Complementary Metal-Oxide Semiconductor (CMOS) Image Sensor ...

ANALYTICAL SCIENCES DECEMBER 2010, VOL. 26

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2010 © The Japan Society for Analytical Chemistry

Rapid Communications

Complementary Metal-Oxide Semiconductor (CMOS) Image Sensor: An Insight as a Point-of-Care Label-Free Immunosensor Karthikeyan KANDASAMY,* Mohana MARIMUTHU,* Gun Yong SUNG,** Chang Geun AHN,** and Sanghyo KIM*† *College of Bionanotechnology, Kyungwon University, San 65, Bokjeong dong, Sujeong gu, Seongnam si, Gyeonggi do 461-701, Republic of Korea **BT Convergence Technology Research Department, Electronics and Telecommunications Research Institute (ETRI), Daejeon 305-700, Republic of Korea The present paper examines the efficiency of a complementary metal-oxide semiconductor (CMOS) using an indium nanoparticle (InNP) substrate for the high-sensitivity detection of antigen/antibody interactions at concentrations as low as 100 pg/ml under normal light. Metal NPs coated with antigen/antibody layers act as a dielectric layer on the conducting sphere, which enhances the number of photons hitting the sensor surface through a light-scattering effect. This photon number is proportional to the digital number observed with the CMOS sensor for detecting antigen/antibody interactions. (Received October 27, 2010; Accepted November 1, 2010; Published December 10, 2010) The development of an optimized immunosensor with high sensitivity and selectivity is crucial for the label-free detection of biological entities. Normally, the maximum range of sensitivity using conventional immunoassays, such as ELISA, is reportedly between 1 – 10 ng/ml. There have been various types of immunosensors used in research related to electric, optic, or magnetic technologies to increase the sensitivity.1–3 Among such approaches, complementary metal oxide semiconductor (CMOS) technology retains its high impact in immunosensing applications.4,5 Recently, DNA detection was also successfully demonstrated using a fully integrated CMOS biochip.6,7 A detailed summary of the progress of biosensor development based on CMOS technology has been offered elsewhere.8,9 Despite the increased development of CMOS biochips, CMOS image sensors based on optical signaling have also received much attention in the biomedical field due to their high potential in personal healthcare development.10,11 The main characteristic of this sensor is a high signal-to-noise ratio, low static power consumption, on-chip functionality and compatibility with standard technology, miniaturization, random access, and speed imaging. Indeed, Wang et al. demonstrated a CMOS active pixel sensor for DNA detection down to 10 pM concentration using ordinary room light.11 Despite such tremendous development, there is still the lack of a suitable point-of-care immunosensor product. Giaever investigated metal nanoparticle (NP) (Nb, Ta, Ni, Au, In, and Si)-coated glass surfaces that adsorb an antigen and form a monolayer of about 30 Å thick, which increases up to 100 Å when the antigen binds with an appropriate antibody.12 Based on this principle, Giaever successfully reported a method for the detection of immunoreactions with the unaided eye using an InNP substrate with a diameter of 1000 Å. This concept could be exploited as a new point-of-care analytical system if incorporated with a CMOS image sensor for the detection of biological entities in smartphones and other portable clinical

diagnostic devices. The present research introduces a novel and simple method, with potential as a point-of-care application, that detects the interaction between a recombinant interferon gamma (IF-γ) protein and selective primary and secondary IF-γ antibodies based on a subsequent variation in the photon number measured using a CMOS image sensor. This method incorporates the concept of antigen and consecutive antibody adsorption to the InNP-coated substrate, and their step-by-step exposure to the CMOS image sensor under normal room light produces a subsequent decrease in the electric signal which is proportional to the number of photons converted by ADC (Fig. 1). When the antigen and respective antibodies are adsorbed onto the InNP substrate, the thickness of the substrate is increased by a nanoscalar amount, which causes a decrease in the light intensity, as determined by the number of photons counted by the CMOS image sensor. The CMOS image sensor used was kindly donated by Siliconfile Technologies Inc. NOON010PC30L is an 110000 pixel single chip CMOS image sensor that was

To whom correspondence should be addressed. E-mail: [email protected]

Fig. 1 Schematic representation of the CMOS image sensor-based detection of antigen-antibody interactions.

