- Email: [email protected]
Attomolar detection of cytokines using a chemiluminescence immunoassay based on an antibody-arrayed CMOS image sensor
Accepted Manuscript Title: Attomolar detection of cytokines using a chemiluminescence immunoassay based on an antibody-arrayed CMOS image sensor Author: Donggu Hong Hyou-Arm Joung Do Young Lee Sanghyo Kim Min-Gon Kim PII: DOI: Reference:
S0925-4005(15)30098-8 http://dx.doi.org/doi:10.1016/j.snb.2015.07.042 SNB 18773
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
3-4-2015 8-7-2015 12-7-2015
Please cite this article as: D. Hong, H.-A. Joung, D.Y. Lee, S. Kim, M.-G. Kim, Attomolar detection of cytokines using a chemiluminescence immunoassay based on an antibody-arrayed CMOS image sensor, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.07.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Research Highlights
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Highlights
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Attomolar detection for interleukins was achieved with directly performing sandwich immunoassay onto a surface of CMOS image sensor chip. A CMOS image sensor was used as a detector for CL imaging and substrate for the sandwich immunoassay.
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The sensor specific data calibration method was used to obtain stable and reproducible results.
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This assay system was evaluated with detection of interleukin-5 with a limit of detection of 0.074 fg mL ,
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Multiple target detection was simultaneously performed.
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which corresponds to a 50 aM, and with dynamic range in 1 fg mL-1 to 20 ng mL-1.
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*Manuscript
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Attomolar detection of cytokines using a chemiluminescence immunoassay based
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on an antibody-arrayed CMOS image sensor
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Donggu Honga+, Hyou-Arm Jounga+, Do Young Leec, Sanghyo Kimd* and Min-Gon Kima,b*
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gwagiro, Gwangju 500-712, Republic of Korea
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b
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Cheomdan-gwagiro, Gwangju 500-712, Republic of Korea
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School of Physics and chemistry, Gwangju Institute of Science & Technology, 261 Cheomdan-
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Advanced Photonics Research Institute, Gwangju Institute of Science & Technology, 261
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Republic of Korea
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Seognam-si, Gyeonggi-do 461-701, Republic of Korea
OPTOLANE Inc., 633 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, Korea 463-400,
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Department of bionanotechnology, Gachon university, San 65, Bokjeong-dong, Sujeong-gu,
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Corresponding authors: [email protected], [email protected]
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These authors are equally contributed to this paper
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Tel.: +82(31)750-8554
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E-mail: [email protected]
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Tel.: +82(62)715-3330
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Fax: +82(62)715-3419
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E-mail: [email protected]
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ABSTRACT
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A chemiluminescence immunoassay based on a CMOS image sensor has been developed for simultaneous
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attomolar detection of cytokines. Attomolar level detection sensitivity is achieved through direct immobilization
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of antibodies on the surface of the CMOS image sensor and using a simple data accumulation process with noise
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calibration after the sandwich immunoassay. Here, the proposed method was showed that the logarithmic linear
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range for interleukin-5 (IL-5) detection is from 1 fg mL-1 to 20 ng ml-1 and the limit of detection (LOD) is 0.074
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fg mL-1, corresponding to a concentration of 7 aM. The LOD represents ca. 1105-fold improvement compared
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with use of conventional ELISA. Furthermore, the multiple targets including IL-2, IL-4, IL-5, and IL-6 can be
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detected with simultaneous and low-cost manner on array type CMOS image sensor, and we hope that this
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sensor system will be a powerful tool in biological studies.
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Keywords
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CMOS image sensor, attomolar detection, cytokines, chemiluminescence, sandwich immunoassay
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1. Introduction Antibody array technology enables simultaneous detection and analysis of protein antigens present in
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samples. In a protein microarray format, the method can be used to track interactions and activities of proteins in
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a large scale and high throughput manner [Templin et al., 2002]. Additionally, the technique requires only small
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quantities of samples and, as a result, it can be employed to generate abundant data sets in a single and relatively
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cost-effective experiment [Melton, 2004]. For this reason, antibody arrays have been applied in a wide range of
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biomarker discovery and diagnostic studies [Devarajan et al., 2003; Srinivas et al., 2002; Fang et al., 2003].
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Although many commercial products are available for qualitatively profiling protein expression and identifying
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candidate biomarkers, almost all of these platforms have detection sensitivities that are less than that of the
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enzyme-linked immunosorbent assay (ELISA) system, which has limit of detection (LOD) in the picomolar
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region.
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New strategies are required in order to develop much more sensitive sensing systems that can be utilized for
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simultaneous detection and analysis of unknown biomarkers. Studies aimed at this goal have led to the
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development of sensors that have LODs at the attomolar level. For example, new techniques have been
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developed that are based on surface plasmon resonance (SPR) or SPR imaging [Lee et al., 2006; Hu et al.,
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2014], electrochemical immunosensors [Chikkaveeraiah et al., 2012; Tang et al., 2012], and photonic crystal
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arrays [Deng et al., 2013]. In particular, various approaches such as carbon-nanotube-based sensors and
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nanowire-based field effect transistor (FET) sensors with various signal amplification strategies were used for
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highly sensitive biomarker measurements [Elnathan et al., 2012; Zheng et al., 2005; Yu et al., Byon at al.,
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2006]. Additionally, several sensitive approaches, such as those that employ rolling circle amplification (RCA),
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electrochemiluminescence (ECL), chemiluminescence (CL) and phtoluminescence have been devised and are
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compatible with the antibody array format [Konry et al., 2009; Sardesai et al., 2013; Zong et al., 2013; Liu et
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al., 2013]. Despite their strong multiplex detection ability and high sensitivity, attomolar sensitivity still remains
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a challenge in simultaneous biomarker measurement. Accordingly, simple, low-cost methods are still required
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for the detection of biomolecules at attomolar levels.
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Detection methods based on CL are among the most sensitive and they have wide dynamic ranges [Kricka,
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1993]. As a result, these types of sensors have been widely used in the biological research [Brien et al., 1991;
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Constantine et al., 1994; Nori et al., 2008]. Light emission in an enzyme-catalyzed CL system is a
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consequence of a chemical oxidation reaction of a reagent catalyzed by peroxidase. For example, horseradish
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peroxidase (HRP) catalyzes oxidation of luminol in the presence of hydrogen peroxide to produce CL. However,
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the catalytic ability of this peroxidase is sometimes insufficient for specific experimental requirements and, as a
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result, supplements are added to enhance CL signals [Aslan and Geddes, 2009]. In addition, light emission
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efficiencies can be maximized by carrying out the CL reaction on the surface of the detector in this system. Recent developments have been made in the design of highly sensitive light detectors. For example,
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complementary metal oxide semiconductor (CMOS) image sensor has become good alternatives to those based
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on charge coupled device (CCD). CMOS based sensors have many attractive features including minimal power
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dissipation [Fossum, 1997], improved image quality [Takayanagi and Nakamura, 2013], and lower power
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consumption [Gamal and Eltoukhy, 2005], operation voltage and cost. For this reason, several bio-sensing
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applications of CMOS image sensor have been described. For example, biofluorescence imaging inside a brain
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tissue phantom has been carried out using a dedicated CMOS image sensor along with CL detection of nucleic
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acids hybridized onto a probe-immobilized CMOS photodetector array [Ng et al., 2006; Mallard et al., 2005].
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Although utilized in in selected cases, CMOS image sensors have not been employed extensively in
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biomolecular studies.
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In the investigation described below, we have successfully implemented a CL based system for the
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simultaneous and attomolar level detection of cytokines on an antibody arrayed CMOS image sensor. The
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CMOS image sensor in this system not only serves as a detector for capturing images but it also acts as a
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substrate for the immunoreaction in order to reduce CL light loss entering to a CMOS image sensor. In this way,
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CL light emitted near the surface of the sensor is directly transmitted to photodiodes which convert light into an
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electric and then digital signal with minimal loss. A calibration method is used to analyze digital data obtained
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from images in order to reduce variations between the default digital signals from chips and between different
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chips, and it is also used for correcting background signals arising from electrothermal effects on the sensors.
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Interleukin-5 (IL-5), one of the cytokines that is present at levels below several pg ml-1, is used as a model target
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to evaluate the sensitivity of the new detector. Furthermore, simultaneous detection of several cytokines on one
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chip is carried out using this system.
