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Multi-wavelength fluorescence detection of submicromolar concentrations using a filter-free fluorescence sensor
Accepted Manuscript Title: Multi-wavelength fluorescence detection of submicromolar concentrations using a filter-free fluorescence sensor Authors: Yong Joon Choi, Kazuhiro Takahashi, Nobuo Misawa, Takeshi Hizawa, Tatsuya Iwata, Kazuaki Sawada PII: DOI: Reference:
S0925-4005(17)31743-4 https://doi.org/10.1016/j.snb.2017.09.077 SNB 23168
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
25-3-2017 7-9-2017 13-9-2017
Please cite this article as: Yong Joon Choi, Kazuhiro Takahashi, Nobuo Misawa, Takeshi Hizawa, Tatsuya Iwata, Kazuaki Sawada, Multi-wavelength fluorescence detection of submicromolar concentrations using a filter-free fluorescence sensor, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2017.09.077 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.
Multi-wavelength fluorescence detection of submicromolar concentrations using a filter-free fluorescence sensor Yong Joon Choi, Kazuhiro Takahashi, Nobuo Misawa, Takeshi Hizawa, Tatsuya Iwata, Kazuaki Sawada
Email: [email protected] Department of Electrical and Electronic Information Engineering, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan.
Graphical abstract
Research Highlights
・We presented a performance analysis of a filter-free fluorescence sensor using an LED light source and fluorescent solution without using an optical filter.
・The intensities of the excitation light and fluorescence were determined by proposed parameters based on output current.
・The excitation light and fluorescence from submicromolar concentrations of FITC, Texas Red, and a mixed solution were successfully separated and simultaneously detected.
Abstract
In this paper, we report on the development of a filter-free fluorescence sensor for the detection of multiple fluorescence wavelengths from a solution of fluorescent dyes with submicromolar concentrations. The filter-free fluorescence sensor has a photo-gate structure, and the intensities of excitation light and fluorescence can be calculated from the photocurrents for the different gate voltages. This sensor makes it possible to measure various wavelengths without the need for optical filters or mirrors. The separation ability of excitation light and fluorescence in the developed sensor was more than 1,000:1 for an LED light source. Fluorescence measurements were carried out for fluorescein isothiocyanate isomer (FITC) and sulforhodamine 101 acid chloride (Texas Red) dyes. Based on our calculations, the excitation light and fluorescence from submicromolar concentrations of FITC, Texas Red, and a mixed solution were successfully separated and simultaneously detected. Keywords: filter-free, fluorescence sensor, biosensor, multi-wavelength detection, FITC, Texas Red
1. Introduction Fluorescence analysis is widely used in biochemical analysis and medical diagnosis procedures because it is easy to use and can provide large amounts of data [1,2]. Fluorescence microscopes are often used to detect fluorescence [3]; however, they consist of an optical filter and other parts, and are generally expensive and bulky [4]. At the same time, on-chip measurements of fluorescence are required for field diagnosis and point of care testing systems [5,6], which requires the development of miniature fluorescence detection devices. Recently, on-chip fluorescence sensors have been developed that consist of optical filters integrated on photodiodes [7–12]. In addition, a fluorescence sensor using buried triple p-n junction photodiodes has also been developed [13]. Although these devices exhibit high sensitivity, the fabrication processes required to integrate the optical filters are complicated. In addition, it is difficult to deal with changes in the fluorescent solution because the detectable fluorescence wavelength is fixed. A filter-free fluorescence sensor with a photo-gate structure has been developed that can measure a wide range of wavelengths without filters by considering the depth of the light absorption in the silicon [14,15]. It is also possible to measure multiple wavelengths at the same time [16]. The separation ability of excitation light and fluorescence in this sensor was 500:1. However, fluorescence detection requires an intensity ratio of more than 1,000:1 between the excitation light and fluorescence. Various techniques have been developed to enhance the separation ability, such as the body-biasing technique [17] and the surface planarization of polycrystalline-silicon gates [18], which have improved the separation ability of the filter-free fluorescence sensor from 500:1 to 1,200:1. In this paper, we describe the detection of fluorescence from fluorescein isothiocyanate isomer (FITC) [19,20] and sulforhodamine 101 acid chloride (Texas Red) [21] using the filterfree fluorescence sensor in which the separation ability of the sensor is 1,200:1 between the excitation light (470 nm) and fluorescence (530 nm). To improve the detection performance, we propose a parameter calculation method, and investigate the detection limit of the sensor for concentrations of FITC and Texas Red. Finally, we also describe the simultaneous detection of three wavelengths using the sensor for the measurement of mixed fluorescent solutions [22–24].
2. Filter-free fluorescence sensor 2.1 Structure
Figure 1a shows an optical microscopy image of the filter-free fluorescence sensor. The sensor was fabricated using 5-μm-rule, N-substrate, 1P2M (1-poly and 2-metal), single well modified complementary metal-oxide semiconductor fabrication technology. The size of the sensing area is 200 × 200 μm. Figure 1b shows a schematic cross section and diagram of the sensor operation. The sensor uses a n-type silicon substrate to fabricate a photogate structure on the p-well, and a pn junction is formed in the p-well. An n+ diffusion layer is placed adjacent to the photogate to measure the photoelectron. The photogate and diffusion layer are surrounded by a p-well contact, which functions as a p-well potential setting and forms holes near the drain in the p-well. The substrate potential is set using a substrate contact, and provides an outlet for the photoelectron deeper than the potential depth. The outside of the photogate is shaded with Al to suppress the generation of photoelectrons. The sensing area of the device consists of five layers: SiO2 (60 nm), n-type polysilicon (350 nm), thermal SiO2 (60 nm), p-well (4 μm), and n-type silicon substrate.
2.2 Sensing principle The developed filter-free fluorescence sensor measures the light intensity for different photogate voltages. When a voltage is applied to the gate, a depletion layer is formed at the SiO2/Si interface with the depth of W. When light is irradiated onto the sensing area, photoelectrons are generated by the light absorption, although the light absorbed in the depletion layer only contributes photocurrent. Equation (1) can be used to calculate the photocurrent generated by the light absorbed in the depletion layer.