†

1216 developed for mobile-phone cameras. This chip incorporates a 376 × 314 pixel array with an on-chip 10-bit ADC. Only the integration time and the analog gain value can be manually modified in order to maximize the background light intensity. Once the background intensity has been maximized and fixed before the immunoassay, the light intensity is measured after treating the samples. For an experiment, 100 nm InNP was deposited on thin overhead projector (OHP) sheet substrates by a thermalevaporation system (Daeki Hi-Tech) to a thickness of 100 nm using the principle of vacuum deposition of thin metal particle films.13 The detailed experimental conditions are given in Supporting Information (Fig. S1). Then the substrates were cut into nine pieces (5 × 5 mm size), followed by washing with deionized water and air-drying. All substrates were analyzed by exposure to the CMOS image sensor in order to measure the photon number. The IF-γ antigen was diluted with a 0.85% w/v NaCl solution to make final concentrations of 100, 10, 1, and 0.1 ng/ml in two sets of individual Petri dishes (total of 8 Petri dishes: two Petri dishes for the single antigen concentration). Eight washed InNP substrates were immersed in two sets of Petri dishes containing four different concentrations of antigen, and then incubated for 60 min. Then, the substrates were washed with phosphate buffered saline (PBS) and dried. The remaining InNP substrate was immersed in a 1% BSA solution and incubated for 60 min, and was used as a negative control. Again, all substrates were analyzed by the CMOS image sensor for the number for photons blocked by the antigen layer. A selective primary rabbit polyclonal IF-γ antibody was diluted in 1% BSA solution to final concentrations of 100 and 0.1 ng/ml, after which an aliquot each concentration was put into four Petri dishes (total of 8 Petri dishes). Eight antigen-coated substrates were immersed in four Petri dishes containing different concentrations of the primary antibody, followed by incubation with occasional shaking for 3 h. The substrates were then washed with PBS and air-dried for photon analysis. The ninth substrate was immersed again in a 1% BSA solution, and then incubated for 3 h, followed by washing with PBS and drying for photon analysis. A selective secondary antibody (FITCconjugated goat anti-globulin) was diluted with 1% BSA to a final concentration of 20 μg/ml, and then aliquots at equal volumes into eight Petri dishes in the dark. Eight primary antibody-coated substrates were immersed in the secondary antibody solution and incubated for 1 h in the dark. Then, the substrates were washed with PBS and dried. After fluorescence analysis, the substrates were subjected to photon analysis. The ninth substrate was processed similarly as the previous protocol by incubation with a 1% BSA solution, and then analyzed for the photon number. Figure 2 shows the experimental results represented as the number of photons analyzed by the CMOS image sensor for the subsequent layers of antigen, primary antibody, and secondary antibody on InNP substrates. Normally, a CMOS image sensor contains a photosite comprised of a light-sensitive area contained in a single photodiode, which helps in the adsorption of photons and conversion into electrons by the photoelectric concept. The converted electrons accumulate in the form of electrical charge, the amount of which is proportional to the number of photons hitting the image sensor.14 Later by ADC, the stored electric charge in the form of an amplified analog voltage is converted to digitized number. The recorded digital number is proportional to the number of photons exposed to the image sensor. Therefore, any objects that block the hitting photons reduce the digital number. The digital number counted when the image sensor is exposed to an InNP substrate will always be higher


Fig. 2 Digital output number obtained for substrates before and after antigen/antibody binding for (A) 100 ng/ml primary antibody concentration and (B) 100 pg/ml primary antibody concentration. Similarly, the relative sensitivity for antigen and antibody binding was given for (C) 100 ng/ml primary antibody concentration and (D) 100 pg/ml primary antibody concentration.