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2. Material and Methods
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2.1. Reagents and apparatus
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CMOS image sensor chips and a reader module were provided by Optolane Inc. (Korea). 4-
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Aminoantipyrine (AAP), luminol sodium salt, 4-iodophenol (4-IP), p-coumaric acid, N,N-dimethylformamide
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(DMF), glutaraldehyde (GA) solution (Grade I, 50%), sodium bicarbonate (NaHCO3) and sodium carbonate
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(Na2CO3), hydrogen peroxide (H2O2), Tween 20, (3-aminopropyl)trimethoxysilane (APTMS) were obtained
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from Sigma Aldrich (Korea). Bovine serum albumin (BSA) was obtained from Fitzgerald. 1X Phosphate
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buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4) and Tris-HCl (1.5 M,
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pH 8.8) were obtained from BIOSESANG, Inc. The TMB substrate reagent set was obtained from BD
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Biosciences (United State). Supersignal west femto maximum sensitivity substrate and supersignal west pico
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chemiluminescent substrate were obtained from Thermo Acientific (United State). Cytokine ELISA kits for
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interleukin-2, -4, -5 and -6 (IL-2, IL-4, IL-5, IL-6), which include antigen capture antibody, biotinylated
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detection antibody and streptavidin-conjugated horseradish peroxidase (STA-HRP), were obtained from BD
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biosciecnces (United State). CL intensities were integrated using a chemiluminescence imaging system
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(ChemiDoc, BIO-RAD). The IR spectrums were obtained using FTIR spectrometer (FT/IT-4200, JASCO).
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2.2. Preparation of the capture antibody immobilized CMOS image sensor The chip was modified to introduce aldehyde groups needed for immobilization of the capture antibodies.
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The aldehyde functionalized surface was prepared by using the following procedure. The 4 µL of 1% BSA/PBS
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solution was applied on the chip, a cover glass was placed on the chip and the assembly was allowed to stand for
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1 h at room temperature (RT). The chip was then washed with PBST (0.05% tween 20 in 1X PBS) and distilled
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water (DW) twice. The aldehyde-functionalized chip surface was prepared by adding 4 µL of 2% (v/v)
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glutaraldehyde in PBS solution onto BSA adsorbed chip surface, a cover glass was placed on the chip and the
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assembly was allowed to stand for 4 h at RT, and the chip surface was washed with PBST and DW twice. The 4
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µL of capture antibody (4 µg mL-1 in 0.1 M carbonate buffer (pH 9.5)) is manually applied to the surface of a
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chip with a micro pipette, then chip is incubated at 4 °C overnight. Finally, after twice wishing, the 1% BSA in
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PBS solution was introduced on CMOS image senor for blocking the residual GA. The volume (4 µL) was
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enough to cover the surface of CMOS image sensor (3 mm 4 mm). The IR spectrums were obtained BSA,
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GA immobilization steps respectably.
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2.3. Sandwich immunoassay for detection of interleukin-5 As depicted in Scheme 1A, the IL-5 sandwich immunoassay is performed on the capture antibodies-
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functionalized chip surface. Solutions of the IL-5 antigen and biotinylated detection antibody are prepared in 1%
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BSA/PBS. The sandwich immunoassay based on the general ELISA procedure is then performed by adding a
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solution (4 µL) of the IL-5 antigen, installing a cover glass for 2 h, and then adding 4 µL of 1 µg mL-1 of the
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biotinylated detection antibody and 4 µg mL-1 of STA-HRP mixture, followed by installation of a cover glass for
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1 h. Between each step in this procedure, the surface is washed with PBST twice. Finally, the chip is incubated
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in a sealed petri dish in order to prevent sample evaporation. To measure IL-5 using the CMOS image sensor, an
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optimized CL solution was used. After introducing the CL reaction solution to the CMOS image sensor at the
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end of the immunoassay, 1-sec exposure images were obtained for 120 sec before and after the reactions. Finally,
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the images were analyzed using the proposed calibration method. The detailed CL optimization procedures are
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described in the following section, and the data processing and calibration methods are described in detail in
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section 3.4.
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2.4. CL optimization
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In order to establish stable conditions for carrying out the HRP catalyzed CL reaction in our system, an
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optimum buffer pH and enhancer concentration must be identified and a method was needed for reducing
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interference of incident photon rate by phenolic compounds produced in the CL reaction. For this purpose, the
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CL characterization test was performed with fixed compositions of 2 mM luminol in 50 mM sodium hydroxide
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(NaOH) and 2 mM H2O2. CL detection was performed after STA-HRP reacts with biotinylated antibody, and its
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intensity is measured using the chemiluminescence imaging system. All composition of CL solution were
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optimized with 96-wellplate in order to follow standard method and the optimized condition was applied to
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CMOS image sensor.
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2.4.1. Optimization of CL buffer pH
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Various pH buffer conditions were tested using a 96-well microtiter plate. STA-HRP (1 µg mL-1) was added
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to each well followed by incubation for 30 min and washing with PBST and DW twice. A fixed composition CL
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solution containing 0.5 mM 4-IP in DMF along with various buffers including pH 7.2 (1X PBS), 7.4 (0.1M
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phosphate buffer), 8.3 (0.1M borate buffer) 8.8 (0.1 M Tris-HCl buffer) and 10.3 (0.1 M carbonate buffer), were
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used. The CL intensity was measured for 7 min.
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2.4.2. Optimization of 4-IP concentration
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4-IP concentration optimization was also carried out. This was performed in a manner that is similar to that
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used for buffer pH optimization. Specifically, 10 ng mL-1 STA-HRP was added to each well of a 96-well
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microtiter plate, followed by addition of a fixed composition of CL solution in 0.1 M Tris-HCl buffer (pH 8.8)
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and various concentrations (1 nM–0.01 mM) of 4-IP. The CL intensity was measured for 7 min.
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2.4.3. Characterization of AAP effect
An improvement of the CL intensity by AAP was observed in the following way. First, 100 ng mL-1 of STA-
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HRP was adsorbed to each well of a 96-well microtiter plate, followed by addition of fixed compositions of CL
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solution in 0.1 M Tris-HCl (pH 8.8) buffer with DMF and various concentrations (1 mM–100 nM) of AAP. The
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CL intensity was measured for 7 min.
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In order to evaluate the optimized CL composition, comparison was made between the CL solution
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containing AAP and commercial CL solution on the CMOS image sensor chip. For this comparison, CL
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solutions, consisting of 50 mM sodium hydroxide (NaOH), 2 mM H2O2, and 0.5 mM enhancer (4-IP or p-
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coumaric acid) with and without 100 nM AAP, were prepared. SuperSignal West Femto Maximum Sensitivity
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Substrate and SuperSignal West Pico Chemiluminescent Substrate were prepared for comparison purposes. The
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IL-5 immunoassay was carried out on six CMOS image sensors for each CL solution. The image sensors were
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first modified with aldehyde functionalized surfaces. Then, IL-5 sandwich immunoassay was performed with an
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antigen concentration of 1 ng mL-1. For comparison, six different CL solutions were applied to each chip at the
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final step. The CL response was measured using a CMOS image sensor reader module and analyzed using the
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noise calibration method.
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2.5. Selectivity test
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The four cytokines IL-2, IL-4, IL-5, and IL-6 were used to evaluate the selectivity of the new assay system
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(Supplementary Fig. S1). Capture antibodies were manually applied to the chip surface with a micro pipette, and
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then blocking step was proceeded with 1% BSA solution. For this purpose, IL-2, IL-4, IL-5 and IL-6 antigens
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serving as multiple detection targets were dissolved in 1% BSA/PBS. Solutions of the four biotinylated
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detection antibodies and STA-HRP in 1% BSA/PBS were prepared in advance. The stepwise procedure was
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initiated by first preparing the aldehyde functionalized surface, and then 1 µL of the four capture antibodies
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were manually spotted and immobilized on each designated region for 2 h. After the blocking step using 1%
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BSA/PBS, 4 µ L of a mixture of the target antigens (10 pg mL-1) was applied to the surface of the chip and
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covered with a cover glass. Then, 4 µL of the detection antibodies and STA-HRP mixture were applied with a
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cover glass onto the chip surface for 1 h. During each of these steps, the surface was washed with PBST twice.
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The subsequent CL reaction was measured by using the chip reader module for 2 min. The detection time was
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enough to distinguish the signal difference among several signals (data not shown).