I
qS
(1 e
W
)
(1)
hc
where q is the elementary charge, S is the sensing area, λ is the wavelength, h is the Planck constant, c is the speed of light in vacuum, W is the depletion depth, and is the light absorption coefficient. If the relationship between the light intensity ϕ and the photocurrent I is known in advance, then ϕ can be calculated from I . Figure 2 shows the operating principle of the filter-free fluorescence sensor using a potential diagram. When a positive bias voltage V1 is applied to the photo gate, the depletion depth is W1. The photocurrent IW1 is generated for W1 and output from the output contact. If a different positive voltage V2 is applied to the photo-gate, the photocurrent IW2 is generated for the depletion depth W2. Thus, different relationships between I and ϕ can be obtained by changing the photo-gate voltage. When light consisting of different wavelengths and intensities (λ1, ϕ1 and λ2, ϕ2) is simultaneously irradiated onto the sensing area, the photocurrent for the depletion depth of W1 can be expressed as in Eq. (2). In a similar manner, the photocurrent for W2 can be calculated using Eq. (3). When the coefficient of the light intensity in each term in Eqs. (2-3) is known, ϕ1 and ϕ2 can be calculated by solving the following simultaneous equations. IW 1
qS 1 hc
(1 e
1W 1
) 1
qS 2 hc
(1 e
2W 1
) 2
(2)
IW
2
qS 1 hc
(1 e
1W 2
) 1
qS 2 hc
(1 e
2W 2
) 2
(3)
3. Experimental results 3.1 Single wavelength current characteristics To measure the output current of the filter-free fluorescence sensor, light sources of various wavelengths were used to irradiate the sensor. The wavelength of a light source is adjusted by passing it through a band-pass filter with the full width at half maximum(FWHM) of 10 to 25 nm. The light sources were irradiated onto the sensing area with a diameter of 150 μm area and an intensity of 5 μW/cm2. Figure 3a shows a schematic diagram of the signal readout manner circuitry. To operate the sensor, 0 and 5 V are applied to the p-well electrode and the n-substrate electrode, respectively. The input voltage of the photogate was -1 to 4 V, and the output current was measured by the semiconductor parameter analyzer (Agilent, 4155A). To measure the noise of the measurement system, the photo-gate voltage was applied at 1, 3 V and the light source (530 nm) was irradiated at 10 μW/cm2. The photocurrent was measured 100 times for each photo-gate voltage. When voltages of 1 and 3 V were applied to the photo-gate, the minimum and maximum range of the photocurrent were 7 and 4 pA, respectively. Table. 1 shows the average values of the photocurrents and their standard deviations. Figure 3b shows the photocurrent-voltage characteristics of the single-wavelength. The photogate was in the accumulation state at voltages up to -0.5 V, the inversion state from -0.5 to 0.5 V, and the deep depletion state at voltages above 0.5 V. For our fabricated sensor, when voltages of 1 and 4 V were applied to the photo-gate, the depths of the potential peak W were 1.15 and 1.52 μm, respectively. The potential peak W was calculated by a SPECTRA (Link Research Corporation), which is a simulation program for three-dimensional electric transient analysis of CCD and CMOS image sensors using real impurity profile device simulator. Figure 3c shows the output photocurrents in the positive direction of x-axis(vertical) of Si substrate depending on the photogate voltage, when the wavelength and intensity of the input light source were 470 nm and 10 μW/cm2, respectively. The positive photocurrent was detected by the output contact, and the negative photocurrent was output by the n-substrate. The potential peak W can be estimated in the section where the photocurrent value changes from positive to negative. At a wavelength of 450 nm, 90% of the light was absorbed into the silicon substrate at a depth of 0.9 μm. When the incident light was mostly absorbed by the shallow silicon surface, the current difference was relatively small. In contrast, when the 90 % of the light penetrated to a silicon substrate at a depth of 6.45 μm at a wavelength of 625 nm, a current difference of 80 nA was generated by the photogate voltage from 1 to 4 V. In general, a high photocurrent was expected at the wavelength of 450 nm where the silicon absorption coefficient α was higher than the wavelength of 550 nm. However, the output photocurrent at the wavelength of 450 nm became smaller than that at the wavelength of 550 nm due to the absorbed by polysilicon. The optical transmittance of polysilicon at wavelengths 450 and 550 nm are 7 and 42 %, respectively. Although short wavelength of 450 nm has a large absorption coefficient, it is mostly absorbed by the polysilicon. Therefore, it is considered that the output photocurrent became smaller than that at the wavelength of 550 nm. As a result, the components of the wavelength can be derived by changing the photogate voltage based on the absorption coefficient of the silicon. It is also possible to determine the wavelength intensities of the light source by calculating the differences in the output current. Figure 3 (d) shows the spectral response of the sensor and used a laser driven light source (Energetiq, EQ-99). The responsivity was measured from 400 to 900 nm and
the FWHM was 10 nm. The absorption by the photogate for the short-wavelengths is relatively high due to the high absorption coefficient of silicon. It is considered that the responsivity of a wavelength of 400 nm is relatively low. The reflectance of the sensing area for a wavelength of 700 nm is about 55 %. Therefore, since the wavelength of 700 nm absorbed in silicon is reduced, a decrease in the response rate of the sensor is expected. Wavelengths above 750 nm are absorbed in regions deeper than the potential depth and are expected to decrease responsivity. By adjusting the p-well layer and the potential depth, high response can be expected in the long wavelength. The emission wavelength of a commonly used fluorescent dye is reported to range from about 420 to 875 nm (eg DyLight 405, DyLight 830). it is considered that the filter-free fluorescence sensor is applicable to most fluorescent dyes. 3.2 Separation ability of the LED light Figure 4a shows the setup used for measurements of the separation ability of the fluorescence sensor. In order to reduce the intensity error, the irradiated light was minimized using a microscope. Two optical fibers were connected to the microscope for irradiation purposes, and light sources with two wavelengths were used: 470 nm to mimic the excitation light and 530 nm for the fluorescence. The FWHM of irradiated light source 470 and 530 nm were25 and 32nm, respectively. The connected light sources passed through the x5 objective lens, and were irradiated with a diameter of 80 μm onto the sensing area. The standard intensity of the light was measured using an optical power meter. An x-axis stage was used to move the filter-free fluorescence sensor and optical power meter to minimize the error between measurements. The electrical characteristics of the sensor were measured using a semiconductor parameter analyzer. During the measurements, the light intensity at 530 nm was varied while the light intensity at 470 nm was maintained constant. The potential depth was changed by controlling the photogate voltage during the irradiation process. The separation ability was defined as the ratio between the lowest light intensity at 530 nm, which was validated by calculation, to that at 470 nm. In terms of the measurement, the potential depths W1 and W2 of the absorbed light were adjusted by applying voltages of 1 V and 3 V to the photo-gate. At these voltages, the depths of the potential peaks W were 1.15 and 1.52 μm, respectively. Here, the maximum intensity of the 530 nm light and the 470 nm light incident on the sensor were set to 5,000 nW/cm2, and the output current was measured while gradually decreasing the intensity of the 530 nm light from 5,000 to 0.1 nW/cm2. The intensities ϕ1 and ϕ2 for the 470 and 530 nm light, respectively, were determined by solving the simultaneous equations obtained by substituting the measured current into (2) and (3). Figure 4b shows the separation ability of the filter-free fluorescence sensor using the 470 and 530 nm LED light sources. The separation ability of the excitation light (470 nm) and fluorescence (530 nm) was 1,200:1. Figure 4c shows the separation ability of the sensor using the 550 and 590 nm LED light source with the FWHM of 20 and 25 nm, which was determined to be 1,000:1. In this case, the separation ability was reduced rather than the wavelengths of 470 and 530 nm. Since the spacing of the wavelengths was relatively close to each other, the separation ability was reduced due to the crosstalk. In the case of 470 and 530 nm, the reflectance of the sensing area is 23.5 and 11.2 %. On the other hand, the reflectance at wavelengths 550 and 590 nm were 21.5 and 39.1 %, which the amount of light at 590 nm wavelength absorbed by silicon was relatively smaller than 550 nm. Therefore, it is considered that the fluorescence (550 nm) was relatively small compared to the excitation light (590 nm), leading to a decrease in the separation ability. 3.3 Analysis of the fluorescent solution
In this study, the detection performance for quantitative measurements of fluorescent dye were carried out using FITC and Texas Red. FITC has the amino-group reactivity of Fluorescein and is a combination of the isothiocyanate reactive group, which dissolves in water and emits a strong yellow-green fluorescence (λex = 495 nm, λem = 520 nm). Since it reacts easily with the amino group, it can be applied to fluorescent labels on amino acids, enzymes, hormones, peptides, and viruses, and is used for diagnostic purposes in collagen disease, syphilis, antigen-antibody, and fluorescent antibody methods. Texas Red binds to proteins and amino acids with high efficiency and emits a deep red fluorescence (λex = 568 nm, λem = 590 to 630 nm). It has an advantage in that it can be labeled, while maintaining the activity of enzymes and without interfering with the fluorescent substances contained in biological samples. For these reasons, Texas Red is generally considered to be the more useful of the two dyes.
