In situ bio-sensing system based on microwave photonics filter (MPF) interrogation method with improved resolution is proposed and experimentally demonstrated. A microfiber Bragg grating (mFBG) is used as sensing probe for DNA hybridization detection. Different from the traditional wavelength monitoring technique, we use the frequency interrogation scheme for resolution-improved bio-sensing detection. Experimental results show that the frequency shift of MPF notch presents a linear response to the surrounding refractive index (SRI) change over the range of 1.33 to 1.38, with a SRI resolution up to 2.6 × 10−5 RIU, which has been increased for almost two orders of magnitude compared with the traditional fundamental mode monitoring technique (~3.6 × 10−3 RIU). Due to the high Q value (about 27), the whole process of DNA hybridization can be in situ monitored. The proposed MPF-based bio-sensing system provides a new interrogation method over the frequency domain with improved sensing resolution and rapid interrogation rate for biochemical and environmental measurement.
© 2015 Optical Society of America
The DNA hybridization detection has found many applications in biotechnology, genetics, medical diagnostics, pathogen detection and drug research [1–6]. Fiber-optic sensors offer many advantages due to their high sensitivity, compact size, immunity to electromagnetic interference and possibility of distributed measurements. Traditional methods of DNA detection introduce the labeling of fluorescence to signal the binding event, which makes the process expensive and time consuming, and it is hard to achieve the in situ monitoring of the whole process of DNA hybridization. Nowadays the label-free DNA detection by measuring the change of surrounding refractive index (SRI) due to the molecular interactions on fiber surface has been attracting much attention. To achieve precise detection of the DNA hybridization interaction, optical fiber sensors need to achieve high SRI sensitivity. Traditional ways include using etched fiber Bragg grating , long period fiber gratings [8–10], tilted fiber Bragg gratings , Fabry-Perot interferometry  and fiber taper technology [13–15]. Recently, microfiber Bragg grating (mFBG) has been proposed and rapidly developed in SRI measurement . It provides strong evanescent fields out of fiber cladding and extremely decreases the sensor device in size. In 2014, D. D. Sun et al proposed an in situ DNA hybridization detection biosensor with a reflective mFBG at concentration of 0.5 μM of ssDNA target solution . All above reported optical fiber bio-sensor is based on wavelength monitoring (part of them converted the wavelength shift to intensity variation), which means that an expensive optical spectrum analyzer (OSA) is necessary.
With the development of photonic technology, the realization of microwave photonic signal processing has become more and more convenient and effective. Microwave photonic filter (MPF) is a powerful technique to process microwave signals directly in the optical domain [18–20]. Besides the well known radio over fiber system and military phased array radar system domain, MPF has a potential application in radio astronomy, Terahertz wave technologies and high accuracy sensing system. Owing to its fast signal processing speed, MPF has been attracted much research attentions in the applications of fiber optic sensing system. However, reported achievements in sensing field are almost focused in the measurement of conventional physical parameters such as stress, temperature, acceleration and vibration [21–23]. Designing novel biomedical sensors (and system) with better quality and lower cost encourages further research in this field.
In this paper, we propose and experimentally demonstrate a resolution-improved DNA hybridization sensing system based on a new interrogation method over the frequency domain together with a surface functionalized mFBG. The mFBG not only works as the sensing probe but also performs the function of wavelength selecting component of MPF system. The proposed DNA hybridization sensing system has the advantages of high sensing resolution and improved Q value compared to traditional wavelength interrogation schemes. The DNA hybridization was clearly monitored in situ for demonstration and its high specificity was also confirmed.
The sensing system is based on a typical 2-tap microwave photonic notch filter scheme. The optical source of the MPF consists of a probe light and a reference light. The probe light source was integrated with an mFBG that sensitive to SRI changes. The calibrated frequency response of the MPF sensing system can be expressed as
The free spectrum range (FSR) of the proposed MPF is
Taking the analyzer resolutions of wavelength demodulation ROSA (by OSA) and analyzer resolutions of frequency domain demodulation RVNA (by vector network analyzer, VNA) into consideration, the sensing resolutions of wavelength shifting monitoring (rλ) and n-th notch frequency shifting (rf) can be expressed as
Notice that the first term in (6) is about 10−2 magnitude order, the MPF-based sensing system has much higher sensing resolution than traditional wavelength demodulation owing to the huge difference between ROSA (109 Hz magnitude order) and RVNA (101~105 Hz magnitude order). The sensing resolution can also be adjusted by tuning reference wavelength and the length of time delay fiber. It should also be noticed that the utilization of higher ordinal notch can be more sensitive but also time-consuming due to the re-calibration step. Thus lower ordinal notches are good options in real-time observation of biomedical process when monitoring time interval should be as short as possible.