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3. Results and discussion
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3.1. Conceptual description of immunoassay on CMOS image sensor
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In this new sandwich immunoassay system, the entire process is carried out directly on the surface of a chip
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coated by a silicon nitride layer as a top surface. Generally, the immobilization method of biomolecules on
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surface plays a key role in various biosensors [Samanta and Sarkar, 2011]. Diverse methods for surface
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modification are known to enhance biocompatibility [Samanta and Sarkar, 2011, Mallard et al., 2005; Wang
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and Jin, 2004]. Currently, more efficient protein immobilization methods such as polymer coating and layer-by-
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layer assembly are used for the improvement of stability and performance with high versatility and flexibility
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manner [Wei et al., 2014; Ariga et al., 2014]. However, since the CMOS image sensor in the new assay system
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is directly attached to the printed circuit board (PCB), these conventional surface modification methods, which
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typically use strong reagents such as sulfuric acid in piranha cleaning, APTMS used in silanization, and plasma
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modification by using strong impact gases such as O2 plasma, cannot be performed [Wang and Jin, 2004; Lan
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et al., 2005]. Thus, a BSA protein-based aldehyde functionalization method, which causes less damage to the
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chip surface, was used in the assay system. The target protein, IL-5, was detected using HRP labeled detection
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antibody and a CL reagent. The CL reaction involving HRP promoted oxidation of luminol is initiated by
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applying the optimized CL reagents. CL light generated on the chip surface then enters the detector and is
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converted to a digital signal. The digital signal, generated before and after the CL reaction is then subjected to
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the calibration method to produce the detection results in a highly sensitive manner.
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3.2. Optimization of capture antibody immobilization on CMOS image sensor
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To immobilize the capture antibodies, a BSA protein-based aldehyde functionalization was conducted.
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Initially, the BSA was adsorbed on the CMOS image sensor and GA was reacted to form the aldehyde-activated
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surface. All modification steps were analyzed by IR spectra. As shown in Fig. S2A, The FTIR reflectance peak
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of GA on CMOS image sensor surface present at 2820 cm-1 (C-H stretch) and 1730 cm-1 (C=O stretch). By
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confirmation of these two peaks on GA treated CMOS image sensor, it was found that the GA for immobilizing
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capture antibody was successfully applied to the chip surface. The capture antibody was then reacted on the
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aldehyde-functionalized surface. Finally, 1% BSA solution was used as a blocking agent instead of
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ethanolamine or sodium borohydride in order to reduce potential damage. The effect of the aldehyde-modified
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surface on the integrity of the immunoassay was estimated using IL-5 and the CL imaging system. Compared to
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a normal, untreated surface, the aldehyde-functionalized surface provided higher sensitivity of the sandwich
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immunoassay for concentrations ranging from 10 ng mL-1 to 100 pg mL-1. The sensitivity of surface
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immobilized antibodies generally depends on the amount of immobilization and orientation [Samanta and
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Sarkar, 2011].
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In addition, the proposed method showed similar intensities for up to 6 times surface washing steps (Fig.
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S2B). However, the signal decreased with increasing the number of the regeneration steps, and the CMOS
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image sensor was not functional after the fourth regeneration (Fig. S2C). Based on this result, this sensor is
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suitable for disposable use.
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3.3. Optimization of chemiluminescence reaction
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Environmental conditions, such as pH, solution composition, and concentration, are very important factors
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that affect the oxidizing conditions of luminol and hydrogen peroxide [Lee and Seliger, 1972; Lind et. al.,
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1983; Faulkner et al., 1993]. In particular, the reaction pH and the selection and optimization of the enhancer
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concentration are important factors in enzyme-catalyzed CL reactions [Iwata et al., 1995; Dotsikas et al., 2007].
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The reaction pH, enhancer concentration, and 4-AAP concentration were optimized 96-well plates, and these
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optimized conditions were applied to the CMOS image sensor. The 4-idophenol (4-IP), which is a sensitive CL
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enhancer [Chen et al., 2012; Wu and Liu, 2009], was used, and the AAP concentration was optimized to
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improve the CL reaction efficiency using the CMOS image sensor. Generally, several phenolic compounds
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including 4-IP are converted to hydrophobic polyphenol in the presence of HRP and hydrogen peroxide as HRP
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substrates. Conversely, soluble dimers are formed in the presence of 4-AAP. Therefore, 4-AAP is possible to
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improve the light efficiency of the CMOS image sensor.
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Initially, we tested the pH and 4-IP concentration. As a result, the optimum pH was obtained as 8.8 (0.1 M
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Tris-HCl buffer) (Fig. S3A) and the highest CL intensity is generated when the concentration of 4-IP was 0.5
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mM (Fig. S3B). After that the effect of AAP on the CL reaction was evaluated on the optimized pH and IP
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concentration. The results show that the CL intensity is highest when 100 nM of the AAP concentration was
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employed (Fig. S3C). The observation that the decrease of CL intensity at high concentrations of AAP is likely
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the reaction of this substance with hydrogen peroxide, which lowers the effective concentration of hydrogen
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peroxide that is needed for the CL reaction. In order to confirm the optimized condition on CMOS image sensor,
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we designed six different optimized conditions (see Fig. S4) to confirm the operation of optimized condition in
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CMOS image sensor simply. A comparison of the results arising from experiments in which six different
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chemiluminescence solutions are used shows that the highest CL intensity arises when 4-IP and AAP are
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employed in combination (Fig. S4). According to the 4-AAP characterization test, the optimized conditions in
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96-well plate were applied to the CMOS image sensor. As a result, we used these optimized condition in
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following experiments.
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3.4. Characterization of the default digital value
In order for the assay system to have a high sensitivity, variations of the digital values arising from
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measurements utilizing a single chip or different chips need to be taken into account. A calibration method was
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developed for reducing the variations. Accordingly, digital intensity data collected before and after the CL
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reaction for 120 s were analyzed using the calibration method as shown in Scheme 1B. The subtractive method
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is embodies in the equation:
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_ _ _ _
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where ACL and CCL are respective average data of integrated digital intensity in reacted pixels and background in
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non-reacted pixels after CL reaction during i sec in presence of chemiluminescent reagents. ABuffer and CBuffer are
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also respective average data of integrated digital intensity in reacted pixels and background in non-reacted pixels
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before CL reaction during i sec in presence of buffer solution (0.1 M Tris-HCl, pH 8.8) only.
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In order to obtain sensitive result, the slight difference of default digital value in different regions even on a
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single chip was considered for multiple detections of cytokines. The correction process, in which the average
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pixel data obtained before reaction (ABR) is subtracted from the average pixel data obtained after CL reaction
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(AAR), gave a highly invariant default digital value. As shown in Fig. 1A, the standard deviation (SD) of AAR
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(black bar) is approximately 15-fold larger than that of AAR-ABR (grey bar), which shows that the deviation in
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a single chip is nearly offset by utilizing the AAR-ABR correction. Additionally, another factor that can lead to a
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non-specific signal in absence of the CL reaction is associated with the electrothermal effect caused by operating
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chip reader module during measurement. Inspection of the plot of AAR data shows that the intensity of the
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digital signal increases substantially upon increasing the measurement time (Fig. 1B). However, the corrected
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(AAR-ABR) data is close to zero, which means that the electrothermal effect of a chip is easily accounted for
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using this correction process. Even though they are made by using the same mechanical process, chips could
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have variable electrical properties that cause variation in their digital output intensities. This phenomenon is
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exemplified by the results of experiments (Fig. 2A) which show that differences exist in the digital intensities
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between IL-5 immunoassays performed on three different chips with the same IL-5 antigen concentration of 1
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ng mL-1. Importantly, the data in Fig. 2B show that these differences nearly disappear when the background
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correction method involving subtraction of background noise is applied.
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3.5. Attomolar detection of interleukin-4 and interleukin-5
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In order to evaluate the new performance of this proposed system, comparison tests were made using a 96-
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well plate, slide glass and the CMOS image sensor. The sandwich immunoassay was performed using the
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standard ELISA procedure with CL, and the glass slide and CMOS image sensor were prepared using same
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surface modification method. As the data in Fig. 3A show, IL-5 can be detected by employing this technique
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with 96-well plate at a concentration as low as 1 pg mL-1 and the calculated LOD of 0.91 pg mL-1 with
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correlation coefficient (r) of 0.9856 (n = 3). Additionally, the assay carried out utilizing a glass slide has a lower
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10 pg mL-1 sensitivity for detection of IL-5 and the LOD of 41.7 pg mL-1 (r = 0.8702, n = 2). In order to further
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explore the sensitivity of the new system, digital intensities (I) arising from analysis of various IL-5
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concentrations were recorded as a function of time (s). The results (Fig. 3A) show that the digital intensity
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increases with increasing IL-5 concentration from 0.001 to 20000 pg mL-1. IL-5 can be detected at a
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concentration as low as 1 fg mL-1 with a calculated LOD of 0.074 fg mL-1 (r = 0.9891, n = 3), which
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corresponds to 50 aM. This higher sensitivity of CMOS image sensor than which of glass slide and 96-well plate
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is attributed to distance between detector and sample as well as the CMOS image sensor optimized calibration
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method. This LOD of immunoassay on the CMOS image sensor is the most sensitive simultaneous
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immunoassay result among the biosensors described in Table 1.