3.3.1 Parameters of the filter-free fluorescence sensor for fluorescent dyes The filter-free fluorescence sensor can be calculated the light intensity for a single wavelength by Equation (1). However, a width of FWHM increases, the reduction in sensitivity and errors in the calculation are expected. To measure the intensity of the weak light generated and light with spectrum width by fluorescent solutions, accurate calculations of the intensity are required. The filter-free fluorescence sensor consists of four layers (SiO2, Polysilicon, thermal SiO2, and Silicon) as shown in Fig. 1. Since the thickness of each layer differs depending on the process, the wavelength intensity of the light source incident in the silicon differs. Consequently, it is difficult to accurately derive the depth and nonlinear absorption coefficient α of the light incident in the silicon, and there is expected to the calculations error and a decrease in sensitivity of the light intensity. Therefore, a parametric method was required to minimize the absorption coefficient and the effect of constants on a light source with spectrum width. In this study, we propose a method of calculating the light intensity using parameters based on the output photocurrent of the sensor versus the wavelengths in the range from 450 to 625 nm. With the exception of the current I and light intensity ϕ components in Eqs. (2) and (3), parameters A, B, C and D, can be expressed as follows.
A
qS 1 hc
(1 e
a 1W 1
)
B
qS 2
(1 e
a 2W 1
)
hc
C
qS 1
(1 e
a 1W
2
)
D
hc
qS 2
(1 e
a 2W
2
)
hc
(4)
The components of the output current can be represented by the sum of the respective currents, as in Eq. (5). IW A 1 B 2 1
IW
2
C 1 D 2
Thus, the light intensities ϕ1 and ϕ2 can be calculated by the inverse matrix.
(5)
1 A C 2
B D
1
(6)
I W1 I W2
In the same way, the equation for the intensities of the three wavelengths can be calculated by substituting the parameters A to I into Eq. (7). 1 2 3
A D G
B E H
C F I
1
IW 1 I W2 I W3
(7)
Figure 5 shows the calculated parameters and spectra of the FITC and Texas Red solution. The FITC parameter values can be represented by the contact points of the photo-gate voltages of 2 and 4 V located at the peaks of the excitation light (470 nm) and fluorescence (520 nm). In the same way, the Texas Red parameter values can be obtained at the peaks of the excitation light (550 nm) and fluorescence (610 nm).
3.3.2 Measurement of fluorescence solution Figure 6 shows the measurement setup in a dark room for assessing the device using a semiconductor parameter analyzer. To evaluate the FITC and Texas Red, 4 ml of solution was deposited into disposable cuvettes. Excitation light with wavelengths of 470 and 550 nm (FITC and Texas Red) were irradiated into the side of the cuvettes. Then, a semiconductor parameter analyzer was used to control the sensor and measure the photocurrents. Excitation light with wavelengths of 470 and 550 nm was then irradiated at intensities of 10 and 4 mW/cm2, respectively, while the concentrations of the FITC and Texas Red in the solution were individually varied from 0.01 to 50 μM. Each fluorescent solution was prepared using diluted ethanol. Figure 7 illustrates the photocurrent of the sensor when voltages of 1 to 4 V were applied to the photo-gate. The current values were based on five experiments. In comparison to the excitation light, the fluorescence was relatively weak, and when the concentration of the FITC and Texas Red increased, the photocurrent decreased. When excitation light with wavelengths of 470 and 550 nm was used, 90% of the light was absorbed into the silicon substrate at depths of 0.8 and 1.7 μm, respectively. Above a concentration of 50 μM, the FITC and Texas Red solution were saturated. The calculation results of the light intensity versus the FITC and Texas Red concentration are shown in Fig. 8. The intensities of the excitation light and fluorescence were determined by solving the equations using the photocurrents and parameters with photo-gate voltages of 1 and 4 V. The calculation results for the FITC solution are shown in Fig. 8a. The excitation light was reduced from 10 to 3 mW/cm2 depending on the concentration, and the fluorescence at 520 nm was detectable up to a concentration of 0.01 μM. The calculation results for the Texas Red solution are shown in Fig. 8b. The excitation light was reduced from 4 to 1 mW/cm2 depending on the concentration, and the fluorescence at 610 nm was detectable up to a concentration of 0.01 μM. As a result, the minimum detection level was determined to be in the submicromolar concentration range. This result is similar to the limit of detection (LOD) for the FITC solution of the fluorescence sensors, which developed by integrating the interference filter and the
absorption filter. However, these sensors are difficult to deal with changes in the fluorescence solution. Table III provides a comparison of the fluorescence sensor with other imaging systems reported in the literature. The fluorescence and excitation light intensities were measured simultaneously.
3.4 Detection of three-wavelengths The filter-free fluorescence sensor is able to measure light intensities without using a filter, and various wavelengths can be measured simultaneously. We used this sensor to detect a mixed solution of FITC and Texas Red using the proposed parameters. A mixed solution of FITC and Texas Red parameter values can be represented by the contact points of the photo-gate voltages of 2, 3 and 4 V located at the peaks of the excitation light (470 nm), fluorescence of FITC (520 nm) and fluorescence of Texas Red(590 nm). Figure 9a shows the spectra versus the concentration of a mixed solution and the parameter. The scattered light at an angle of 90 degrees from the incident light was measured using a spectrometer. In the case of the FITC (1 μM) and Texas Red (1 μM), only the excitation light at 470 nm was emitted by the low concentration of the fluorescence solution. In the case of the FITC (10 μM) and Texas Red (10 μM), the spectral peak values of 470, 520, and 610 nm are shown. The light intensity at 470 nm was decreased by the 10 μM concentration of the FITC solution, and emitted at 520 nm. The wavelength of 610 nm emitted by the Texas Red was excited by the FITC due to the Fluorescence resonance energy transfer (FRET). In the case of the FITC (1 μM) and Texas Red (10 μM), the wavelength of 470 nm was absorbed by the Texas Red as the excitation light, and emitted fluorescence at 610 nm. Compared to those of the FITC (10 μM) and Texas Red (10 μM), this was a relatively small intensity. The reason is that the fluorescence emitted from the fluorescence of the FITC had a large influence. For the measurements of the three wavelengths, the currents were measured by mixing the FITC and Texas Red at concentrations of 1, 5, and 10 μM, respectively. The wavelength and intensity of the input light source were 470 nm and 1 mW/cm2, respectively, and the input voltages of the photo-gate were 2, 3, and 4 V, respectively. The average value of the current was measured five times for each solution, and the results are shown in Fig. 9b. The measured current value was highly dependent on the intensity of the excitation light due to the FITC concentration. In the case of the same FITC concentration, the current was changed by the concentration of the Texas Red. Figure 9c shows the intensity as calculated using Eq. (7). As the FITC concentration increased, the intensity at 470 nm decreased while the intensities at 520 and 610 nm increased. Because most of the excitation light was absorbed by the FITC, the light intensities at 520 and 610 nm changed less than that at 470 nm. This is because, as was mentioned in Section 3.3.2, the emitted fluorescence was very weak compared to the intensity of the excitation light directly incident on the sensor, and there was a limit to the ability to perform simultaneous measurements of the spectrum. In addition, the fluorescence emitted from the FITC had a relatively wide full width at half maximum (FWHM) parameter, which may overlap with the wavelength of 610 nm and make it difficult to calculate the exact intensity. Therefore, it will be necessary to study the calculation of the light intensity based on the FWHM determination. When the concentration of the FITC was 10 μM, the light intensity at 620 nm increased with increasing concentration of Texas Red. Based on these results, we can separate the three wavelengths using the proposed parameters while simultaneously measuring them. It is also possible to calculate the intensity of each wavelength.