3. Experimental setup
Figure 1 shows the experimental setup of the proposed sensing interrogation scheme. In the optical source part of the MPF, we introduced an erbium-doped fiber ring laser (EDFL, as shown in the dot-line box) as the probe light. The mFBG was inserted into ring cavity by a circulator and functions as a narrow bandwidth optical filter. A polarization controller (PC, PC3) is used to optimize the polarization state. In order to increase the cavity length and to stabilize the output of the probe laser, a 660 m length of dispersion shifted fiber was placed inside the ring cavity. The longitudinal modes around the peak of mFBG’s reflective spectrum (fundamental mode peak) lased out eventually. The reference light generated by a tunable laser and the probe light from the EDFL were coupled to one route to provide the taps of the MPF.
In the MPF part, 80% of the optical source was used as carrier wave and other 20% part was directly sent to an OSA for optical spectrum monitoring. The radio frequency signal was modulated to the probe laser by an intensity modulator and another two PCs (PC1 and PC2) were used before the intensity modulator for optimum modulation efficiency. The modulated signals were launched into a 10-km length of single mode fiber acting as a wideband dispersive medium, and were finally detected by a photodetector (PD). The corresponding calibrated frequency response of the MPF was measured by the VNA.
The micro fiber we used is a commercial multimode fiber drawn and tapered with a butane flame brushing. The mFBG is inscribed by 193 nm excimer laser and phase mask method. The reflective spectrum (fundamental mode peak) of the mFBG in air and pure water are both shown in Fig. 2.
It should be noted that, we chose the EDFL instead of mFBG reflection spectrum filtering from a broadband source (BBS) as the optical source of the MPF for the reason that the latter has power ripple problem. Since the spectrum profile of BBS output is not flat, the probe light power will change when its wavelength shifts. The variation of SRI will also introduce the power descending of spectrum filtering. Moreover, the reflective spectrum may contain many side lobes for the non-uniform of mFBG diameter which will bring much noise to the MPF system. While the EDFL has a narrower bandwidth compared with the spectrum filtering of BBS and also free from the power fluctuation and side lobe induced noise.
4. Results and discussion
4.1. SRI sensing characters of MPF based bio-sensing system
The change of SRI is achieved by adjusting the concentration of sodium chloride solution. The SRI of the aqueous solutions ranges from 1.335 to 1.375. The wavelength of the reference laser was set at 1548.8 nm. The SRI sensing characteristics of probe laser monitoring and MPF-based sensing system are shown in Fig. 3.
The SRI sensitivity of the MPF system is 11.9 GHz/RIU. The comparison of SRI sensing resolution between probe laser monitoring and MPF sensing system is shown in Table 1.
The SRI resolution of the MPF sensing system is about two order of magnitude higher than that of probe laser monitoring method (by using the same sensor probe). The sensitivity and resolution of MPF sensing system can be further improved by measuring the frequency shift of higher order notch and utilizing VNA with higher detecting resolution respectively. The probe laser monitoring spectrum (when SRI is 1.3507, 1.3632 and 1.3751) and frequency responses of the MPF-based SRI system are shown in Fig. 4. The frequency response in full frequency range when SRI is 1.3751 is shown in the inset of Fig. 4(b).
The separation between EDFL spectrums under different SRI is not obvious while the separation of notches for frequency responses of the MPF is clearly shown in Fig. 4 (b). The comparison of Q value (defined as the ratio between resonance shift and its 3 dB bandwidth) for probe laser monitoring and MPF sensing system (from RI = 1.3632 to 1.3751) are shown in Tab.2.
As shown in Table 2, it is clearly that MPF sensing system has a much higher Q value (value of 27.6) than the laser spectrum monitoring method (value of 0.4). It should also be noted that for traditional spectrum monitoring method in which only the passive component spectrum are measured, the Q value is even lower than the probe laser monitoring method owing to a larger 3 dB bandwidth (as shown in Fig. 2).
4.2 DNA hybridization monitoring
The immobilization of probe single stranded DNA (ssDNA) was realized by covalent method (reported in  which has shown good reproducibility). Firstly we used the piranha solution to clean the surface of mFBG and add negative-charge to mFBG for half an hour. Then the mFBG was immerged into the poly-L-Lysine solution (PLL, 0.1% w/v in water) with positive charges. A monolayer of PLL with positive charges was immobilized onto the negatively charged fiber surface after an hour. Finally the probe ssDNA with negative-charged phosphate groups were immobilized on the surface of PLL. Between each step above, the mFBG was rinsed by deionized (DI) water. The ssDNA was dissolved with saline sodium citrate buffer and the concentration of probe ssDNA is 10 μM. The sequence of ssDNA is 5′-TCC AGA CAT GAT AAG ATA CAT TGA TG-3′. The MPF frequency response and the probe laser spectrum in DI water after immobilization were recorded.