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Another target, IL-4, was assayed using the new system. As shown in Fig. S5, inspection of the data shows
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that the LOD for detection of IL-4 is 1.74 fg mL-1, corresponding to 980 aM (r = 0.9051, n = 3). Although it
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appears that IL-4 detection using the CMOS image sensor indicated is less sensitive and reproducible than that
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for IL-5 detection, the results show that the possibility exists for using this system to detect other targets with
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attomolar level sensitivity. Noteworthy points are that this system requires only a small 4 µL volume of a
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solution containing IL-5 antigen to cover the chip surface and the concentration data collected at early time
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points (1 min) are highly linear. Most importantly, the new the CMOS image sensor-based system is
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approximately 1105-fold more sensitive than the conventional ELISA method. Furthermore, we measured the
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1 pg mL-1 IL-5 spiked human serum samples, and compared with above assay result of IL-5 antigen diluted in 1%
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BSA/PBS. From this experiment, we obtained similar intensity results of serum sample with digital intensity
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value compared with IL-5 immunoassay result (Fig. S7). Although, this result was not enough to evaluate the
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real sample application, we thought that this result showed the possibility of real sample application.
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3.6. Selectivity and simultaneous detection of multiple cytokines
The selectivity and multiple detection ability of the new assay were evaluated with a mixture of 10 pg mL-1
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of each of four different cytokines, IL-2, -4, -5 and -6, using a 9 well-arrayed CMOS image sensor in which the
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surfaces of a chip contained one of the four different immobilized capture antibodies. As the data in in Fig. 3B
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demonstrate, the digital intensities selectively increased in response to IL-2, -4, -5 and -6 in each detection
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region. Thus, only the complimentary antigen in each case reacts with its specific capture antibody to generate a
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detectable CL signal.
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Simultaneous multiple cytokines detection was also performed on a 9 well-arrayed chip surface. When no
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antigen sample is introduced, the digital intensity is close to zero in all detection areas. However, a highly
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intense signal arises when the sample containing 10 pg mL-1 of all antigens is tested. Because the four cytokine
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samples were selected from eight types by using a cross-activity test, the ability to perform multiple detection
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demonstrates that little non-specific reaction takes place during the sandwich immunoassay. The fact that this
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test was also successfully conducted using the well-type CMOS image sensor suggests that multiple detection
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for various proteins is possible by applying micro-dispensing technique on the CMOS image sensor surface.
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Finally, the multiple cytokines spiked samples depend on various concentrations was measured, the LODs
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were obtained as 0.72 fg mL-1, 0.29 fg mL-1, 0.17 fg mL-1, 0.55 fg mL-1 for IL-4, IL-2, IL-5 and IL-6 respectably,
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and all antigens successfully measured from the 1000 pg mL-1 to 1 fg mL-1 (Fig. S6). Although, some results
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showed the relative higher standard deviations, it is showed the possibility to use as an alternative ELISA
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method with ultra-sensitive manner.
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4. Conclusion In the investigation described above, we have implemented a chemiluminescence-based simultaneous
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attomolar level detection for cytokines that utilizes a CMOS image sensor. Critical factors associated with the
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chemiluminescence process that generates the signal in this assay, such as buffer pH and enhancer concentration,
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were optimized, and the ability of AAP to reduce surface interference arising from byproducts was confirmed.
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Optimized amounts of the components for the CL process were applied to the surface on which the sandwich
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immunoassay was performed. Most importantly, a background signal calibration method was devised to correct
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for variations in data caused by electrothermal effect and mechanical production in a single chip and between
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chips. Use of the data calibration method enables the cytokine to reach an attomolar level sensitivity and to have
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a high specificity for protein detection. Evaluation of the assay system showed that it can be employed to detect
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IL-5 with a limit of detection of 0.074 fg mL-1, which corresponds to a 50 aM, and with dynamic range in 1 fg
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mL-1 to 20 ng mL-1. In addition, the system successfully used to detect four different cytokines in a simultaneous
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manner, and simply confirmed the similar CL intensities between buffer and human serum spiked IL-5 (Figure
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S7). Although, this result was not enough to evaluate the real sample measurement, this result showed the
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possibility of real sample application. The attractive features of the strategy used to devise the new
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immunoassay suggest that it can be utilized to design portable, cost-effective and simple point-of-care detection
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devices and potentially be applied to creating more efficient multiple detection methods in microarray based
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assays for unknown and important biomarkers.
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Acknowledgements
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This work was financially supported by grants from the GRL Program (NRF-2013K1A1A2A02050616), the
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NLRL Program (NRF-2011-0028915), and the R&D Joint Venture Program funded by the Ministry of Science,
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ICT and Future Planning.
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Figure captions
465 Scheme 1. Overall schematic representation of the interleukin-5 detection on a CMOS image sensor (not to scale).
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Chemiluminescence emission light passes through the pixel after specific sandwich immunoassay is performed and is
468
converted to a digital value (from 0 to 255) depending on the intensity of the emission (A). Detail information for the data
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acquisition and calibration steps. The correction process for stabilizing the default digital intensity variation, which subtracts
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the average pixel data obtained before reaction (ABR) from average pixel data obtained after CL reaction (AAR). ABR data
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are obtained using 0.1 M Tri-HCl buffer (a, step 1). AAR data are obtained using optimized chemiluminescence reagents.
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120 images per each reaction (step 1 and 2) are acquired by the CMOS image sensor and processed utilizing this calibration
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method (B).
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Fig. 1. (A) AAR-ABR data calibration method reduces the variation of the default digital values among pixels in a single
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chip. The standard deviation (SD) was investigated in randomly selected five regions on a single chip. The correction
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process, in which the average pixel data obtained before reaction (ABR) is subtracted from the average pixel data obtained
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after CL reaction (AAR), gave a highly invariant default digital value. The big difference among several regions appears
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when only AAR was calculated (black bar), and it nearly disappears when AAR-ABR correction was applied (grey bar). The
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SD in AAR-ABR correction is approximately 15 times lower than AAR data. (B) Electrothermal effect during chip operation
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causes a distortion of data analysis. Increase of default digital intensity in absence of CL reaction can interfere in the data
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analysis after CL reaction, and the increase phenomenon can be removed through the correction using the AAR-ABR
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method.
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Fig. 2. The data calibration method is necessary for reducing the variations among different chips. (A) The different intensity
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result among three chips that exhibited the same concentration of IL-5 antigen was present when the digital data were
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calculated with data after CL reaction only. (B) The differences nearly disappear by applying a data calibration method.
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Fig. 3. Immunoassay on CMOS image sensor provides a good sensitivity and selectivity result. (A) Calibration curve for
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quantitative detection of IL-5 on a CMOS image sensor (△ △, left ordinate), slide glass (○, left ordinate) and 96-well plate (□,
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right ordinate). Data points and each error bar represent averages and standard deviations of the two (slide glass) and three
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(CMOS image sensor and 96-well plate) independent measurements. (B) Selectivity and simultaneous multiple target
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detection ability were evaluated with four different cytokines, IL-2, 4, 5 and 6. All data bars and each error bar in the
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histogram represent the average and the standard deviations of the two independent measurements.
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Figures
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Scheme 1.
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Fig. 1.
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Fig. 2.
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Fig. 3.
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Table 1. Biomarker detection limits using various methods for simultaneous detection described in this article
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*Author Biographies
Biographies Donggu Hong is a Ph.D. candidate at the School of Physics and chemistry, Gwangju Institute of Science & Technology Hyou-Arm Joung is a Ph.D. candidate at the School of Physics and chemistry, Gwangju Institute of Science & Technology
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Do Young Lee is Chief Executive Officer in OPTOLANE Inc., Sanghyo Kim obtained a Ph.D. in Pohang University of science and technology and is a professor in the department of bionanotechnology at Gachon University.
