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Label-free biosensing using cascaded double-microring resonators integrated with microfluidic channels
Optics Communications 344 (2015) 129–133
Contents lists available at ScienceDirect
Optics Communications journal homepage: www.elsevier.com/locate/optcom
Label-free biosensing using cascaded double-microring resonators integrated with microfluidic channels Yangqing Chen, Fang Yu, Chang Yang, Jinyan Song, Longhua Tang, Mingyu Li n, Jian-Jun He State Key Laboratory of Modern Optical Instrumentations, Department of Optical Engineering, Zhejiang University, Hangzhou 310027, China
art ic l e i nf o
a b s t r a c t
Article history: Received 14 September 2014 Received in revised form 19 November 2014 Accepted 8 January 2015 Available online 9 January 2015
Fast and accurate quantitative measurement of biologically relevant molecules has been demonstrated for medical diagnostics and drug applications in photonic integrated circuits. Herein, we reported a highly-sensitive optical biosensor based on cascaded double-microring resonators. The sensor was integrated with microfluidic channels and investigated with its label-free detection capability. With a wavelength resolution of 0.47 nm, the measured binding capacity of the antibody on the surface exhibits reliable detection limit down to 7.10 μg/mL using human immunoglobulin G (hIgG). & 2015 Elsevier B.V. All rights reserved.
Keywords: Cascaded double-microring resonators Biosensor Label-free
1. Introduction Integrated optical waveguide biosensors offer the promise of fast and accurate solutions for detection and analysis in biomedical diagnostics, environment monitoring and food safety applications. Great attention has been attracted owing to their superior advantages such as immune to electromagnetic interference, label free detection and integration of multifunctional devices on one chip [1,2]. Recently, special interest was focused on silicon-oninsulator (SOI) sensor based on evanescent field. With its natural benefit of high sensitivity, ultra-compactness and manufacture compatibility with the standard complementary metal–oxide– semiconductor (CMOS) process [3,4], advanced SOI sensors were able to be fabricated with low cost and mature industrial infrastructure [5]. Under this category, many relevant results have been reported, including surface plasmon resonance(SPR) [6], photonic crystals [7], Mach–Zehnder interferometer [8], micro-disk [9] and microring resonator [10]. Among various types of optical sensors, microring resonators have been regarded as a promising solution for biomolecular recognition and chemical analysis [11,12]. The sensing light is coupled into the resonators under resonant condition and confined near the waveguide surface of the resonators with an evanescent field exponentially decaying into the surrounding medium. The propagation of light in the resonator is thus influenced by the refractive index (RI) of the reagent contact to the waveguide surface. Compared with Mach–Zehnder interferometer, the degree of n
Corresponding author. E-mail address: [email protected] (M. Li).
http://dx.doi.org/10.1016/j.optcom.2015.01.028 0030-4018/& 2015 Elsevier B.V. All rights reserved.
interaction of light in microring resonators is determined by the amount of rotations of light, and not restricted by the physical length [13]. The high-Q microring resonator can thus provide a higher sensitivity due to the sharper resonance peak. However, the interrogation of high-Q optical microring resonator requires a narrow line width tunable laser or a high-resolution optical spectrum analyzer. These equipments are very expensive and cannot be integrated into one chip. To address these problems, our group has previously proposed and demonstrated a highly sensitive optical sensor based on cascaded double-microring resonators with Vernier effect [14–17]. The method was then adopted by several other research groups and respectable results have been achieved [18–20]. We have previously reported a double-microring resonator based sensor exhibiting sensitivity up to 24,300 nm per refractive index unit (RIU) using different concentration solutions of NaCl [17], far surpassing the sensitivity of 135 nm/RIU for a SOI single ring resonator [21] and 12.7 nm/RIU for the polymer dual ring resonators [22]. To detect streptavidin, the cascaded double-microring resonators were further made biotin film immobilized on the surface [23]. However, this type of sensor cannot be applied for kinetic measurement of molecular interaction as it is not integrated with microfluidic channels. In this paper, we further demonstrate the label-free detection of cascaded double-ring sensor in biosensing by measuring the refraction index change induced by the biorecognition molecular interaction. The sensor was fabricated by conventional photolithography and integrated with microfluidic channels to detect the concentration of human immunoglobulin G (IgG). By using the standard silicon surface functionalization procedure, the anti-human IgG is immobilized on the surface of the sensing area in the
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optical waveguide sensor. The measuring results demonstrate this cascaded double-microring resonator to be a promising real time sensor of measuring the molecules binding kinetics for biomedical and chemical applications.