The frequency shift of first notch was used to monitor the DNA hybridization process to achieve in situ observation, since counting the notch orders and then choosing the corresponding frequency span is time costing and makes the sensing process less efficient. Thus we chose the first notch as the sensing point. The wavelength of the reference laser was set at 1552.1 nm. During the DNA hybridization process, the mFBG was immerged into the target complementary ssDNA solution (5′-CA TCA ATG TAT CTT ATC ATG TCT GGA-3′) for an hour. Meanwhile, the non-complementary ssDNA (5′-C TCA CGT TAA TGC ATT TTG GTC-3′) has also been tested to evaluate the specificity of the proposed sensing system. The concentration of the target complementary ssDNA solution and the non-complementary ssDNA solution are the same (10 μM for both). As shown in Fig. 5, in situ DNA hybridization processes were monitored by traditional wavelength interrogation method (red curve) together with our proposed MPF-based frequency interrogation method (blue curve) under the same sensing condition (using the same mFBG sensing probe). The measurements were repeated three times from the piranha solution cleaning step with the same mFBG sensing probe.
As shown in Fig. 5(a), the probe laser monitoring method (red curve) can only distinguish the DNA hybridization process and the sensing wavelength vibrated irregularly during the DNA hybridization. It should be noted that the minimum OSA detecting resolution is 0.02 nm (OSA: YOKOGAWA AQ6370B) which is the same order of magnitude of the standard deviation of the monitoring curve (the error bar). Thus the vibration of DNA hybridization monitoring curve is due to the limited resolution of the OSA, and the error bar cannot indicate the reproducibility range of the wavelength interrogation method. The same variation trend occurs in the non-specificity testing group (as shown in Fig. 5(b)), which means that it also fails in the specific recognition of DNA hybridization.
For the same sensing condition, the MPF-based frequency modulation method shows two stages associated with the clearly discriminated hybridization reaction process within 55 minutes. The hybridization process includes a rapid reaction occurred in the first 10 minutes, showing a notch frequency shift rate of 3.3 × 107 Hz/min and a much slower reaction process with a rate of 3.5 × 106 Hz/min from 10 to 55 minutes as shown in Fig. 5(a). The VNA detecting resolution we used is 0.5 MHz (Agilent PN N5222A) which is much better than the standard deviation of monitoring curve. The error bar shows the reproducibility range of the interrogation sensing system which is primarily limited by the immobilization of the mFBG sensing probe. While in the non-specificity testing process (as shown in Fig. 5(b)), the MPF-based frequency modulation method shows a stationary process in contrast to the vibration trend of the probe laser monitoring methods. Thus high specificity DNA hybridization can be clearly confirmed by using MPF-based frequency of interrogation method.
To identify the existence of DNA hybridization over fiber surface, Fig. 6 presents the experimental spectrum of the sensor output before and after DNA hybridization, in which Fig. 6(a) is achieved by probe laser monitoring and Fig. 6(b) is got by MPF-based interrogation method, and all of them are detected in the same DI water. Clearly, the traditional wavelength monitoring method fails to discriminate the existence of hybridization with target ssDNA (the wavelength shift of probe laser is 0.02 nm equal to the minimum detecting resolution of OSA). While the proposed MPF-based interrogation method present an unambiguous difference over frequency domain. The notch frequency shift of MPF is 100 MHz which is higher than the detecting resolution of VNA (the minimum detecting resolution of VNA is 1 Hz, while we chose a resolution of 0.5 MHz in experiment to achieve high measuring efficiency). The separation between notches is obvious which means the MPF-based bio-sensing method has higher distinguish ability. Moreover, the detecting resolution of VNA could be further smaller to achieve higher sensing accuracy. Figure 7 shows the surface of the mFBG sensor probe after DNA hybridization with fluorescently labeled ssDNA (5′NH2 C6-CA TCA ATG TAT CTT ATC ATG TCT GGA-3′Cy3) by confocal microscopy with 543 nm laser source for the excitation of the Cy3 fluorophore. Thus the non-specific absorption effect can be removed and the DNA hybridization process is confirmed by traditional fluorescent label method.
Here the sample volume used in experiment is 1 mL. We used taper centrifuge tube and pipettor (Accuracy is 0.1 μL) to contain and measure the sample liquid. The system can be implemented on a microchip so that less sample volume is needed. Meanwhile, the mFBG can be reutilized after cleaning step by piranha solutions.
In conclusion, the feasibility of DNA hybridization sensing system with improved resolution based on MPF technique with a surface functionalized mFBG has been experimentally demonstrated. Compared with traditional spectrum monitoring methods, the MPF-based SRI sensing system provides an improved resolution of 2.6 × 10−5 RIU together with higher Q value. The surface functionalization of mFBG probe was achieved by a monolayer of PLL and the DNA hybridization reaction was in situ monitored unambiguously by MPF interrogation method with good specificity, making it a good potential for biochemical and environmental measurement.
This work was funded by the National Natural Science Foundation of China (No. 61475065 and No. 61205080), Guangdong Natural Science Foundation of China (No. 2015A030313322, No. 2014A030313387 and No. 2014A030310419).
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