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Min-Gon Kim obtained a Ph.D. in Pohang University of science and technology and is a professor in the department of chemistry at Gwangju Institute of Science & Technology
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S0925-4005(15)30098-8 http://dx.doi.org/doi:10.1016/j.snb.2015.07.042 SNB 18773
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
3-4-2015 8-7-2015 12-7-2015
Please cite this article as: D. Hong, H.-A. Joung, D.Y. Lee, S. Kim, M.-G. Kim, Attomolar detection of cytokines using a chemiluminescence immunoassay based on an antibody-arrayed CMOS image sensor, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.07.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Research Highlights
1
Highlights
2 3 4
Attomolar detection for interleukins was achieved with directly performing sandwich immunoassay onto a surface of CMOS image sensor chip. A CMOS image sensor was used as a detector for CL imaging and substrate for the sandwich immunoassay.
6
The sensor specific data calibration method was used to obtain stable and reproducible results.
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This assay system was evaluated with detection of interleukin-5 with a limit of detection of 0.074 fg mL ,
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Multiple target detection was simultaneously performed.
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which corresponds to a 50 aM, and with dynamic range in 1 fg mL-1 to 20 ng mL-1.
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*Manuscript
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Attomolar detection of cytokines using a chemiluminescence immunoassay based
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on an antibody-arrayed CMOS image sensor
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Donggu Honga+, Hyou-Arm Jounga+, Do Young Leec, Sanghyo Kimd* and Min-Gon Kima,b*
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a
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gwagiro, Gwangju 500-712, Republic of Korea
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b
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Cheomdan-gwagiro, Gwangju 500-712, Republic of Korea
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School of Physics and chemistry, Gwangju Institute of Science & Technology, 261 Cheomdan-
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Advanced Photonics Research Institute, Gwangju Institute of Science & Technology, 261
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c
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Republic of Korea
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d
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Seognam-si, Gyeonggi-do 461-701, Republic of Korea
OPTOLANE Inc., 633 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, Korea 463-400,
M
Department of bionanotechnology, Gachon university, San 65, Bokjeong-dong, Sujeong-gu,
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Corresponding authors: [email protected], [email protected]
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+
These authors are equally contributed to this paper
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Tel.: +82(31)750-8554
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E-mail: [email protected]
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Tel.: +82(62)715-3330
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Fax: +82(62)715-3419
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E-mail: [email protected]
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ABSTRACT
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A chemiluminescence immunoassay based on a CMOS image sensor has been developed for simultaneous
32
attomolar detection of cytokines. Attomolar level detection sensitivity is achieved through direct immobilization
33
of antibodies on the surface of the CMOS image sensor and using a simple data accumulation process with noise
34
calibration after the sandwich immunoassay. Here, the proposed method was showed that the logarithmic linear
35
range for interleukin-5 (IL-5) detection is from 1 fg mL-1 to 20 ng ml-1 and the limit of detection (LOD) is 0.074
36
fg mL-1, corresponding to a concentration of 7 aM. The LOD represents ca. 1105-fold improvement compared
37
with use of conventional ELISA. Furthermore, the multiple targets including IL-2, IL-4, IL-5, and IL-6 can be
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detected with simultaneous and low-cost manner on array type CMOS image sensor, and we hope that this
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sensor system will be a powerful tool in biological studies.
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Keywords
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CMOS image sensor, attomolar detection, cytokines, chemiluminescence, sandwich immunoassay
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1. Introduction Antibody array technology enables simultaneous detection and analysis of protein antigens present in
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samples. In a protein microarray format, the method can be used to track interactions and activities of proteins in
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a large scale and high throughput manner [Templin et al., 2002]. Additionally, the technique requires only small
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quantities of samples and, as a result, it can be employed to generate abundant data sets in a single and relatively
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cost-effective experiment [Melton, 2004]. For this reason, antibody arrays have been applied in a wide range of
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biomarker discovery and diagnostic studies [Devarajan et al., 2003; Srinivas et al., 2002; Fang et al., 2003].
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Although many commercial products are available for qualitatively profiling protein expression and identifying
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candidate biomarkers, almost all of these platforms have detection sensitivities that are less than that of the
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enzyme-linked immunosorbent assay (ELISA) system, which has limit of detection (LOD) in the picomolar
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region.
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New strategies are required in order to develop much more sensitive sensing systems that can be utilized for
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simultaneous detection and analysis of unknown biomarkers. Studies aimed at this goal have led to the
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development of sensors that have LODs at the attomolar level. For example, new techniques have been
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developed that are based on surface plasmon resonance (SPR) or SPR imaging [Lee et al., 2006; Hu et al.,
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2014], electrochemical immunosensors [Chikkaveeraiah et al., 2012; Tang et al., 2012], and photonic crystal
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arrays [Deng et al., 2013]. In particular, various approaches such as carbon-nanotube-based sensors and
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nanowire-based field effect transistor (FET) sensors with various signal amplification strategies were used for
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highly sensitive biomarker measurements [Elnathan et al., 2012; Zheng et al., 2005; Yu et al., Byon at al.,
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2006]. Additionally, several sensitive approaches, such as those that employ rolling circle amplification (RCA),
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electrochemiluminescence (ECL), chemiluminescence (CL) and phtoluminescence have been devised and are
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compatible with the antibody array format [Konry et al., 2009; Sardesai et al., 2013; Zong et al., 2013; Liu et
79
al., 2013]. Despite their strong multiplex detection ability and high sensitivity, attomolar sensitivity still remains
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a challenge in simultaneous biomarker measurement. Accordingly, simple, low-cost methods are still required
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for the detection of biomolecules at attomolar levels.
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Detection methods based on CL are among the most sensitive and they have wide dynamic ranges [Kricka,
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1993]. As a result, these types of sensors have been widely used in the biological research [Brien et al., 1991;
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Constantine et al., 1994; Nori et al., 2008]. Light emission in an enzyme-catalyzed CL system is a
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consequence of a chemical oxidation reaction of a reagent catalyzed by peroxidase. For example, horseradish
86
peroxidase (HRP) catalyzes oxidation of luminol in the presence of hydrogen peroxide to produce CL. However,
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the catalytic ability of this peroxidase is sometimes insufficient for specific experimental requirements and, as a
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result, supplements are added to enhance CL signals [Aslan and Geddes, 2009]. In addition, light emission
89
efficiencies can be maximized by carrying out the CL reaction on the surface of the detector in this system. Recent developments have been made in the design of highly sensitive light detectors. For example,
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complementary metal oxide semiconductor (CMOS) image sensor has become good alternatives to those based
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on charge coupled device (CCD). CMOS based sensors have many attractive features including minimal power
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dissipation [Fossum, 1997], improved image quality [Takayanagi and Nakamura, 2013], and lower power
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consumption [Gamal and Eltoukhy, 2005], operation voltage and cost. For this reason, several bio-sensing
95
applications of CMOS image sensor have been described. For example, biofluorescence imaging inside a brain
96
tissue phantom has been carried out using a dedicated CMOS image sensor along with CL detection of nucleic
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acids hybridized onto a probe-immobilized CMOS photodetector array [Ng et al., 2006; Mallard et al., 2005].
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Although utilized in in selected cases, CMOS image sensors have not been employed extensively in
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biomolecular studies.
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In the investigation described below, we have successfully implemented a CL based system for the
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simultaneous and attomolar level detection of cytokines on an antibody arrayed CMOS image sensor. The
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CMOS image sensor in this system not only serves as a detector for capturing images but it also acts as a
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substrate for the immunoreaction in order to reduce CL light loss entering to a CMOS image sensor. In this way,
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CL light emitted near the surface of the sensor is directly transmitted to photodiodes which convert light into an
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electric and then digital signal with minimal loss. A calibration method is used to analyze digital data obtained
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from images in order to reduce variations between the default digital signals from chips and between different
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chips, and it is also used for correcting background signals arising from electrothermal effects on the sensors.
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Interleukin-5 (IL-5), one of the cytokines that is present at levels below several pg ml-1, is used as a model target
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to evaluate the sensitivity of the new detector. Furthermore, simultaneous detection of several cytokines on one
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chip is carried out using this system.