2. Device fabrication The sensor includes two layers: the integrated optical waveguide structure layer and the microfluidic system layer. The optical waveguide structure based on SOI substrate with a 220 nm-thick top silicon layer and 2 μm-thick buried oxide layer is composed of an input waveguide, a reference ring and a sensing ring cascaded by a common bus waveguide, and an output waveguide, as shown in Fig. 1(a). The optical waveguide wafer is covered by SU-8 upper cladding layer, while the sensing ring is exposed to the analyzed sample in the sensing window. The diameters of the reference and sensing ring of the sensor are 230 μm and 252.2 μm, respectively and directional couplers of 50 μm length with a gap of 1 μm are employed in all the ring resonators. All the ridge waveguides are designed with 30 nm shallow etched depth and 1 μm width enabling traditional contact photolithography patterning with single mode operation. The fabrication started by spin-coating a thin film of photoresist on the SOI wafer. After conventional photolithography, the pattern of the photoresist was transferred to the SOI wafer by inductively coupled plasma reactive ion etching. The wafer was then spin coated with SU-8 as the upper cladding of the optical waveguide, and sensing window was opened by photolithography. Improved performance could be further realized with smaller patterns by employing a stepper or electronic beam lithography. More details of the operational principle and fabrication process of cascaded double-rings sensor were described in the reference [24]. The microfluidic channels are fabricated with polydimethylsiloxane (PDMS) [13]. The PDMS (Sylgard 184, DowCorning, USA) is degassed to remove air bubbles using vacuum pump. Then the PDMS is casted on the master to a thickness of 2 mm, and cured at 85 °C for 30 min in an oven. The fluidic structure is then peeled off from the master and cut out, and holes are punched for liquid input and output ports. The optical sensor is integrated into a chip by bonding with the microfluidic channels using epoxy glue, as shown in Fig. 1(b). The microfluidic system can control and manipulate small volumes of liquid samples or reagents required for testing and analysis quickly and reliably.
3. Measurement setup The sensor was tested under the end-fire system. As shown in Fig. 2, the measurement setup mainly includes two parts: the controllers of liquids and the controllers of optics. Liquid flow is
Fig. 2. The end-fire system. Liquid flow is maintained by syringe pumps. Laser wavelength tuning and signal read out are automated by computer control. (1) Input fiber; (2) fiber polarization controller; (3) sensor; (4) output fiber; (5, 6, 7) three-dimensional nanopositioning stages; (8) pipe for vacuum pump; (9) pipe for sample input; (10) pipe for sample output; and (11) waste reservoir.
controlled by syringe pumps (MICROINFUSION PUMP WZS-50F6), which supply a continuous flow of buffer or samples through the microfluidic channels passing by the sensing window and then to the waste solution bottle. The transverse electric (TE) mode light from the tunable light source (Agilent 81600B) is coupled into input waveguide of the sensor by the polarization maintaining lensed fibers. The output light is collected by the power meter (Agilent 81635A). For the optical measurement system, three three-dimensional nanopositioning stages (Thorlabs MAX312) are employed to accurately place the optical input fiber, output fiber and the chip. In addition, an infrared camera is used to assist the fiber alignment with the input waveguide. A fiber polarization controller is applied to adjust the TE polarization with a polarization filter. The whole measurement setup is automated by the computer.