4. Conclusions In this paper, we presented a performance analysis of a filter-free fluorescence sensor using an LED light source and fluorescent solution. The intensities of the excitation light and fluorescence were determined by solving several equations using the measured photocurrent and proposed parameters. The separation ability of the filter-free fluorescence sensor was more than 1,000:1 when an LED light source was used. To measure the fluorescence solution, we proposed a parameter to minimize the effect of the interference introduced by each layer. Fluo rescent solution measurements were carried out using FITC and Texas Red dyes. The sensor was able to separate the excitation light and fluorescence of the fluorescent solution, and the intensity of the light could be measured at the same time. In addition, the fluorescence of submicromolar concentrations of solutions could be detected. We determined it was possible to use FRET analysis with the filter-free fluorescence sensor to simultaneously measure the intensities of three wavelengths without using an optical filter.
ACKNOWLEDGMENT This work was supported by JSPS KAKENHI Grant Number 24226010 and CREST, Japan Science and Technology Agency.
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Biography
Yong-Joon Choi received his B.S. degree in Information Technology and Electronic Education and a M.S. in Bio-electronic Engineering from the Andong National University, Gyeongbuk, Korea, in 2010, and 2013, respectively. He is a Ph.D. student at the Department of the Electrical and Electronic Information Engineering, Toyohashi University of Technology, Aichi, Japan. His research interests focus on the development of fluorescence sensors and biosensing systems.
Kazuhiro Takahashi received the B.S. degree in mechanical engineering from Nagoya University,
Japan in 2003 and the M.S. and Ph.D. degrees in electrical engineering from the University of Tokyo, Japan, in 2005 and 2008, respectively. From 2008 to 2009, he was a post-doc researcher with Institute of Industrial Science (IIS), the University of Tokyo. From 2009 to 2013, he was an Assistant Professor with electrical and electronic information engineering, Toyohashi University of Technology. Since 2013, he has been a tenure track lecturer with electrical and electronic information engineering, Toyohashi University of Technology. His research interest is the development of CMOS-MEMS sensors and actuators for optical and biological applications.
Nobuo Misawa received the Ph.D. degree from The Graduate University for Advanced Studies in 2006. In the same year, he was a research associate at the Institute for Molecular Science. From Nov. 2006 to Mar. 2009, he was a project assistant professor of Information and Robot Technology Research Initiative at The University of Tokyo. From Apr. 2009 to Mar. 2010, he was a project assistant professor at the Institute of Industrial Science at The University of Tokyo and a research associate of the Bio Electromechanical Autonomous Nano Systems project. From Apr. 2010 to Mar. 2015, he was a tenuretrack assistant professor of Electronics-Inspired Interdisciplinary Research Institute at Toyohashi University of Technology. From Apr. to Dec. 2015, he was a project lecturer at the same institute. Since Jan. 2016, he is a research associate of Kanagawa Academy of Science and Technology. His research interests are BioMEMS and surface science, especially microfabrication, lipid membrane and membrane proteins.
Takeshi Hizawa received the B.A. and M.S. degrees in Electrical and Electronic Engineering and his Ph.D. in Electronic and Information Engineering from the Toyohashi University of Technology, Aichi, Japan, in 2002, 2004, and 2007, respectively. From 2007 to 2013, he worked at Toshiba Corp. He was engaged in the development of semiconductor process technology. In 2013, he joined Toyohashi University of Technology, where he is now serving as a technical support staff of semiconductor devices and process technology.
Tatsuya Iwata received his Ph.D. from Kyoto University in 2014. Since then, he is an assistant professor at the Department of the Electrical and Electronic Information Engineering of Toyohashi University of Technology. His research interests focus on integrated chemical and bio-sensors.
Kazuaki Sawada was born in Kumamoto, Japan, in 1963. He received his B.A. and M.S. in electrical and electronic engineering and a Ph.D. in system and information engineering from the Toyohashi University of Technology, Aichi, Japan, in 1986, 1988, and 1991, respectively. From 1991 to 1998, he worked as a Research Associate with the Research Institute of Electronics, Shizuoka University, Shizuoka, Japan. In 1998, he joined the Department of Electrical and Electronic Engineering, Toyohashi University of Technology, Aichi, Japan, and he is currently a Professor and Director of Electronics-Inspired Interdisciplinary Research Institute (EIIRIS). In 2005, he was a Guest Researcher with the Technical University of Munich, Munich, Germany. His current research interests focus on the development of ultrahigh-sensitivity biosensing and microfluidic devices.
(a)
(b) Fig. 1. (a) Photomicrograph of the filter-free fluorescence sensor, and (b) a schematic illustrating a cross section of the sensor.
Fig. 2. Potential diagram of the filter-free fluorescence sensor. (PG: photogate)
(a)
(b)
(c)
Fig. 3. (a) A schematic diagram of the signal readout manner circuitry (b) Photocurrent-voltage characteristics for the single-wavelength; (c) the output photocurrents and potential depth of Si substrate depending on the photogate voltage using the SPECTRA.
(a)
(b)
(c) Fig. 4. (a) Configuration of the measuring device, (b) separation ability of filterfree fluorescence sensor using the 470 and 530 nm LED light source, and (c) the 550 and 590 nm LED light source.
Fig. 5. Calculated parameters and spectra of the FITC and Texas Red solution.
Fig. 6. Schematic view of the measurement system using the FITC and Texas Red.
(a)
(b)
Fig. 7. Output photo current of filter-free fluorescence sensor for (a) FITC, and (b) Texas Red concentrations.
(a)
(b) Fig. 8. (a) Separation ability of the filter-free fluorescence sensor using the FITC and (b) Texas Red solution.
(a)
(b)
(c) Fig. 9. (a) Spectra of the mixed solution and parameter of FITC and Texas Red, (b) output photo current of the filter-free fluorescence sensor, and (c) the calculated intensity of the three wavelengths based on the output photo current.