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2. Material and Methods
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2.1. Reagents and apparatus
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CMOS image sensor chips and a reader module were provided by Optolane Inc. (Korea). 4-
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Aminoantipyrine (AAP), luminol sodium salt, 4-iodophenol (4-IP), p-coumaric acid, N,N-dimethylformamide
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(DMF), glutaraldehyde (GA) solution (Grade I, 50%), sodium bicarbonate (NaHCO3) and sodium carbonate
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(Na2CO3), hydrogen peroxide (H2O2), Tween 20, (3-aminopropyl)trimethoxysilane (APTMS) were obtained
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from Sigma Aldrich (Korea). Bovine serum albumin (BSA) was obtained from Fitzgerald. 1X Phosphate
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buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4) and Tris-HCl (1.5 M,
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pH 8.8) were obtained from BIOSESANG, Inc. The TMB substrate reagent set was obtained from BD
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Biosciences (United State). Supersignal west femto maximum sensitivity substrate and supersignal west pico
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chemiluminescent substrate were obtained from Thermo Acientific (United State). Cytokine ELISA kits for
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interleukin-2, -4, -5 and -6 (IL-2, IL-4, IL-5, IL-6), which include antigen capture antibody, biotinylated
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detection antibody and streptavidin-conjugated horseradish peroxidase (STA-HRP), were obtained from BD
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biosciecnces (United State). CL intensities were integrated using a chemiluminescence imaging system
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(ChemiDoc, BIO-RAD). The IR spectrums were obtained using FTIR spectrometer (FT/IT-4200, JASCO).
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2.2. Preparation of the capture antibody immobilized CMOS image sensor The chip was modified to introduce aldehyde groups needed for immobilization of the capture antibodies.
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The aldehyde functionalized surface was prepared by using the following procedure. The 4 µL of 1% BSA/PBS
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solution was applied on the chip, a cover glass was placed on the chip and the assembly was allowed to stand for
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1 h at room temperature (RT). The chip was then washed with PBST (0.05% tween 20 in 1X PBS) and distilled
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water (DW) twice. The aldehyde-functionalized chip surface was prepared by adding 4 µL of 2% (v/v)
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glutaraldehyde in PBS solution onto BSA adsorbed chip surface, a cover glass was placed on the chip and the
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assembly was allowed to stand for 4 h at RT, and the chip surface was washed with PBST and DW twice. The 4
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µL of capture antibody (4 µg mL-1 in 0.1 M carbonate buffer (pH 9.5)) is manually applied to the surface of a
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chip with a micro pipette, then chip is incubated at 4 °C overnight. Finally, after twice wishing, the 1% BSA in
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PBS solution was introduced on CMOS image senor for blocking the residual GA. The volume (4 µL) was
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enough to cover the surface of CMOS image sensor (3 mm 4 mm). The IR spectrums were obtained BSA,
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GA immobilization steps respectably.
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2.3. Sandwich immunoassay for detection of interleukin-5 As depicted in Scheme 1A, the IL-5 sandwich immunoassay is performed on the capture antibodies-
Page 6 of 24
functionalized chip surface. Solutions of the IL-5 antigen and biotinylated detection antibody are prepared in 1%
146
BSA/PBS. The sandwich immunoassay based on the general ELISA procedure is then performed by adding a
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solution (4 µL) of the IL-5 antigen, installing a cover glass for 2 h, and then adding 4 µL of 1 µg mL-1 of the
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biotinylated detection antibody and 4 µg mL-1 of STA-HRP mixture, followed by installation of a cover glass for
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1 h. Between each step in this procedure, the surface is washed with PBST twice. Finally, the chip is incubated
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in a sealed petri dish in order to prevent sample evaporation. To measure IL-5 using the CMOS image sensor, an
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optimized CL solution was used. After introducing the CL reaction solution to the CMOS image sensor at the
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end of the immunoassay, 1-sec exposure images were obtained for 120 sec before and after the reactions. Finally,
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the images were analyzed using the proposed calibration method. The detailed CL optimization procedures are
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described in the following section, and the data processing and calibration methods are described in detail in
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section 3.4.
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2.4. CL optimization
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In order to establish stable conditions for carrying out the HRP catalyzed CL reaction in our system, an
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optimum buffer pH and enhancer concentration must be identified and a method was needed for reducing
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interference of incident photon rate by phenolic compounds produced in the CL reaction. For this purpose, the
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CL characterization test was performed with fixed compositions of 2 mM luminol in 50 mM sodium hydroxide
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(NaOH) and 2 mM H2O2. CL detection was performed after STA-HRP reacts with biotinylated antibody, and its
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intensity is measured using the chemiluminescence imaging system. All composition of CL solution were
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optimized with 96-wellplate in order to follow standard method and the optimized condition was applied to
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CMOS image sensor.
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2.4.1. Optimization of CL buffer pH
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Various pH buffer conditions were tested using a 96-well microtiter plate. STA-HRP (1 µg mL-1) was added
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to each well followed by incubation for 30 min and washing with PBST and DW twice. A fixed composition CL
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solution containing 0.5 mM 4-IP in DMF along with various buffers including pH 7.2 (1X PBS), 7.4 (0.1M
171
phosphate buffer), 8.3 (0.1M borate buffer) 8.8 (0.1 M Tris-HCl buffer) and 10.3 (0.1 M carbonate buffer), were
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used. The CL intensity was measured for 7 min.
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2.4.2. Optimization of 4-IP concentration
Page 7 of 24
4-IP concentration optimization was also carried out. This was performed in a manner that is similar to that
176
used for buffer pH optimization. Specifically, 10 ng mL-1 STA-HRP was added to each well of a 96-well
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microtiter plate, followed by addition of a fixed composition of CL solution in 0.1 M Tris-HCl buffer (pH 8.8)
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and various concentrations (1 nM–0.01 mM) of 4-IP. The CL intensity was measured for 7 min.
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2.4.3. Characterization of AAP effect
An improvement of the CL intensity by AAP was observed in the following way. First, 100 ng mL-1 of STA-
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HRP was adsorbed to each well of a 96-well microtiter plate, followed by addition of fixed compositions of CL
183
solution in 0.1 M Tris-HCl (pH 8.8) buffer with DMF and various concentrations (1 mM–100 nM) of AAP. The
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CL intensity was measured for 7 min.
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In order to evaluate the optimized CL composition, comparison was made between the CL solution
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containing AAP and commercial CL solution on the CMOS image sensor chip. For this comparison, CL
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solutions, consisting of 50 mM sodium hydroxide (NaOH), 2 mM H2O2, and 0.5 mM enhancer (4-IP or p-
190
coumaric acid) with and without 100 nM AAP, were prepared. SuperSignal West Femto Maximum Sensitivity
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Substrate and SuperSignal West Pico Chemiluminescent Substrate were prepared for comparison purposes. The
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IL-5 immunoassay was carried out on six CMOS image sensors for each CL solution. The image sensors were
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first modified with aldehyde functionalized surfaces. Then, IL-5 sandwich immunoassay was performed with an
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antigen concentration of 1 ng mL-1. For comparison, six different CL solutions were applied to each chip at the
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final step. The CL response was measured using a CMOS image sensor reader module and analyzed using the
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noise calibration method.
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2.5. Selectivity test
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The four cytokines IL-2, IL-4, IL-5, and IL-6 were used to evaluate the selectivity of the new assay system
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(Supplementary Fig. S1). Capture antibodies were manually applied to the chip surface with a micro pipette, and
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then blocking step was proceeded with 1% BSA solution. For this purpose, IL-2, IL-4, IL-5 and IL-6 antigens
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serving as multiple detection targets were dissolved in 1% BSA/PBS. Solutions of the four biotinylated
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detection antibodies and STA-HRP in 1% BSA/PBS were prepared in advance. The stepwise procedure was
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initiated by first preparing the aldehyde functionalized surface, and then 1 µL of the four capture antibodies
Page 8 of 24
were manually spotted and immobilized on each designated region for 2 h. After the blocking step using 1%
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BSA/PBS, 4 µ L of a mixture of the target antigens (10 pg mL-1) was applied to the surface of the chip and
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covered with a cover glass. Then, 4 µL of the detection antibodies and STA-HRP mixture were applied with a
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cover glass onto the chip surface for 1 h. During each of these steps, the surface was washed with PBST twice.
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The subsequent CL reaction was measured by using the chip reader module for 2 min. The detection time was
210
enough to distinguish the signal difference among several signals (data not shown).