4. Experimental results and discussions 4.1. Volume sensing Volume sensing experiment is performed to prove the cascaded double-microring resonators sensing ability and eliminate the influence of background refractive index, where non-functionalized sensor was used. The optical sensitivity of the sensor was calibrated by using aqueous solutions of NaCl with different concentrations (0%, 2%, 4%, 6%, 8%). As shown in Fig. 3, the measured refractive index sensitivity is 1804 nm/RIU. Different solutions of proteins were prepared by serial dilutions in 10 mM PBS and injected to the microfluidic channel. The transmission spectrum and real time resonance wavelength of the cascaded double-microring resonators are shown in Fig. 4(a) and (b), respectively. From Fig. 4
Fig. 1. (a) Optical microscope image of the cascaded double-microring. The reference ring and the waveguide are covered with an upper cladding layer SU-8 while the sensing ring is exposed to the air. (b) Photograph of one of the fabricated sensor. The steel tubes make fluidic flow to the microfluidic network below.
Y. Chen et al. / Optics Communications 344 (2015) 129–133
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Fig. 3. Measured wavelength shift versus refractive index change of aqueous solutions of NaCl with different concentrations.
(a), a remarkable resonance wavelength shift can be clearly observed when changing the concentration of PBS from 10 mM to 100 mM, induced by the change of refractive index. The shift of the resonance wavelengths returns to baseline as the flow returns to DI water. During the injection of different protein solutions, the resonance wavelength remains the same with constant injection of 10 mM PBS, indicating that the influence of background refractive index could be ignored during the detection of biomolecule. 4.2. Surface derivatization and antigen recognition To remove potential residual organic contaminants introduced from fabrication, the wafer is first cleaned with a piranha solution (1:1H2SO4: 30% H2O2), followed by copious rinsing with water, isopropanol and dried by nitrogen. As illustrated by the schematic in Fig. 5, the chip surface is subsequently modified using standard silanization chemistry [25], and then exposed to 2.5% solution of APTES (1:100 deionized water/ ethanol) for 12 h at 4 °C. After silanization, the wafer is rinsed with ethanol and dried by nitrogen. Covalent attachment of biomolecules to the wafer surface is achieved using glutaraldehyde cross-linking. The silanized surface is immersed in 5% solution of glutaraldehyde for 1 h, and cleaned with 10 mM PBS using centrifugal separation to remove excess cross-linking agent. The chip is then exposed to 0.1 mg/mL solution of streptavidin for 1 h and 0.1 mg/mL solution of BSA for 1 h to close nonspecific binding sites. The functionalized surface is next exposed to solution of biotinylated antibodies (usually 0.05 mg/mL or higher) for at least 30 min to maximize the number of covalently immobilized antibody. For human IgG detection experiments, PBS (10 mM, PH:7.4) is used as buffer. The solution of human IgG is made by serial dilution in PBS, which then flows through the microfluidic channel and over the sensor surface at a rate of 25 μL/min. Each reaction could be monitored owing to the refractive index change induced by covalently attaching organic molecules. A large resonance wavelength shift of 4.16 nm was induced by the addition of anti-IgG antibodies to the functionalized surface, as shown in the real time data in Fig. 6. A rinse with PBS helps remove any non-covalently bound antibodies, and the shift remains no change. Following the immobilization of anti-hIgG antibodies, the sensor was measured to verify that immobilized antibodies were functional and the sensor was responsive to IgG antigen. As expected, Fig. 6 showed that specific recognition of
Fig. 4. (a) Transmission spectra when injecting different solutions. (b) Measured wavelength shift of the double-microring sensor when injecting different solutions (※ indicating DI water. Standard concentrations of the proteins were prepared from the stock solution by serial dilutions in 10 mM PBS).
anti-hIgG functionalized surface only happened for a 0.5 mg/mL solution of hIgG, but not for the 0.1 mg/mL solution of casein. 4.3. Quantitative detection of human IgG To make quantitative measurements of human IgG, the chip was functionalized with anti-hIgG antibodies as described above. To demonstrate the dynamic range for the biosensor, solutions of human IgG were sequentially flowed over the surface at concentrations of 12.5, 25, 50, 100, 125, 250, 500 μg/ml, respectively, and the shift of resonance wavelength was monitored in real time.
