriers and cannot detect fluorescence from the microchambers. One of the systems comprised a light guide made of a silicon microhole array and an interference filter (Fig. 2(a)) [3]. The other system comprised a combination of total reflection structure and an absorption filter (Fig. 2(b)) [4]. The two systems could effectively attenuate the excitation light. However, the systems also attenuated a considerable amount of fluorescence simultaneously, which was a problem. The main cause of the low intensity of fluorescence that reaches the photodetector is the distance between the fluorescence source (microchamber) and the photodetector (CMOS image sensor). (a)
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ELISAs are good candidates for realizing compact diagnosis systems, but a space-consuming and expensive fluorescence microscope for detecting the fluorescence is still needed.
Fig. 3. Coupling efficiency for fluorescent light dependence on F = d / a. The curves are calculated on the assumption that fluorescence is emitted isotropically from a point light source.
Figure 4 shows the structure of the micro light pipe array. Every micro light pipe has a dedicated microchamber in close proximity. There is only a thin silicon dioxide film between them. The micro light pipes are made of metal which has high reflectivity. Each micro light pipe is filled with an absorption filter for attenuating excitation light. The absorption filter is made of a transparent epoxy resin doped with a yellow dye. We selected fluorescein as the fluorescent compound for demonstrating our system. The central fluorescence wavelength of fluorescein is 525 nm, and its excitation wavelength is approximately 470 nm. The yellow dye absorbs the excitation light efficiently and transmits the fluorescent light. Fluorescent reaction chamber array and micropillar absorption filter array
Excitation light Fluorescent reaction chamber (5 µmφ) Hydrophobic peripheral (CYTOP) Hydrophilic bottom (SiO2) Light pipe (metal) Absorption filter (dye-doped resin) Attenuation of excitation light
Absorption filter
Image sensor
Fig. 2. Previous lensless digital ELISA systems. (a) Silicon light guide array and interference filter, (b) total reflection and absorption filter.
B. Micro Light Pipe Array Structure The detectable fraction of total fluorescence is defined as its coupling efficiency (CE) for fluorescence. A low CE for fluorescence hinders high-sensitivity detection. The side length of a photodiode in a pixel of a CMOS image sensor is several micro-meters, while the distance between the microchamber and the photodiode is several tens of micrometers in our previous systems. As shown in Fig. 3, if the ratio of the distance (d) and the side length (a) equals to 10, the corresponding CE decreases to less than one thousandth [5]. Owing to the thickness of the filters and the substrates of the microchamber array, it is difficult to position the microchambers close to the photodetectors. Our micro light
A pixel of CMOS image sensor
Fig. 4. Structure of micro light pipe absorption filter with microchamber.
C. Coupling Efficency Simulation The CE for fluorescent light of the proposed system can be predicted by using a simple model assuming (1) the microchamber as a point fluorescent light source allocated at the center of the entrance of the micro light pipe, (2) the rightcircular-cylinder-shaped micro light pipes have completely smooth inner surfaces, and (3) the fluorescent light is absorbed by the absorption filter according to Beer-Lambert’s law. Because the fluorescent light is emitted isotropically, the intensity is proportional to the solid angle ω. The reflection and the absorption (related to the light path length) are also functions of the solid angle. The fraction of fluorescent light can then be calculated from equation (1). The integration range is from 0 to 2π, which corresponds to the bottom hemisphere,
ଶగ
ܫ௧௦௧ ൌ න ܫ ܶఠ ܴఠഘ ݀߱
(1)
where Itransmit is the intensity of fluorescent light transmitted through the micro light pipe, I0 is the initial intensity of fluorescent light, Tω is the solid-angle-dependent fraction of transmission, Rω is the solid-angle-dependent reflectance, and nω is the number of reflections on the inner surface of the micro light pipe, which is also a function of the solid angle. When copper is used for the light pipe metal, light pipe has 30 µm in height and 8 µm in diameter, and the normal transmittance is 0.8 for a 10-µm-thick absorption filter, the fraction of transmitted fluorescent light was calculated to be 2.9%. This calculation result suggests that this structure improves the CE significantly compared to our previous systems. D. CMOS Image Sensor Having Stacked Photodiodes The micro light pipe structure might cause variation in the intensity of the fluorescence and leaked excitation light. For example, variation in the yellow dye concentration in the absorption filter, variation in the dimensions of the micro light pipes, and autofluorescence of the epoxy resin can change the light intensity. If the CMOS image sensor can discriminate the wavelength of the light, the small signal of the fluorescence is (a)
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the same dimensions as a single unit of the micro light pipe array. The CMOS image sensor is a three-transistor active pixel sensor (3Tr-APS). We are improving the sensitivity and the noise performance by utilizing a foure-transistor APS architecture in a stacked photodiode CMOS image sensor. III.