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3. Results and discussion
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3.1. Conceptual description of immunoassay on CMOS image sensor
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In this new sandwich immunoassay system, the entire process is carried out directly on the surface of a chip
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coated by a silicon nitride layer as a top surface. Generally, the immobilization method of biomolecules on
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surface plays a key role in various biosensors [Samanta and Sarkar, 2011]. Diverse methods for surface
217
modification are known to enhance biocompatibility [Samanta and Sarkar, 2011, Mallard et al., 2005; Wang
218
and Jin, 2004]. Currently, more efficient protein immobilization methods such as polymer coating and layer-by-
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layer assembly are used for the improvement of stability and performance with high versatility and flexibility
220
manner [Wei et al., 2014; Ariga et al., 2014]. However, since the CMOS image sensor in the new assay system
221
is directly attached to the printed circuit board (PCB), these conventional surface modification methods, which
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typically use strong reagents such as sulfuric acid in piranha cleaning, APTMS used in silanization, and plasma
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modification by using strong impact gases such as O2 plasma, cannot be performed [Wang and Jin, 2004; Lan
224
et al., 2005]. Thus, a BSA protein-based aldehyde functionalization method, which causes less damage to the
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chip surface, was used in the assay system. The target protein, IL-5, was detected using HRP labeled detection
226
antibody and a CL reagent. The CL reaction involving HRP promoted oxidation of luminol is initiated by
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applying the optimized CL reagents. CL light generated on the chip surface then enters the detector and is
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converted to a digital signal. The digital signal, generated before and after the CL reaction is then subjected to
229
the calibration method to produce the detection results in a highly sensitive manner.
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3.2. Optimization of capture antibody immobilization on CMOS image sensor
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To immobilize the capture antibodies, a BSA protein-based aldehyde functionalization was conducted.
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Initially, the BSA was adsorbed on the CMOS image sensor and GA was reacted to form the aldehyde-activated
Page 9 of 24
surface. All modification steps were analyzed by IR spectra. As shown in Fig. S2A, The FTIR reflectance peak
235
of GA on CMOS image sensor surface present at 2820 cm-1 (C-H stretch) and 1730 cm-1 (C=O stretch). By
236
confirmation of these two peaks on GA treated CMOS image sensor, it was found that the GA for immobilizing
237
capture antibody was successfully applied to the chip surface. The capture antibody was then reacted on the
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aldehyde-functionalized surface. Finally, 1% BSA solution was used as a blocking agent instead of
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ethanolamine or sodium borohydride in order to reduce potential damage. The effect of the aldehyde-modified
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surface on the integrity of the immunoassay was estimated using IL-5 and the CL imaging system. Compared to
241
a normal, untreated surface, the aldehyde-functionalized surface provided higher sensitivity of the sandwich
242
immunoassay for concentrations ranging from 10 ng mL-1 to 100 pg mL-1. The sensitivity of surface
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immobilized antibodies generally depends on the amount of immobilization and orientation [Samanta and
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Sarkar, 2011].
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In addition, the proposed method showed similar intensities for up to 6 times surface washing steps (Fig.
246
S2B). However, the signal decreased with increasing the number of the regeneration steps, and the CMOS
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image sensor was not functional after the fourth regeneration (Fig. S2C). Based on this result, this sensor is
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suitable for disposable use.
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3.3. Optimization of chemiluminescence reaction
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Environmental conditions, such as pH, solution composition, and concentration, are very important factors
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that affect the oxidizing conditions of luminol and hydrogen peroxide [Lee and Seliger, 1972; Lind et. al.,
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1983; Faulkner et al., 1993]. In particular, the reaction pH and the selection and optimization of the enhancer
255
concentration are important factors in enzyme-catalyzed CL reactions [Iwata et al., 1995; Dotsikas et al., 2007].
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The reaction pH, enhancer concentration, and 4-AAP concentration were optimized 96-well plates, and these
257
optimized conditions were applied to the CMOS image sensor. The 4-idophenol (4-IP), which is a sensitive CL
258
enhancer [Chen et al., 2012; Wu and Liu, 2009], was used, and the AAP concentration was optimized to
259
improve the CL reaction efficiency using the CMOS image sensor. Generally, several phenolic compounds
260
including 4-IP are converted to hydrophobic polyphenol in the presence of HRP and hydrogen peroxide as HRP
261
substrates. Conversely, soluble dimers are formed in the presence of 4-AAP. Therefore, 4-AAP is possible to
262
improve the light efficiency of the CMOS image sensor.
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Initially, we tested the pH and 4-IP concentration. As a result, the optimum pH was obtained as 8.8 (0.1 M
Page 10 of 24
Tris-HCl buffer) (Fig. S3A) and the highest CL intensity is generated when the concentration of 4-IP was 0.5
265
mM (Fig. S3B). After that the effect of AAP on the CL reaction was evaluated on the optimized pH and IP
266
concentration. The results show that the CL intensity is highest when 100 nM of the AAP concentration was
267
employed (Fig. S3C). The observation that the decrease of CL intensity at high concentrations of AAP is likely
268
the reaction of this substance with hydrogen peroxide, which lowers the effective concentration of hydrogen
269
peroxide that is needed for the CL reaction. In order to confirm the optimized condition on CMOS image sensor,
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we designed six different optimized conditions (see Fig. S4) to confirm the operation of optimized condition in
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CMOS image sensor simply. A comparison of the results arising from experiments in which six different
272
chemiluminescence solutions are used shows that the highest CL intensity arises when 4-IP and AAP are
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employed in combination (Fig. S4). According to the 4-AAP characterization test, the optimized conditions in
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96-well plate were applied to the CMOS image sensor. As a result, we used these optimized condition in
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following experiments.
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3.4. Characterization of the default digital value
In order for the assay system to have a high sensitivity, variations of the digital values arising from
279
measurements utilizing a single chip or different chips need to be taken into account. A calibration method was
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developed for reducing the variations. Accordingly, digital intensity data collected before and after the CL
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reaction for 120 s were analyzed using the calibration method as shown in Scheme 1B. The subtractive method
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is embodies in the equation:
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_ _ _ _
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where ACL and CCL are respective average data of integrated digital intensity in reacted pixels and background in
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non-reacted pixels after CL reaction during i sec in presence of chemiluminescent reagents. ABuffer and CBuffer are
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also respective average data of integrated digital intensity in reacted pixels and background in non-reacted pixels
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before CL reaction during i sec in presence of buffer solution (0.1 M Tris-HCl, pH 8.8) only.
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In order to obtain sensitive result, the slight difference of default digital value in different regions even on a
288
single chip was considered for multiple detections of cytokines. The correction process, in which the average
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pixel data obtained before reaction (ABR) is subtracted from the average pixel data obtained after CL reaction
290
(AAR), gave a highly invariant default digital value. As shown in Fig. 1A, the standard deviation (SD) of AAR
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(black bar) is approximately 15-fold larger than that of AAR-ABR (grey bar), which shows that the deviation in
Page 11 of 24
a single chip is nearly offset by utilizing the AAR-ABR correction. Additionally, another factor that can lead to a
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non-specific signal in absence of the CL reaction is associated with the electrothermal effect caused by operating
294
chip reader module during measurement. Inspection of the plot of AAR data shows that the intensity of the
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digital signal increases substantially upon increasing the measurement time (Fig. 1B). However, the corrected
296
(AAR-ABR) data is close to zero, which means that the electrothermal effect of a chip is easily accounted for
297
using this correction process. Even though they are made by using the same mechanical process, chips could
298
have variable electrical properties that cause variation in their digital output intensities. This phenomenon is
299
exemplified by the results of experiments (Fig. 2A) which show that differences exist in the digital intensities
300
between IL-5 immunoassays performed on three different chips with the same IL-5 antigen concentration of 1
301
ng mL-1. Importantly, the data in Fig. 2B show that these differences nearly disappear when the background
302
correction method involving subtraction of background noise is applied.
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3.5. Attomolar detection of interleukin-4 and interleukin-5
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In order to evaluate the new performance of this proposed system, comparison tests were made using a 96-
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well plate, slide glass and the CMOS image sensor. The sandwich immunoassay was performed using the
307
standard ELISA procedure with CL, and the glass slide and CMOS image sensor were prepared using same
308
surface modification method. As the data in Fig. 3A show, IL-5 can be detected by employing this technique
309
with 96-well plate at a concentration as low as 1 pg mL-1 and the calculated LOD of 0.91 pg mL-1 with
310
correlation coefficient (r) of 0.9856 (n = 3). Additionally, the assay carried out utilizing a glass slide has a lower
311
10 pg mL-1 sensitivity for detection of IL-5 and the LOD of 41.7 pg mL-1 (r = 0.8702, n = 2). In order to further
312
explore the sensitivity of the new system, digital intensities (I) arising from analysis of various IL-5
313
concentrations were recorded as a function of time (s). The results (Fig. 3A) show that the digital intensity
314
increases with increasing IL-5 concentration from 0.001 to 20000 pg mL-1. IL-5 can be detected at a
315
concentration as low as 1 fg mL-1 with a calculated LOD of 0.074 fg mL-1 (r = 0.9891, n = 3), which
316
corresponds to 50 aM. This higher sensitivity of CMOS image sensor than which of glass slide and 96-well plate
317
is attributed to distance between detector and sample as well as the CMOS image sensor optimized calibration
318
method. This LOD of immunoassay on the CMOS image sensor is the most sensitive simultaneous
319
immunoassay result among the biosensors described in Table 1.