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Fig. 5. Sample treatment procedure. (A) Silicon surface of microring sensors prior to modification. (B) APTES reacts with the surface siloxane groups to generate an aminoterminated surface. (C) Glutaraldehyde reacts with primary amines to cross-linking. (D) Streptavidin cross link to amines. (E) BSA close nonspecific binding sites. (F) AntihIgG/bio reacts with streptavidin. (G) Addition of hIgG results in antigen recognition.
Fig. 6. The resonance wavelength shift when measuring the binding capacity of the antibody on the surface and specific recognition (*buffer).
At each concentration, the solution of human IgG was flowed for 20 min of binding followed by a quick (5 min) rinse with PBS to remove any nonbinding antigen. Fig. 7(a) shows a response from cascaded double-microring resonators during the entire
concentration exposure series. The detection resolution of the readout system must be taken into account when calculating the detection limit for the cascaded double-microring resonators-based sensor. To monitor resonance wavelength, the readout system used in the present study had a low detection resolution of R¼0.47 nm. From Fig. 7(b), the sensitivity S ¼0.0668 nm/μg ml 1 and detection limit L¼R/ S¼ 7.1 μg/mL were obtained. The concentration of antigen was measured to be 7.1–125 μg/mL in the linear detection range. To further increase the sensitivity of the cascaded double-microring resonator sensors, we can change the operating mode to TM (transverse magnetic) mode. The SOI waveguides have high refractive index contrast and thus most of the electronic field is distributed in the cladding of the waveguide. As a result, the electronic field E of the TM mode of the strip waveguide could be enhanced by the large discontinuity of field at the core/cladding vertical interface, as the normal component of D (where D ¼ εE, ε is the dielectric constant of the material) is continuous across the boundary according to the electromagnetic boundary condition. On the contrary, the electronic field of TE mode of the ridge waveguide is confined in the core of the waveguide. As a result, TE mode used for sensing has lower sensitivity compared to TM mode, due to the smaller mode-field overlap with the analyte [15]. Therefore, the sensitivity could be greatly improved by operating at TM mode. We can incorporate stepper or electron beam lithography to fabricate deep etched narrow width waveguide with low
Fig. 7. (a) The resonance wavelength shift of different concentrations of hIgG (the concentrations of 12.5, 25, 50, 100, 125, 250 and 500 μg/ml of PBS buffer). (b) Calibration curves for hIgG determination.
Y. Chen et al. / Optics Communications 344 (2015) 129–133
propagating loss in TM mode. Currently, the waveguides were designed with 1 μm width and 30 nm shallow etched depth considering the resolution limitation of conventional photolithography and single mode condition. This led to high lateral leakage loss for TM mode [26]. Sensitivity of the cascaded doublemicroring resonator sensors can be further improved by utilizing stepper or electron beam lithography to fabricate deep etched narrow width waveguide to reduce propagating loss in TM mode.
5. Conclusion In this paper, we reported the design, fabrication, and characterization of a highly-sensitive optical biosensor based on cascaded double-microring resonators integrated with microfluidic channels. The optical chip was modified by a layer of molecules using the standard silicon surface functionalization procedure. Successful detection of human IgG demonstrated the biosensor to be of great potential in portable, multiplexed biosensing devices. These devices suggest promising applications for label-free detection and quantitative measurements of various biologically relevant molecules. Further integration of light source and photodiodes on the SOI chip would open up new perspectives to make portable devices for on-site testing.
Acknowledgments This work was supported by National High Technology Research and Development Program of China ( No. 2014AA06A504 ), Science and Technology Department of Zhejiang Province(No. 2014C31030), Fundamental Research Funds for Central Universities (No. 2014QNA5018), National Natural Science Foundation of China (Grant no. 61307073) and by Samsung Electronics Co. Ltd..