TABLE. I. AVERAGE VALUES OF THE PHOTOCURRENTS AND THEIR STANDARD DEVIATIONS. Photo-gate : 1 V
Photo-gate : 3 V
12.08
12.98
Average current [nA]
-3
0.94 × 10-3
1.48 × 10
Standard deviation
Table. II. Comparative analysis of fluorescence sensors. [8]
[9]
This work
Filter type
Absorption
Interference
Filter-free
Excitation ranges
< 520 nm
488 nm
Detection ranges
> 490 nm
510 - 600 nm
LOD (FITC)
1 - 10 nM
680 pM
400 - 900 nm 10 nM
S0925-4005(17)31743-4 https://doi.org/10.1016/j.snb.2017.09.077 SNB 23168
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
25-3-2017 7-9-2017 13-9-2017
Please cite this article as: Yong Joon Choi, Kazuhiro Takahashi, Nobuo Misawa, Takeshi Hizawa, Tatsuya Iwata, Kazuaki Sawada, Multi-wavelength fluorescence detection of submicromolar concentrations using a filter-free fluorescence sensor, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2017.09.077 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.
Multi-wavelength fluorescence detection of submicromolar concentrations using a filter-free fluorescence sensor Yong Joon Choi, Kazuhiro Takahashi, Nobuo Misawa, Takeshi Hizawa, Tatsuya Iwata, Kazuaki Sawada
Email: [email protected] Department of Electrical and Electronic Information Engineering, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan.
Graphical abstract
Research Highlights
・We presented a performance analysis of a filter-free fluorescence sensor using an LED light source and fluorescent solution without using an optical filter.
・The intensities of the excitation light and fluorescence were determined by proposed parameters based on output current.
・The excitation light and fluorescence from submicromolar concentrations of FITC, Texas Red, and a mixed solution were successfully separated and simultaneously detected.
Abstract
In this paper, we report on the development of a filter-free fluorescence sensor for the detection of multiple fluorescence wavelengths from a solution of fluorescent dyes with submicromolar concentrations. The filter-free fluorescence sensor has a photo-gate structure, and the intensities of excitation light and fluorescence can be calculated from the photocurrents for the different gate voltages. This sensor makes it possible to measure various wavelengths without the need for optical filters or mirrors. The separation ability of excitation light and fluorescence in the developed sensor was more than 1,000:1 for an LED light source. Fluorescence measurements were carried out for fluorescein isothiocyanate isomer (FITC) and sulforhodamine 101 acid chloride (Texas Red) dyes. Based on our calculations, the excitation light and fluorescence from submicromolar concentrations of FITC, Texas Red, and a mixed solution were successfully separated and simultaneously detected. Keywords: filter-free, fluorescence sensor, biosensor, multi-wavelength detection, FITC, Texas Red
1. Introduction Fluorescence analysis is widely used in biochemical analysis and medical diagnosis procedures because it is easy to use and can provide large amounts of data [1,2]. Fluorescence microscopes are often used to detect fluorescence [3]; however, they consist of an optical filter and other parts, and are generally expensive and bulky [4]. At the same time, on-chip measurements of fluorescence are required for field diagnosis and point of care testing systems [5,6], which requires the development of miniature fluorescence detection devices. Recently, on-chip fluorescence sensors have been developed that consist of optical filters integrated on photodiodes [7–12]. In addition, a fluorescence sensor using buried triple p-n junction photodiodes has also been developed [13]. Although these devices exhibit high sensitivity, the fabrication processes required to integrate the optical filters are complicated. In addition, it is difficult to deal with changes in the fluorescent solution because the detectable fluorescence wavelength is fixed. A filter-free fluorescence sensor with a photo-gate structure has been developed that can measure a wide range of wavelengths without filters by considering the depth of the light absorption in the silicon [14,15]. It is also possible to measure multiple wavelengths at the same time [16]. The separation ability of excitation light and fluorescence in this sensor was 500:1. However, fluorescence detection requires an intensity ratio of more than 1,000:1 between the excitation light and fluorescence. Various techniques have been developed to enhance the separation ability, such as the body-biasing technique [17] and the surface planarization of polycrystalline-silicon gates [18], which have improved the separation ability of the filter-free fluorescence sensor from 500:1 to 1,200:1. In this paper, we describe the detection of fluorescence from fluorescein isothiocyanate isomer (FITC) [19,20] and sulforhodamine 101 acid chloride (Texas Red) [21] using the filterfree fluorescence sensor in which the separation ability of the sensor is 1,200:1 between the excitation light (470 nm) and fluorescence (530 nm). To improve the detection performance, we propose a parameter calculation method, and investigate the detection limit of the sensor for concentrations of FITC and Texas Red. Finally, we also describe the simultaneous detection of three wavelengths using the sensor for the measurement of mixed fluorescent solutions [22–24].
2. Filter-free fluorescence sensor 2.1 Structure
Figure 1a shows an optical microscopy image of the filter-free fluorescence sensor. The sensor was fabricated using 5-μm-rule, N-substrate, 1P2M (1-poly and 2-metal), single well modified complementary metal-oxide semiconductor fabrication technology. The size of the sensing area is 200 × 200 μm. Figure 1b shows a schematic cross section and diagram of the sensor operation. The sensor uses a n-type silicon substrate to fabricate a photogate structure on the p-well, and a pn junction is formed in the p-well. An n+ diffusion layer is placed adjacent to the photogate to measure the photoelectron. The photogate and diffusion layer are surrounded by a p-well contact, which functions as a p-well potential setting and forms holes near the drain in the p-well. The substrate potential is set using a substrate contact, and provides an outlet for the photoelectron deeper than the potential depth. The outside of the photogate is shaded with Al to suppress the generation of photoelectrons. The sensing area of the device consists of five layers: SiO2 (60 nm), n-type polysilicon (350 nm), thermal SiO2 (60 nm), p-well (4 μm), and n-type silicon substrate.
2.2 Sensing principle The developed filter-free fluorescence sensor measures the light intensity for different photogate voltages. When a voltage is applied to the gate, a depletion layer is formed at the SiO2/Si interface with the depth of W. When light is irradiated onto the sensing area, photoelectrons are generated by the light absorption, although the light absorbed in the depletion layer only contributes photocurrent. Equation (1) can be used to calculate the photocurrent generated by the light absorbed in the depletion layer.