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Another target, IL-4, was assayed using the new system. As shown in Fig. S5, inspection of the data shows
321
that the LOD for detection of IL-4 is 1.74 fg mL-1, corresponding to 980 aM (r = 0.9051, n = 3). Although it
Page 12 of 24
appears that IL-4 detection using the CMOS image sensor indicated is less sensitive and reproducible than that
323
for IL-5 detection, the results show that the possibility exists for using this system to detect other targets with
324
attomolar level sensitivity. Noteworthy points are that this system requires only a small 4 µL volume of a
325
solution containing IL-5 antigen to cover the chip surface and the concentration data collected at early time
326
points (1 min) are highly linear. Most importantly, the new the CMOS image sensor-based system is
327
approximately 1105-fold more sensitive than the conventional ELISA method. Furthermore, we measured the
328
1 pg mL-1 IL-5 spiked human serum samples, and compared with above assay result of IL-5 antigen diluted in 1%
329
BSA/PBS. From this experiment, we obtained similar intensity results of serum sample with digital intensity
330
value compared with IL-5 immunoassay result (Fig. S7). Although, this result was not enough to evaluate the
331
real sample application, we thought that this result showed the possibility of real sample application.
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3.6. Selectivity and simultaneous detection of multiple cytokines
The selectivity and multiple detection ability of the new assay were evaluated with a mixture of 10 pg mL-1
335
of each of four different cytokines, IL-2, -4, -5 and -6, using a 9 well-arrayed CMOS image sensor in which the
336
surfaces of a chip contained one of the four different immobilized capture antibodies. As the data in in Fig. 3B
337
demonstrate, the digital intensities selectively increased in response to IL-2, -4, -5 and -6 in each detection
338
region. Thus, only the complimentary antigen in each case reacts with its specific capture antibody to generate a
339
detectable CL signal.
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Simultaneous multiple cytokines detection was also performed on a 9 well-arrayed chip surface. When no
341
antigen sample is introduced, the digital intensity is close to zero in all detection areas. However, a highly
342
intense signal arises when the sample containing 10 pg mL-1 of all antigens is tested. Because the four cytokine
343
samples were selected from eight types by using a cross-activity test, the ability to perform multiple detection
344
demonstrates that little non-specific reaction takes place during the sandwich immunoassay. The fact that this
345
test was also successfully conducted using the well-type CMOS image sensor suggests that multiple detection
346
for various proteins is possible by applying micro-dispensing technique on the CMOS image sensor surface.
347
Finally, the multiple cytokines spiked samples depend on various concentrations was measured, the LODs
348
were obtained as 0.72 fg mL-1, 0.29 fg mL-1, 0.17 fg mL-1, 0.55 fg mL-1 for IL-4, IL-2, IL-5 and IL-6 respectably,
349
and all antigens successfully measured from the 1000 pg mL-1 to 1 fg mL-1 (Fig. S6). Although, some results
350
showed the relative higher standard deviations, it is showed the possibility to use as an alternative ELISA
351
method with ultra-sensitive manner.
Page 13 of 24
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4. Conclusion In the investigation described above, we have implemented a chemiluminescence-based simultaneous
354
attomolar level detection for cytokines that utilizes a CMOS image sensor. Critical factors associated with the
355
chemiluminescence process that generates the signal in this assay, such as buffer pH and enhancer concentration,
356
were optimized, and the ability of AAP to reduce surface interference arising from byproducts was confirmed.
357
Optimized amounts of the components for the CL process were applied to the surface on which the sandwich
358
immunoassay was performed. Most importantly, a background signal calibration method was devised to correct
359
for variations in data caused by electrothermal effect and mechanical production in a single chip and between
360
chips. Use of the data calibration method enables the cytokine to reach an attomolar level sensitivity and to have
361
a high specificity for protein detection. Evaluation of the assay system showed that it can be employed to detect
362
IL-5 with a limit of detection of 0.074 fg mL-1, which corresponds to a 50 aM, and with dynamic range in 1 fg
363
mL-1 to 20 ng mL-1. In addition, the system successfully used to detect four different cytokines in a simultaneous
364
manner, and simply confirmed the similar CL intensities between buffer and human serum spiked IL-5 (Figure
365
S7). Although, this result was not enough to evaluate the real sample measurement, this result showed the
366
possibility of real sample application. The attractive features of the strategy used to devise the new
367
immunoassay suggest that it can be utilized to design portable, cost-effective and simple point-of-care detection
368
devices and potentially be applied to creating more efficient multiple detection methods in microarray based
369
assays for unknown and important biomarkers.
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Acknowledgements
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This work was financially supported by grants from the GRL Program (NRF-2013K1A1A2A02050616), the
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NLRL Program (NRF-2011-0028915), and the R&D Joint Venture Program funded by the Ministry of Science,
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ICT and Future Planning.
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Figure captions
465 Scheme 1. Overall schematic representation of the interleukin-5 detection on a CMOS image sensor (not to scale).
467
Chemiluminescence emission light passes through the pixel after specific sandwich immunoassay is performed and is
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converted to a digital value (from 0 to 255) depending on the intensity of the emission (A). Detail information for the data
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acquisition and calibration steps. The correction process for stabilizing the default digital intensity variation, which subtracts
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the average pixel data obtained before reaction (ABR) from average pixel data obtained after CL reaction (AAR). ABR data
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are obtained using 0.1 M Tri-HCl buffer (a, step 1). AAR data are obtained using optimized chemiluminescence reagents.
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120 images per each reaction (step 1 and 2) are acquired by the CMOS image sensor and processed utilizing this calibration
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method (B).
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Fig. 1. (A) AAR-ABR data calibration method reduces the variation of the default digital values among pixels in a single
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chip. The standard deviation (SD) was investigated in randomly selected five regions on a single chip. The correction
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process, in which the average pixel data obtained before reaction (ABR) is subtracted from the average pixel data obtained
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after CL reaction (AAR), gave a highly invariant default digital value. The big difference among several regions appears
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when only AAR was calculated (black bar), and it nearly disappears when AAR-ABR correction was applied (grey bar). The
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SD in AAR-ABR correction is approximately 15 times lower than AAR data. (B) Electrothermal effect during chip operation
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causes a distortion of data analysis. Increase of default digital intensity in absence of CL reaction can interfere in the data
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analysis after CL reaction, and the increase phenomenon can be removed through the correction using the AAR-ABR
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method.
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Fig. 2. The data calibration method is necessary for reducing the variations among different chips. (A) The different intensity
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result among three chips that exhibited the same concentration of IL-5 antigen was present when the digital data were
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calculated with data after CL reaction only. (B) The differences nearly disappear by applying a data calibration method.
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Fig. 3. Immunoassay on CMOS image sensor provides a good sensitivity and selectivity result. (A) Calibration curve for
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quantitative detection of IL-5 on a CMOS image sensor (△ △, left ordinate), slide glass (○, left ordinate) and 96-well plate (□,
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right ordinate). Data points and each error bar represent averages and standard deviations of the two (slide glass) and three
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(CMOS image sensor and 96-well plate) independent measurements. (B) Selectivity and simultaneous multiple target
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detection ability were evaluated with four different cytokines, IL-2, 4, 5 and 6. All data bars and each error bar in the
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histogram represent the average and the standard deviations of the two independent measurements.
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Figures
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Scheme 1.
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Fig. 1.
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Fig. 2.
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Fig. 3.
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Table 1. Biomarker detection limits using various methods for simultaneous detection described in this article
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*Author Biographies
Biographies Donggu Hong is a Ph.D. candidate at the School of Physics and chemistry, Gwangju Institute of Science & Technology Hyou-Arm Joung is a Ph.D. candidate at the School of Physics and chemistry, Gwangju Institute of Science & Technology
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Do Young Lee is Chief Executive Officer in OPTOLANE Inc., Sanghyo Kim obtained a Ph.D. in Pohang University of science and technology and is a professor in the department of bionanotechnology at Gachon University.
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Min-Gon Kim obtained a Ph.D. in Pohang University of science and technology and is a professor in the department of chemistry at Gwangju Institute of Science & Technology
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