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Contents lists available at ScienceDirect
Optics Communications journal homepage: www.elsevier.com/locate/optcom
Label-free biosensing using cascaded double-microring resonators integrated with microfluidic channels Yangqing Chen, Fang Yu, Chang Yang, Jinyan Song, Longhua Tang, Mingyu Li n, Jian-Jun He State Key Laboratory of Modern Optical Instrumentations, Department of Optical Engineering, Zhejiang University, Hangzhou 310027, China
art ic l e i nf o
a b s t r a c t
Article history: Received 14 September 2014 Received in revised form 19 November 2014 Accepted 8 January 2015 Available online 9 January 2015
Fast and accurate quantitative measurement of biologically relevant molecules has been demonstrated for medical diagnostics and drug applications in photonic integrated circuits. Herein, we reported a highly-sensitive optical biosensor based on cascaded double-microring resonators. The sensor was integrated with microfluidic channels and investigated with its label-free detection capability. With a wavelength resolution of 0.47 nm, the measured binding capacity of the antibody on the surface exhibits reliable detection limit down to 7.10 μg/mL using human immunoglobulin G (hIgG). & 2015 Elsevier B.V. All rights reserved.
Keywords: Cascaded double-microring resonators Biosensor Label-free
1. Introduction Integrated optical waveguide biosensors offer the promise of fast and accurate solutions for detection and analysis in biomedical diagnostics, environment monitoring and food safety applications. Great attention has been attracted owing to their superior advantages such as immune to electromagnetic interference, label free detection and integration of multifunctional devices on one chip [1,2]. Recently, special interest was focused on silicon-oninsulator (SOI) sensor based on evanescent field. With its natural benefit of high sensitivity, ultra-compactness and manufacture compatibility with the standard complementary metal–oxide– semiconductor (CMOS) process [3,4], advanced SOI sensors were able to be fabricated with low cost and mature industrial infrastructure [5]. Under this category, many relevant results have been reported, including surface plasmon resonance(SPR) [6], photonic crystals [7], Mach–Zehnder interferometer [8], micro-disk [9] and microring resonator [10]. Among various types of optical sensors, microring resonators have been regarded as a promising solution for biomolecular recognition and chemical analysis [11,12]. The sensing light is coupled into the resonators under resonant condition and confined near the waveguide surface of the resonators with an evanescent field exponentially decaying into the surrounding medium. The propagation of light in the resonator is thus influenced by the refractive index (RI) of the reagent contact to the waveguide surface. Compared with Mach–Zehnder interferometer, the degree of n
Corresponding author. E-mail address: [email protected] (M. Li).
http://dx.doi.org/10.1016/j.optcom.2015.01.028 0030-4018/& 2015 Elsevier B.V. All rights reserved.
interaction of light in microring resonators is determined by the amount of rotations of light, and not restricted by the physical length [13]. The high-Q microring resonator can thus provide a higher sensitivity due to the sharper resonance peak. However, the interrogation of high-Q optical microring resonator requires a narrow line width tunable laser or a high-resolution optical spectrum analyzer. These equipments are very expensive and cannot be integrated into one chip. To address these problems, our group has previously proposed and demonstrated a highly sensitive optical sensor based on cascaded double-microring resonators with Vernier effect [14–17]. The method was then adopted by several other research groups and respectable results have been achieved [18–20]. We have previously reported a double-microring resonator based sensor exhibiting sensitivity up to 24,300 nm per refractive index unit (RIU) using different concentration solutions of NaCl [17], far surpassing the sensitivity of 135 nm/RIU for a SOI single ring resonator [21] and 12.7 nm/RIU for the polymer dual ring resonators [22]. To detect streptavidin, the cascaded double-microring resonators were further made biotin film immobilized on the surface [23]. However, this type of sensor cannot be applied for kinetic measurement of molecular interaction as it is not integrated with microfluidic channels. In this paper, we further demonstrate the label-free detection of cascaded double-ring sensor in biosensing by measuring the refraction index change induced by the biorecognition molecular interaction. The sensor was fabricated by conventional photolithography and integrated with microfluidic channels to detect the concentration of human immunoglobulin G (IgG). By using the standard silicon surface functionalization procedure, the anti-human IgG is immobilized on the surface of the sensing area in the
130
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optical waveguide sensor. The measuring results demonstrate this cascaded double-microring resonator to be a promising real time sensor of measuring the molecules binding kinetics for biomedical and chemical applications.