than the exposure of subsequent layers of antigen, primary, and secondary antibody layers. Figure 2 (A and B) demonstrates this principle in which the photon number gradually increased upon a decrease in the antigen concentration individually with two primary antibody concentrations (100 ng/ml and 100 pg/ml concentrations). Furthermore, this effect was confirmed by an increased number of photons in the primary and secondary antibody layers with respective antigen concentration. Importantly, the InNP substrate plays an important role in photon analysis. It was reported that conducting particles, such as InNPs, support the scattering of electromagnetic radiation, which can be highly facilitated when the particles coated are with a thin dielectric layer.12 In the present work, the dielectric layer was meant for the double layer of the antigen and antibody adsorption on the conducting InNP substrate. Due to this so-called scattering by the InNP/antigen/antibody substrate, it is possible to detect antigen-antibody interactions at concentrations


1217 possible to improve the sensitivity of the CMOS image sensor (under preparation). Further, this simple method increases the potential for use of CMOS image sensors in smartphones and various clinical diagnostic devices for the detection of several biological entities.

Acknowledgements This research was supported by the Kyungwon University Research Fund in 2010. This work was also partly supported by the ETRI Adventure Research Program (Grant No. 10QC1110, Photosensitive Biosensor Array Chip for Mobile Devices) and further supported by the KOCI (Grant No. ZC1110, Basic Research for the Ubiquitous Life Care Module Development).

Supporting Information

Fig. 3 Calibration curve illustrating the change in the sensor signal (photon count) with respect to the IF-γ antigen concentration. Also calibration plots for further changes in the sensor signal when the antigen interacts with the respective primary and secondary antibodies are given ((A) 100 ng/ml and (B) 100 pg/ml of primary antidody concentrations).

down to 100 pg/ml of the antigen and the antibody. Figure 2 (C and D) describes the relative sensitivity range (number of photons blocked by the InNP/antigen/antibody substrates) obtained by reducing the digital value of InNP/antigen from InNP, InNP/antigen/primary antibody from InNP/antigen, and InNP/antigen/primary/secondary antibody from InNP/antigen/ primary antibody substrates. These results further confirm the ability of the sensor to detect antigen-antibody interactions at concentrations down to 100 pg/ml. Figure 3 demonstrates the rate of change in the photon count as a function of the antigen concentration and the antigenprimary/secondary antibody interactions. The correlation coefficient analyzed for substrates bound with antigen, antigen/primary antibody (100 ng/ml), antigen/primary antibody (100 ng/ml)/secondary antibody were 0.9089, 0.9501, 0.9485, respectively. Similarly, the correlation coefficients analyzed for substrates bound with an antigen, an antigen/primary antibody (100 pg/ml) and, anantigen/primary antibody (100 pg/ml)/ secondary antibody were 0.9001, 0.9191, 0.9333, respectively. The graph displays a linear relationship within the range of the antigen concentration, where the regression line fits the data well. For a confirmation by the conventional method, fluorescence analysis was carried out with the indicated antigen and antibody concentrations shown in Supporting Information. The fluorescence intensity was reduced upon a decrease in the antigen and antibody concentration (Fig. S2). The lowest sensitivity was obtained at about 1 ng/ml of antigen. In summary, the experimental results show the valuable impact of the CMOS image sensor and InNP substrates for the detection of antigen/antibody interactions. A detailed study is in progress to unravel the underlying principle involved in analyzing the selection of the alloy composition as well as the size of NPs for improved scattering with respect to the antigen. Thereby, it is

FE-SEM image of InNP coated OHP substrate (Fig. S1) and fluorescent microscopic images of substrates coated with various concentrations of antigen, followed by primary antibody and FITC-conjugated secondary antibody binding (Fig. S2) can be referred from Supporting Information. This material is available free of charge on the web at http://www.jsac.or.jp/ analsci/.

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