LIGHT PIPE ARRAY MANUFACTURING PROCESS
In this section, the outline of the overall process flow for manufacturing the light pipe with a microchamber array is presented. Some fundamental manufacturing technologies are also introduced. The process starts with the utilization of thermally oxidized silicon substrate as a starting material to realize the structure. The micro light pipe and the microchamber were formed on each side of the thermally grown silicon dioxide film, whose thickness was 1 µm. A. Electroplating Process for Light Pipe Formation The micro light pipe array was formed on the 4-inch oxidized silicon wafer. At first, titanium and copper thin film were deposited by sputtering as seed metals for electroplating. Before the electroplating, a circular pattern array was formed with photolithography and wet etching (Fig. 6(b)). Each circular pattern was located at the same position as a light pipe. The micropillar array patterns (35 µm in height) were then formed by using a thick photoresist (KMPR1035, Nippon Kayaku Co., Ltd.) with photolithography (Fig. 6(c)). Copper was electroplated (30 µm in thickness) around the micropillar patterns (Fig. 6(d)). After the electroplating process, the thick photoresist was removed with a wet process (Fig. 6(e)). The surface of the side wall pillars were made smooth by baking after development. The inner surfaces of the light pipes, which are the replicas of the pillars, are also smooth. (a) Si substrate with thermal oxide (d) Copper electroplating
(g) Removing Si by dry etching
(b) Seed metal pattern
(e) Thick photoresist removal
(h) Thermal oxide appeared
(c) Thick photoresist pattern
(f) Upside down
(i) Hydrophobic film pattern
Fig. 5. Stacked photodiode CMOS image sensor. (a) Cross section and layout of a pixel, (b) photograph of the sensor chip.
detected even when the leaked excitation light intensity is fluctuates. We have demonstrated a stacked photodiode CMOS image sensor for ELISA [6]. The layout and the cross section of a pixel and a photograph of the CMOS image sensor are shown in Fig. 5. The specifications of the CMOS image sensor TABLE I. Specifications of the CMOS image sensor
Process technology Supply voltage Chip size Pixel type Pixel size Photodiode types Fill factor
0.18 µm 1-poly 6-metal CMOS for mixed signal 3.3 V (Analog) / 1.8 V (Digital) 2.2 mm × 2.5 mm 3-transistor active pixel sensor 15 µm × 15 µm Stacked 2 photodiodes ・N+/PW (Top, PD1) ・PW/DNW (Bottom, PD2) 31.8% × 2 layers
are listed in Table I. The pixel size is 15 × 15 µm2, which are
Fig. 6. Illustration of manufacturing process flow.
B. Filling with Absorption Filter An absorption filter was prepared by mixing a yellow dye (Varifast Yellow 3150, Orient Chemical Industries Co., Ltd., an example of transmission spectrum is shown in Fig. 7) dissolved in small amount of cyclopentanone (Tokyo Chemical Industry Co., Ltd.) and a transparent epoxy resin (Z-1, Nissin Resin Co., Ltd.). The epoxy resin is a thermoset resin that initiates a curing reaction immediately after a curing agent is added to the base resin. A small plate of the micro light pipe
array was dipped in an uncured dye-doped epoxy resin under reduced pressure. The micro light pipes were filled with the dye-doped resin by alternating between atmospheric and reduced pressure several times. The excess dye-doped resin was removed by scraping out with a plastic spatula and wiping with ethanol-wetted paper.
Transmittance
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process and operation of fluorescence detection, so that we can align each micro light pipe and a pixel of a CMOS image sensor properly one by one. We are developing a fixture unit for the operation of fluorescent detection. For rapid diagnosis, it is required that the CMOS image sensors have high sensitivity and better noise performance. We are developing a stacked photodiode CMOS image sensor with 4Tr-APS architecture and noise reduction circuits.
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Fig. 7 An example of transmission spectrum of dye-doped resin film.
C. Silicon Substrate Removal The silicon substrate was completely removed by reactive ion etching using SF6 as an etchant gas. Because the etching rate selectivity between silicon and silicon dioxide is very high, the thermally grown silicon dioxide with a smooth surface appeared as a back surface on the micro light pipe array (Fig. 6(h)). D. Microchamber Array Formation A hydrophobic amorphous fluoropolymer (CYTOP 809M, Asahi-glass Co., Ltd.) was spin-coated and cured on the back side surface of the micro light pipe array. The thickness of the CYTOP film was 3 µm. The microchamber array pattern was formed by photolithography and reactive ion etching (Fig. 6(i)). Microphotographs of a thick photoresist pattern and a light pipe array filled with dye-doped resin are shown in Fig. 8. (a)
(b)
ACKNOWLEDGMENT This work was supported by Japan Science and Technology Agency, Core Research for Evolutional Science and Technology (JST-CREST), VLSI Design and Education Center (VDEC), and the University of Tokyo with the collaboration with Cadence Corporation and Mentor Graphics Corporation. Our collaboration with the Foundation for Nara Institute of Science and Technology, and Taiyo Manufacturing Co. Ltd. enabled us to use their electroplating technology. REFERENCES [1]
[2]
[3] Fig. 8. Microphotographs of manufactured micro light pipe array. (a) Thick photoresist pattern for electroplating, (b) micro light pipe array filled with dye-doped resin.
IV.
DISCUSSION
Because copper is a popular and cost-effective for electroplating material, we chose it as the metal for the micro light pipe manufacturing process. Copper can be replaced with a metal with a higher reflectance. If silver is applied instead of copper, the calculated CE for fluorescence is improved more than doubled. The manufactured micro light pipe array filter looks like a thin copper plate. The copper plate might undergo not only elastic deformation but also plastic deformation. Permanent deformation should be avoided throughout the manufacturing
CONCLUSIONS
We developed a lensless digital ELISA system that has a high CE for fluorescent light. A micro light pipe array structure was introduced for lensless fluorescent imaging. Simulation results shows a significant improvement in the CE. Manufacturing processes are also developed. Electroplating around thick photoresist patterns resulted in smooth inner surfaces for the micro light pipes, which helps achieve high reflectance. The micro light pipe structure makes it possible to achieving a high sensitivity that is comparable to the digital ELISA with a fluorescence microscope and provides a miniaturized digital ELISA system.
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