2. Device fabrication The sensor includes two layers: the integrated optical waveguide structure layer and the microfluidic system layer. The optical waveguide structure based on SOI substrate with a 220 nm-thick top silicon layer and 2 μm-thick buried oxide layer is composed of an input waveguide, a reference ring and a sensing ring cascaded by a common bus waveguide, and an output waveguide, as shown in Fig. 1(a). The optical waveguide wafer is covered by SU-8 upper cladding layer, while the sensing ring is exposed to the analyzed sample in the sensing window. The diameters of the reference and sensing ring of the sensor are 230 μm and 252.2 μm, respectively and directional couplers of 50 μm length with a gap of 1 μm are employed in all the ring resonators. All the ridge waveguides are designed with 30 nm shallow etched depth and 1 μm width enabling traditional contact photolithography patterning with single mode operation. The fabrication started by spin-coating a thin film of photoresist on the SOI wafer. After conventional photolithography, the pattern of the photoresist was transferred to the SOI wafer by inductively coupled plasma reactive ion etching. The wafer was then spin coated with SU-8 as the upper cladding of the optical waveguide, and sensing window was opened by photolithography. Improved performance could be further realized with smaller patterns by employing a stepper or electronic beam lithography. More details of the operational principle and fabrication process of cascaded double-rings sensor were described in the reference [24]. The microfluidic channels are fabricated with polydimethylsiloxane (PDMS) [13]. The PDMS (Sylgard 184, DowCorning, USA) is degassed to remove air bubbles using vacuum pump. Then the PDMS is casted on the master to a thickness of 2 mm, and cured at 85 °C for 30 min in an oven. The fluidic structure is then peeled off from the master and cut out, and holes are punched for liquid input and output ports. The optical sensor is integrated into a chip by bonding with the microfluidic channels using epoxy glue, as shown in Fig. 1(b). The microfluidic system can control and manipulate small volumes of liquid samples or reagents required for testing and analysis quickly and reliably.
3. Measurement setup The sensor was tested under the end-fire system. As shown in Fig. 2, the measurement setup mainly includes two parts: the controllers of liquids and the controllers of optics. Liquid flow is
Fig. 2. The end-fire system. Liquid flow is maintained by syringe pumps. Laser wavelength tuning and signal read out are automated by computer control. (1) Input fiber; (2) fiber polarization controller; (3) sensor; (4) output fiber; (5, 6, 7) three-dimensional nanopositioning stages; (8) pipe for vacuum pump; (9) pipe for sample input; (10) pipe for sample output; and (11) waste reservoir.
controlled by syringe pumps (MICROINFUSION PUMP WZS-50F6), which supply a continuous flow of buffer or samples through the microfluidic channels passing by the sensing window and then to the waste solution bottle. The transverse electric (TE) mode light from the tunable light source (Agilent 81600B) is coupled into input waveguide of the sensor by the polarization maintaining lensed fibers. The output light is collected by the power meter (Agilent 81635A). For the optical measurement system, three three-dimensional nanopositioning stages (Thorlabs MAX312) are employed to accurately place the optical input fiber, output fiber and the chip. In addition, an infrared camera is used to assist the fiber alignment with the input waveguide. A fiber polarization controller is applied to adjust the TE polarization with a polarization filter. The whole measurement setup is automated by the computer.