I
qS
(1 e
W
)
(1)
hc
where q is the elementary charge, S is the sensing area, λ is the wavelength, h is the Planck constant, c is the speed of light in vacuum, W is the depletion depth, and is the light absorption coefficient. If the relationship between the light intensity ϕ and the photocurrent I is known in advance, then ϕ can be calculated from I . Figure 2 shows the operating principle of the filter-free fluorescence sensor using a potential diagram. When a positive bias voltage V1 is applied to the photo gate, the depletion depth is W1. The photocurrent IW1 is generated for W1 and output from the output contact. If a different positive voltage V2 is applied to the photo-gate, the photocurrent IW2 is generated for the depletion depth W2. Thus, different relationships between I and ϕ can be obtained by changing the photo-gate voltage. When light consisting of different wavelengths and intensities (λ1, ϕ1 and λ2, ϕ2) is simultaneously irradiated onto the sensing area, the photocurrent for the depletion depth of W1 can be expressed as in Eq. (2). In a similar manner, the photocurrent for W2 can be calculated using Eq. (3). When the coefficient of the light intensity in each term in Eqs. (2-3) is known, ϕ1 and ϕ2 can be calculated by solving the following simultaneous equations. IW 1
qS 1 hc
(1 e
1W 1
) 1
qS 2 hc
(1 e
2W 1
) 2
(2)
IW
2
qS 1 hc
(1 e
1W 2
) 1
qS 2 hc
(1 e
2W 2
) 2
(3)
3. Experimental results 3.1 Single wavelength current characteristics To measure the output current of the filter-free fluorescence sensor, light sources of various wavelengths were used to irradiate the sensor. The wavelength of a light source is adjusted by passing it through a band-pass filter with the full width at half maximum(FWHM) of 10 to 25 nm. The light sources were irradiated onto the sensing area with a diameter of 150 μm area and an intensity of 5 μW/cm2. Figure 3a shows a schematic diagram of the signal readout manner circuitry. To operate the sensor, 0 and 5 V are applied to the p-well electrode and the n-substrate electrode, respectively. The input voltage of the photogate was -1 to 4 V, and the output current was measured by the semiconductor parameter analyzer (Agilent, 4155A). To measure the noise of the measurement system, the photo-gate voltage was applied at 1, 3 V and the light source (530 nm) was irradiated at 10 μW/cm2. The photocurrent was measured 100 times for each photo-gate voltage. When voltages of 1 and 3 V were applied to the photo-gate, the minimum and maximum range of the photocurrent were 7 and 4 pA, respectively. Table. 1 shows the average values of the photocurrents and their standard deviations. Figure 3b shows the photocurrent-voltage characteristics of the single-wavelength. The photogate was in the accumulation state at voltages up to -0.5 V, the inversion state from -0.5 to 0.5 V, and the deep depletion state at voltages above 0.5 V. For our fabricated sensor, when voltages of 1 and 4 V were applied to the photo-gate, the depths of the potential peak W were 1.15 and 1.52 μm, respectively. The potential peak W was calculated by a SPECTRA (Link Research Corporation), which is a simulation program for three-dimensional electric transient analysis of CCD and CMOS image sensors using real impurity profile device simulator. Figure 3c shows the output photocurrents in the positive direction of x-axis(vertical) of Si substrate depending on the photogate voltage, when the wavelength and intensity of the input light source were 470 nm and 10 μW/cm2, respectively. The positive photocurrent was detected by the output contact, and the negative photocurrent was output by the n-substrate. The potential peak W can be estimated in the section where the photocurrent value changes from positive to negative. At a wavelength of 450 nm, 90% of the light was absorbed into the silicon substrate at a depth of 0.9 μm. When the incident light was mostly absorbed by the shallow silicon surface, the current difference was relatively small. In contrast, when the 90 % of the light penetrated to a silicon substrate at a depth of 6.45 μm at a wavelength of 625 nm, a current difference of 80 nA was generated by the photogate voltage from 1 to 4 V. In general, a high photocurrent was expected at the wavelength of 450 nm where the silicon absorption coefficient α was higher than the wavelength of 550 nm. However, the output photocurrent at the wavelength of 450 nm became smaller than that at the wavelength of 550 nm due to the absorbed by polysilicon. The optical transmittance of polysilicon at wavelengths 450 and 550 nm are 7 and 42 %, respectively. Although short wavelength of 450 nm has a large absorption coefficient, it is mostly absorbed by the polysilicon. Therefore, it is considered that the output photocurrent became smaller than that at the wavelength of 550 nm. As a result, the components of the wavelength can be derived by changing the photogate voltage based on the absorption coefficient of the silicon. It is also possible to determine the wavelength intensities of the light source by calculating the differences in the output current. Figure 3 (d) shows the spectral response of the sensor and used a laser driven light source (Energetiq, EQ-99). The responsivity was measured from 400 to 900 nm and
the FWHM was 10 nm. The absorption by the photogate for the short-wavelengths is relatively high due to the high absorption coefficient of silicon. It is considered that the responsivity of a wavelength of 400 nm is relatively low. The reflectance of the sensing area for a wavelength of 700 nm is about 55 %. Therefore, since the wavelength of 700 nm absorbed in silicon is reduced, a decrease in the response rate of the sensor is expected. Wavelengths above 750 nm are absorbed in regions deeper than the potential depth and are expected to decrease responsivity. By adjusting the p-well layer and the potential depth, high response can be expected in the long wavelength. The emission wavelength of a commonly used fluorescent dye is reported to range from about 420 to 875 nm (eg DyLight 405, DyLight 830). it is considered that the filter-free fluorescence sensor is applicable to most fluorescent dyes. 3.2 Separation ability of the LED light Figure 4a shows the setup used for measurements of the separation ability of the fluorescence sensor. In order to reduce the intensity error, the irradiated light was minimized using a microscope. Two optical fibers were connected to the microscope for irradiation purposes, and light sources with two wavelengths were used: 470 nm to mimic the excitation light and 530 nm for the fluorescence. The FWHM of irradiated light source 470 and 530 nm were25 and 32nm, respectively. The connected light sources passed through the x5 objective lens, and were irradiated with a diameter of 80 μm onto the sensing area. The standard intensity of the light was measured using an optical power meter. An x-axis stage was used to move the filter-free fluorescence sensor and optical power meter to minimize the error between measurements. The electrical characteristics of the sensor were measured using a semiconductor parameter analyzer. During the measurements, the light intensity at 530 nm was varied while the light intensity at 470 nm was maintained constant. The potential depth was changed by controlling the photogate voltage during the irradiation process. The separation ability was defined as the ratio between the lowest light intensity at 530 nm, which was validated by calculation, to that at 470 nm. In terms of the measurement, the potential depths W1 and W2 of the absorbed light were adjusted by applying voltages of 1 V and 3 V to the photo-gate. At these voltages, the depths of the potential peaks W were 1.15 and 1.52 μm, respectively. Here, the maximum intensity of the 530 nm light and the 470 nm light incident on the sensor were set to 5,000 nW/cm2, and the output current was measured while gradually decreasing the intensity of the 530 nm light from 5,000 to 0.1 nW/cm2. The intensities ϕ1 and ϕ2 for the 470 and 530 nm light, respectively, were determined by solving the simultaneous equations obtained by substituting the measured current into (2) and (3). Figure 4b shows the separation ability of the filter-free fluorescence sensor using the 470 and 530 nm LED light sources. The separation ability of the excitation light (470 nm) and fluorescence (530 nm) was 1,200:1. Figure 4c shows the separation ability of the sensor using the 550 and 590 nm LED light source with the FWHM of 20 and 25 nm, which was determined to be 1,000:1. In this case, the separation ability was reduced rather than the wavelengths of 470 and 530 nm. Since the spacing of the wavelengths was relatively close to each other, the separation ability was reduced due to the crosstalk. In the case of 470 and 530 nm, the reflectance of the sensing area is 23.5 and 11.2 %. On the other hand, the reflectance at wavelengths 550 and 590 nm were 21.5 and 39.1 %, which the amount of light at 590 nm wavelength absorbed by silicon was relatively smaller than 550 nm. Therefore, it is considered that the fluorescence (550 nm) was relatively small compared to the excitation light (590 nm), leading to a decrease in the separation ability. 3.3 Analysis of the fluorescent solution
In this study, the detection performance for quantitative measurements of fluorescent dye were carried out using FITC and Texas Red. FITC has the amino-group reactivity of Fluorescein and is a combination of the isothiocyanate reactive group, which dissolves in water and emits a strong yellow-green fluorescence (λex = 495 nm, λem = 520 nm). Since it reacts easily with the amino group, it can be applied to fluorescent labels on amino acids, enzymes, hormones, peptides, and viruses, and is used for diagnostic purposes in collagen disease, syphilis, antigen-antibody, and fluorescent antibody methods. Texas Red binds to proteins and amino acids with high efficiency and emits a deep red fluorescence (λex = 568 nm, λem = 590 to 630 nm). It has an advantage in that it can be labeled, while maintaining the activity of enzymes and without interfering with the fluorescent substances contained in biological samples. For these reasons, Texas Red is generally considered to be the more useful of the two dyes.