4. Experimental results and discussions 4.1. Volume sensing Volume sensing experiment is performed to prove the cascaded double-microring resonators sensing ability and eliminate the influence of background refractive index, where non-functionalized sensor was used. The optical sensitivity of the sensor was calibrated by using aqueous solutions of NaCl with different concentrations (0%, 2%, 4%, 6%, 8%). As shown in Fig. 3, the measured refractive index sensitivity is 1804 nm/RIU. Different solutions of proteins were prepared by serial dilutions in 10 mM PBS and injected to the microfluidic channel. The transmission spectrum and real time resonance wavelength of the cascaded double-microring resonators are shown in Fig. 4(a) and (b), respectively. From Fig. 4
Fig. 1. (a) Optical microscope image of the cascaded double-microring. The reference ring and the waveguide are covered with an upper cladding layer SU-8 while the sensing ring is exposed to the air. (b) Photograph of one of the fabricated sensor. The steel tubes make fluidic flow to the microfluidic network below.
Y. Chen et al. / Optics Communications 344 (2015) 129–133
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Fig. 3. Measured wavelength shift versus refractive index change of aqueous solutions of NaCl with different concentrations.
(a), a remarkable resonance wavelength shift can be clearly observed when changing the concentration of PBS from 10 mM to 100 mM, induced by the change of refractive index. The shift of the resonance wavelengths returns to baseline as the flow returns to DI water. During the injection of different protein solutions, the resonance wavelength remains the same with constant injection of 10 mM PBS, indicating that the influence of background refractive index could be ignored during the detection of biomolecule. 4.2. Surface derivatization and antigen recognition To remove potential residual organic contaminants introduced from fabrication, the wafer is first cleaned with a piranha solution (1:1H2SO4: 30% H2O2), followed by copious rinsing with water, isopropanol and dried by nitrogen. As illustrated by the schematic in Fig. 5, the chip surface is subsequently modified using standard silanization chemistry [25], and then exposed to 2.5% solution of APTES (1:100 deionized water/ ethanol) for 12 h at 4 °C. After silanization, the wafer is rinsed with ethanol and dried by nitrogen. Covalent attachment of biomolecules to the wafer surface is achieved using glutaraldehyde cross-linking. The silanized surface is immersed in 5% solution of glutaraldehyde for 1 h, and cleaned with 10 mM PBS using centrifugal separation to remove excess cross-linking agent. The chip is then exposed to 0.1 mg/mL solution of streptavidin for 1 h and 0.1 mg/mL solution of BSA for 1 h to close nonspecific binding sites. The functionalized surface is next exposed to solution of biotinylated antibodies (usually 0.05 mg/mL or higher) for at least 30 min to maximize the number of covalently immobilized antibody. For human IgG detection experiments, PBS (10 mM, PH:7.4) is used as buffer. The solution of human IgG is made by serial dilution in PBS, which then flows through the microfluidic channel and over the sensor surface at a rate of 25 μL/min. Each reaction could be monitored owing to the refractive index change induced by covalently attaching organic molecules. A large resonance wavelength shift of 4.16 nm was induced by the addition of anti-IgG antibodies to the functionalized surface, as shown in the real time data in Fig. 6. A rinse with PBS helps remove any non-covalently bound antibodies, and the shift remains no change. Following the immobilization of anti-hIgG antibodies, the sensor was measured to verify that immobilized antibodies were functional and the sensor was responsive to IgG antigen. As expected, Fig. 6 showed that specific recognition of
Fig. 4. (a) Transmission spectra when injecting different solutions. (b) Measured wavelength shift of the double-microring sensor when injecting different solutions (※ indicating DI water. Standard concentrations of the proteins were prepared from the stock solution by serial dilutions in 10 mM PBS).
anti-hIgG functionalized surface only happened for a 0.5 mg/mL solution of hIgG, but not for the 0.1 mg/mL solution of casein. 4.3. Quantitative detection of human IgG To make quantitative measurements of human IgG, the chip was functionalized with anti-hIgG antibodies as described above. To demonstrate the dynamic range for the biosensor, solutions of human IgG were sequentially flowed over the surface at concentrations of 12.5, 25, 50, 100, 125, 250, 500 μg/ml, respectively, and the shift of resonance wavelength was monitored in real time.