3.3.1 Parameters of the filter-free fluorescence sensor for fluorescent dyes The filter-free fluorescence sensor can be calculated the light intensity for a single wavelength by Equation (1). However, a width of FWHM increases, the reduction in sensitivity and errors in the calculation are expected. To measure the intensity of the weak light generated and light with spectrum width by fluorescent solutions, accurate calculations of the intensity are required. The filter-free fluorescence sensor consists of four layers (SiO2, Polysilicon, thermal SiO2, and Silicon) as shown in Fig. 1. Since the thickness of each layer differs depending on the process, the wavelength intensity of the light source incident in the silicon differs. Consequently, it is difficult to accurately derive the depth and nonlinear absorption coefficient α of the light incident in the silicon, and there is expected to the calculations error and a decrease in sensitivity of the light intensity. Therefore, a parametric method was required to minimize the absorption coefficient and the effect of constants on a light source with spectrum width. In this study, we propose a method of calculating the light intensity using parameters based on the output photocurrent of the sensor versus the wavelengths in the range from 450 to 625 nm. With the exception of the current I and light intensity ϕ components in Eqs. (2) and (3), parameters A, B, C and D, can be expressed as follows.
A
qS 1 hc
(1 e
a 1W 1
)
B
qS 2
(1 e
a 2W 1
)
hc
C
qS 1
(1 e
a 1W
2
)
D
hc
qS 2
(1 e
a 2W
2
)
hc
(4)
The components of the output current can be represented by the sum of the respective currents, as in Eq. (5). IW A 1 B 2 1
IW
2
C 1 D 2
Thus, the light intensities ϕ1 and ϕ2 can be calculated by the inverse matrix.
(5)
1 A C 2
B D
1
(6)
I W1 I W2
In the same way, the equation for the intensities of the three wavelengths can be calculated by substituting the parameters A to I into Eq. (7). 1 2 3
A D G
B E H
C F I
1
IW 1 I W2 I W3
(7)
Figure 5 shows the calculated parameters and spectra of the FITC and Texas Red solution. The FITC parameter values can be represented by the contact points of the photo-gate voltages of 2 and 4 V located at the peaks of the excitation light (470 nm) and fluorescence (520 nm). In the same way, the Texas Red parameter values can be obtained at the peaks of the excitation light (550 nm) and fluorescence (610 nm).
3.3.2 Measurement of fluorescence solution Figure 6 shows the measurement setup in a dark room for assessing the device using a semiconductor parameter analyzer. To evaluate the FITC and Texas Red, 4 ml of solution was deposited into disposable cuvettes. Excitation light with wavelengths of 470 and 550 nm (FITC and Texas Red) were irradiated into the side of the cuvettes. Then, a semiconductor parameter analyzer was used to control the sensor and measure the photocurrents. Excitation light with wavelengths of 470 and 550 nm was then irradiated at intensities of 10 and 4 mW/cm2, respectively, while the concentrations of the FITC and Texas Red in the solution were individually varied from 0.01 to 50 μM. Each fluorescent solution was prepared using diluted ethanol. Figure 7 illustrates the photocurrent of the sensor when voltages of 1 to 4 V were applied to the photo-gate. The current values were based on five experiments. In comparison to the excitation light, the fluorescence was relatively weak, and when the concentration of the FITC and Texas Red increased, the photocurrent decreased. When excitation light with wavelengths of 470 and 550 nm was used, 90% of the light was absorbed into the silicon substrate at depths of 0.8 and 1.7 μm, respectively. Above a concentration of 50 μM, the FITC and Texas Red solution were saturated. The calculation results of the light intensity versus the FITC and Texas Red concentration are shown in Fig. 8. The intensities of the excitation light and fluorescence were determined by solving the equations using the photocurrents and parameters with photo-gate voltages of 1 and 4 V. The calculation results for the FITC solution are shown in Fig. 8a. The excitation light was reduced from 10 to 3 mW/cm2 depending on the concentration, and the fluorescence at 520 nm was detectable up to a concentration of 0.01 μM. The calculation results for the Texas Red solution are shown in Fig. 8b. The excitation light was reduced from 4 to 1 mW/cm2 depending on the concentration, and the fluorescence at 610 nm was detectable up to a concentration of 0.01 μM. As a result, the minimum detection level was determined to be in the submicromolar concentration range. This result is similar to the limit of detection (LOD) for the FITC solution of the fluorescence sensors, which developed by integrating the interference filter and the
absorption filter. However, these sensors are difficult to deal with changes in the fluorescence solution. Table III provides a comparison of the fluorescence sensor with other imaging systems reported in the literature. The fluorescence and excitation light intensities were measured simultaneously.
3.4 Detection of three-wavelengths The filter-free fluorescence sensor is able to measure light intensities without using a filter, and various wavelengths can be measured simultaneously. We used this sensor to detect a mixed solution of FITC and Texas Red using the proposed parameters. A mixed solution of FITC and Texas Red parameter values can be represented by the contact points of the photo-gate voltages of 2, 3 and 4 V located at the peaks of the excitation light (470 nm), fluorescence of FITC (520 nm) and fluorescence of Texas Red(590 nm). Figure 9a shows the spectra versus the concentration of a mixed solution and the parameter. The scattered light at an angle of 90 degrees from the incident light was measured using a spectrometer. In the case of the FITC (1 μM) and Texas Red (1 μM), only the excitation light at 470 nm was emitted by the low concentration of the fluorescence solution. In the case of the FITC (10 μM) and Texas Red (10 μM), the spectral peak values of 470, 520, and 610 nm are shown. The light intensity at 470 nm was decreased by the 10 μM concentration of the FITC solution, and emitted at 520 nm. The wavelength of 610 nm emitted by the Texas Red was excited by the FITC due to the Fluorescence resonance energy transfer (FRET). In the case of the FITC (1 μM) and Texas Red (10 μM), the wavelength of 470 nm was absorbed by the Texas Red as the excitation light, and emitted fluorescence at 610 nm. Compared to those of the FITC (10 μM) and Texas Red (10 μM), this was a relatively small intensity. The reason is that the fluorescence emitted from the fluorescence of the FITC had a large influence. For the measurements of the three wavelengths, the currents were measured by mixing the FITC and Texas Red at concentrations of 1, 5, and 10 μM, respectively. The wavelength and intensity of the input light source were 470 nm and 1 mW/cm2, respectively, and the input voltages of the photo-gate were 2, 3, and 4 V, respectively. The average value of the current was measured five times for each solution, and the results are shown in Fig. 9b. The measured current value was highly dependent on the intensity of the excitation light due to the FITC concentration. In the case of the same FITC concentration, the current was changed by the concentration of the Texas Red. Figure 9c shows the intensity as calculated using Eq. (7). As the FITC concentration increased, the intensity at 470 nm decreased while the intensities at 520 and 610 nm increased. Because most of the excitation light was absorbed by the FITC, the light intensities at 520 and 610 nm changed less than that at 470 nm. This is because, as was mentioned in Section 3.3.2, the emitted fluorescence was very weak compared to the intensity of the excitation light directly incident on the sensor, and there was a limit to the ability to perform simultaneous measurements of the spectrum. In addition, the fluorescence emitted from the FITC had a relatively wide full width at half maximum (FWHM) parameter, which may overlap with the wavelength of 610 nm and make it difficult to calculate the exact intensity. Therefore, it will be necessary to study the calculation of the light intensity based on the FWHM determination. When the concentration of the FITC was 10 μM, the light intensity at 620 nm increased with increasing concentration of Texas Red. Based on these results, we can separate the three wavelengths using the proposed parameters while simultaneously measuring them. It is also possible to calculate the intensity of each wavelength.
4. Conclusions In this paper, we presented a performance analysis of a filter-free fluorescence sensor using an LED light source and fluorescent solution. The intensities of the excitation light and fluorescence were determined by solving several equations using the measured photocurrent and proposed parameters. The separation ability of the filter-free fluorescence sensor was more than 1,000:1 when an LED light source was used. To measure the fluorescence solution, we proposed a parameter to minimize the effect of the interference introduced by each layer. Fluo rescent solution measurements were carried out using FITC and Texas Red dyes. The sensor was able to separate the excitation light and fluorescence of the fluorescent solution, and the intensity of the light could be measured at the same time. In addition, the fluorescence of submicromolar concentrations of solutions could be detected. We determined it was possible to use FRET analysis with the filter-free fluorescence sensor to simultaneously measure the intensities of three wavelengths without using an optical filter.
ACKNOWLEDGMENT This work was supported by JSPS KAKENHI Grant Number 24226010 and CREST, Japan Science and Technology Agency.
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Biography
Yong-Joon Choi received his B.S. degree in Information Technology and Electronic Education and a M.S. in Bio-electronic Engineering from the Andong National University, Gyeongbuk, Korea, in 2010, and 2013, respectively. He is a Ph.D. student at the Department of the Electrical and Electronic Information Engineering, Toyohashi University of Technology, Aichi, Japan. His research interests focus on the development of fluorescence sensors and biosensing systems.
Kazuhiro Takahashi received the B.S. degree in mechanical engineering from Nagoya University,
Japan in 2003 and the M.S. and Ph.D. degrees in electrical engineering from the University of Tokyo, Japan, in 2005 and 2008, respectively. From 2008 to 2009, he was a post-doc researcher with Institute of Industrial Science (IIS), the University of Tokyo. From 2009 to 2013, he was an Assistant Professor with electrical and electronic information engineering, Toyohashi University of Technology. Since 2013, he has been a tenure track lecturer with electrical and electronic information engineering, Toyohashi University of Technology. His research interest is the development of CMOS-MEMS sensors and actuators for optical and biological applications.
Nobuo Misawa received the Ph.D. degree from The Graduate University for Advanced Studies in 2006. In the same year, he was a research associate at the Institute for Molecular Science. From Nov. 2006 to Mar. 2009, he was a project assistant professor of Information and Robot Technology Research Initiative at The University of Tokyo. From Apr. 2009 to Mar. 2010, he was a project assistant professor at the Institute of Industrial Science at The University of Tokyo and a research associate of the Bio Electromechanical Autonomous Nano Systems project. From Apr. 2010 to Mar. 2015, he was a tenuretrack assistant professor of Electronics-Inspired Interdisciplinary Research Institute at Toyohashi University of Technology. From Apr. to Dec. 2015, he was a project lecturer at the same institute. Since Jan. 2016, he is a research associate of Kanagawa Academy of Science and Technology. His research interests are BioMEMS and surface science, especially microfabrication, lipid membrane and membrane proteins.
Takeshi Hizawa received the B.A. and M.S. degrees in Electrical and Electronic Engineering and his Ph.D. in Electronic and Information Engineering from the Toyohashi University of Technology, Aichi, Japan, in 2002, 2004, and 2007, respectively. From 2007 to 2013, he worked at Toshiba Corp. He was engaged in the development of semiconductor process technology. In 2013, he joined Toyohashi University of Technology, where he is now serving as a technical support staff of semiconductor devices and process technology.
Tatsuya Iwata received his Ph.D. from Kyoto University in 2014. Since then, he is an assistant professor at the Department of the Electrical and Electronic Information Engineering of Toyohashi University of Technology. His research interests focus on integrated chemical and bio-sensors.
Kazuaki Sawada was born in Kumamoto, Japan, in 1963. He received his B.A. and M.S. in electrical and electronic engineering and a Ph.D. in system and information engineering from the Toyohashi University of Technology, Aichi, Japan, in 1986, 1988, and 1991, respectively. From 1991 to 1998, he worked as a Research Associate with the Research Institute of Electronics, Shizuoka University, Shizuoka, Japan. In 1998, he joined the Department of Electrical and Electronic Engineering, Toyohashi University of Technology, Aichi, Japan, and he is currently a Professor and Director of Electronics-Inspired Interdisciplinary Research Institute (EIIRIS). In 2005, he was a Guest Researcher with the Technical University of Munich, Munich, Germany. His current research interests focus on the development of ultrahigh-sensitivity biosensing and microfluidic devices.
(a)
(b) Fig. 1. (a) Photomicrograph of the filter-free fluorescence sensor, and (b) a schematic illustrating a cross section of the sensor.
Fig. 2. Potential diagram of the filter-free fluorescence sensor. (PG: photogate)
(a)
(b)
(c)
Fig. 3. (a) A schematic diagram of the signal readout manner circuitry (b) Photocurrent-voltage characteristics for the single-wavelength; (c) the output photocurrents and potential depth of Si substrate depending on the photogate voltage using the SPECTRA.
(a)
(b)
(c) Fig. 4. (a) Configuration of the measuring device, (b) separation ability of filterfree fluorescence sensor using the 470 and 530 nm LED light source, and (c) the 550 and 590 nm LED light source.
Fig. 5. Calculated parameters and spectra of the FITC and Texas Red solution.
Fig. 6. Schematic view of the measurement system using the FITC and Texas Red.
(a)
(b)
Fig. 7. Output photo current of filter-free fluorescence sensor for (a) FITC, and (b) Texas Red concentrations.
(a)
(b) Fig. 8. (a) Separation ability of the filter-free fluorescence sensor using the FITC and (b) Texas Red solution.
(a)
(b)
(c) Fig. 9. (a) Spectra of the mixed solution and parameter of FITC and Texas Red, (b) output photo current of the filter-free fluorescence sensor, and (c) the calculated intensity of the three wavelengths based on the output photo current.
TABLE. I. AVERAGE VALUES OF THE PHOTOCURRENTS AND THEIR STANDARD DEVIATIONS. Photo-gate : 1 V
Photo-gate : 3 V
12.08
12.98
Average current [nA]
-3
0.94 × 10-3
1.48 × 10
Standard deviation
Table. II. Comparative analysis of fluorescence sensors. [8]
[9]
This work
Filter type
Absorption
Interference
Filter-free
Excitation ranges
< 520 nm
488 nm
Detection ranges
> 490 nm
510 - 600 nm
LOD (FITC)
1 - 10 nM
680 pM
400 - 900 nm 10 nM