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Fig. 5. Sample treatment procedure. (A) Silicon surface of microring sensors prior to modification. (B) APTES reacts with the surface siloxane groups to generate an aminoterminated surface. (C) Glutaraldehyde reacts with primary amines to cross-linking. (D) Streptavidin cross link to amines. (E) BSA close nonspecific binding sites. (F) AntihIgG/bio reacts with streptavidin. (G) Addition of hIgG results in antigen recognition.
Fig. 6. The resonance wavelength shift when measuring the binding capacity of the antibody on the surface and specific recognition (*buffer).
At each concentration, the solution of human IgG was flowed for 20 min of binding followed by a quick (5 min) rinse with PBS to remove any nonbinding antigen. Fig. 7(a) shows a response from cascaded double-microring resonators during the entire
concentration exposure series. The detection resolution of the readout system must be taken into account when calculating the detection limit for the cascaded double-microring resonators-based sensor. To monitor resonance wavelength, the readout system used in the present study had a low detection resolution of R¼0.47 nm. From Fig. 7(b), the sensitivity S ¼0.0668 nm/μg ml 1 and detection limit L¼R/ S¼ 7.1 μg/mL were obtained. The concentration of antigen was measured to be 7.1–125 μg/mL in the linear detection range. To further increase the sensitivity of the cascaded double-microring resonator sensors, we can change the operating mode to TM (transverse magnetic) mode. The SOI waveguides have high refractive index contrast and thus most of the electronic field is distributed in the cladding of the waveguide. As a result, the electronic field E of the TM mode of the strip waveguide could be enhanced by the large discontinuity of field at the core/cladding vertical interface, as the normal component of D (where D ¼ εE, ε is the dielectric constant of the material) is continuous across the boundary according to the electromagnetic boundary condition. On the contrary, the electronic field of TE mode of the ridge waveguide is confined in the core of the waveguide. As a result, TE mode used for sensing has lower sensitivity compared to TM mode, due to the smaller mode-field overlap with the analyte [15]. Therefore, the sensitivity could be greatly improved by operating at TM mode. We can incorporate stepper or electron beam lithography to fabricate deep etched narrow width waveguide with low
Fig. 7. (a) The resonance wavelength shift of different concentrations of hIgG (the concentrations of 12.5, 25, 50, 100, 125, 250 and 500 μg/ml of PBS buffer). (b) Calibration curves for hIgG determination.
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propagating loss in TM mode. Currently, the waveguides were designed with 1 μm width and 30 nm shallow etched depth considering the resolution limitation of conventional photolithography and single mode condition. This led to high lateral leakage loss for TM mode [26]. Sensitivity of the cascaded doublemicroring resonator sensors can be further improved by utilizing stepper or electron beam lithography to fabricate deep etched narrow width waveguide to reduce propagating loss in TM mode.
5. Conclusion In this paper, we reported the design, fabrication, and characterization of a highly-sensitive optical biosensor based on cascaded double-microring resonators integrated with microfluidic channels. The optical chip was modified by a layer of molecules using the standard silicon surface functionalization procedure. Successful detection of human IgG demonstrated the biosensor to be of great potential in portable, multiplexed biosensing devices. These devices suggest promising applications for label-free detection and quantitative measurements of various biologically relevant molecules. Further integration of light source and photodiodes on the SOI chip would open up new perspectives to make portable devices for on-site testing.
Acknowledgments This work was supported by National High Technology Research and Development Program of China ( No. 2014AA06A504 ), Science and Technology Department of Zhejiang Province(No. 2014C31030), Fundamental Research Funds for Central Universities (No. 2014QNA5018), National Natural Science Foundation of China (Grant no. 61307073) and by Samsung Electronics Co. Ltd..
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