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Abstract
A new type of cascaded taper integrated ultra-long-period fiber grating (ULPFG) based immunobiologic sensor has been developed that benefits from the self-assembled monolayer of class I hydrophobin HGFI. Due to the cascaded arc, discharge tapers constitute an ultra-long-period and circular symmetrical refractive index modulation along fiber axial direction, and by local integration in one period, the mode coupling would generate to the higher harmonic of LP02, LP03 and LP04 modes in the wavelength range from 1300 nm to 1620 nm. The hydrophobic characteristic of the ULPFG surface is modified employing the HGFI, and the antibody molecule probes could be absorbed strongly on the HGFI nano-film, furthermore, the performances of immunobiologic sensing are investigated employing multiple control groups of matched and mismatched antigen molecule targets. The results show that it possesses higher sensing sensitivity of 4.5 nm/(µg/ml), faster response time about of 35 min, lower stability error of 8.8%, and excellent immuno-specificity. Moreover, this proposed ULPFG sensor has the advantages of low cost, simple fabrication and label-free, which is a powerful tool in the trace biomedical detection field.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Biological detection plays an important role in the areas of modern medicine and biological engineering [1,2]. The capable of specific determination of biological detection technology is essential for therapeutic treatments, and selective determination of certain biological molecules also can improve outcomes for patients [3]. Due to their desirable merits such as high geometric adaptability, anti-interference, reliable and in-situ measurements and resistance to high pressure and corrosion, the technology of fiber-optic biological detection has been widely investigated in the field of clinical diagnosis [4], environment protection [5], and food security [6]. To improve the interaction between light and matter, and avoid polluting biological materials, optofluidic integrated with optical fiber sense unit is a good alternative for biological detection and permits the realization of novel optical waveguides, devices, and sensors. Optical fiber grating is often used in biochemical sensing and detection because of its advantages such as simple fabrication, low insertion loss and easy integration [7–9], however, the onefold resonant wavelength limits the accuracy of sensing demodulation. The ultra-long-period fiber grating (ULPFG) possesses millimeter scale period refractive index modulation along fiber axial direction, causing the fundamental mode coupling to the co-propagating high order harmonic cladding modes, as a result, the transmission spectrum of ULPFG contains multiple wavelength resonances with multiple mode components [10], which makes it more tolerant to the demodulation of biological response signals, and can also weigh the response of each mode resonance to evaluate the error of biosensing.
The immobilization of bio-probe molecules on the optical fiber surface is a key step to enhance the biological detection efficiency. Nowadays, various methods of the fiber surface functionalization have been employed, such as electrostatic incorporation method utilizing Poly-L-Lysine (PLL) with the positive charges of amino group [11], physisorption method by gold nanoparticles [12], and covalent binding by silanization [13], etc. With unique molecular self-assemble characteristics, the hydrophobins have attracted considerable research interest as a functional material [14–16]. It not only possesses robust immobilization effect, but also keeps excellent biocompatibility [17,18]. Our previous works have proved that hydrophobin could self-assemble into nano films on the hydrophobic surface of optical fibers by hydrophobic force, and the hydrophilic groups of hydrophobin nano-film were capable to adsorb antibody molecules to form antibody-coated substrate [19,20].This property is very useful for immune-detection.
In this paper, a class I hydrophobin HGFI functionalized ULPFG sensor integrated by cascaded arc discharge tapers is designed for the immunobiologic specificity determination. Because of the long refractive index modulation of micro-taper in a period should not be considered as perturbation, the coupling modes in the wavelength range from 1300 nm to 1620 nm are analyzed by local integration method. The transmission spectrum of ULPFG consist of multiple high harmonic modes resonances such as 5LP02, 6LP02, 8LP03 and 9LP04 modes, which are tracked for monitoring adsorption and binding events in the bioreaction detection. Depending on the assistance of microfluidic units, the processes of self-assembly of HGFI, antibody coating on the HGFI nano-film, and matched or mismatched antigen targets absorption by the binding antibody are investigated experimentally in real time. Not only the sensor possesses higher sensing sensitivity and faster response time for antigen target detection, but also performs an excellent stability with the standard deviation error of 8.8% and high specificity for the immuno recognition between goat–anti-rabbit immunoglobulin G (IgG) (antibody GAR) and rabbit–anti-hemagglutinin IgG (antigen R). The proposed HGFI assisted ULPFG-based immunobiologic sensor is very suitable for immune detection.
2. Electric arc discharge-taper-based ULPFG and principle of operation
The cascaded micro-tapers constitute ultra-long periodic refractive index modulation along the fiber axial direction as presented in the Fig. 1, the micro-tapers are fabricated through electric arc discharging on single mode fiber (SMF) by using a commercial fusion splicer (FITEL, ver.2), as the micrograph shown each micro-taper range about 690µm in length, the tapering waist diameter is about 45.51µm, and the period Λ is 4000µm, which refers to the distance between adjacent taper waist. The configuration of ULPFG consists of five similar electric arc discharge tapers, and the total length of ULPFG is 16.69 mm.
The light propagating theory of ULPFG is similar to the LPFG. Due to the electric arc discharge taper is circular symmetrical structure, the fundamental mode could couple to the co-propagating multi-high-order circular symmetrical linear polarization modes and resonate at certain wavelengths. Unlike the method of UV-exposure and CO2 laser writing, the refractive index modulation of tapers based ULPFG could not be considered as perturbation. Therefore, the fiber diameter variation in tapering region should be considered for investigating the mechanism of coupling modes, the profile of fiber taper region could be expressed as the equation [21]:
In Eq. (1), R1=62.5µm and R10=22.754 µm, which refer to the cladding radiuses of the original fiber and taper waist, respectively. The length of taper region l is 690µm. The pulling speed of fabrication is v = 0.001k (µm/µs), k = 1,2,3…8. Because of the taper region is zygomorphic around the taper waist, only the fiber radiuses in half taper region are studied, which are plotted in the Fig. 2(a). It is obvious the taper evolution is determined by the pulling speed coefficient k, the higher the pulling speed, the steeper the taper area. Wherein the curve of k = 6 matches the boundary dimension of our electric arc discharge taper, benefiting from the curve of k = 6, the micro-taper region is partitioned equidistantly by 19 cross sections named symmetrically L1, L2, … L10, L9’, L8’ …L1’ respectively, which are indicated in the Fig. 2(b), L1 and L10 denote the cross sections of original fiber and taper waist severally. These gradual changes of fiber diameters in the taper region would result in dramatical variations of mode field boundary conditions, and changing the mode field distributions and modes effective refractive indexes finally. The coupled modes in the wavelength range from 1300 nm to 1620 nm were calculated by the phase matching condition, higher harmonic modes from LP02 to LP04 modes are coupled and resonant in this spectrum. The modes evolutions of LP01, LP02, LP03 and LP04 modes are calculated theoretically, the results at the wavelength of 1340 nm are listed in the bottom of Fig. 2(b), it presents that both the mode fields of fundamental mode and cladding modes spread outward obviously with the fiber thinning from the cross section L1 to L10, the attenuated constraint ability of guided light provides favorable conditions for the formation of evanescent field, which could improve the sensing sensitivity of external environmental matter detection.
Fig. 2. (a) Evolution of arc discharge-taper radius with pulling speed coefficient k in the half taper region; (b) The schematic diagram of equally partitioned micro-taper by 19 cross sections, the bottom is the mode fields of LP01, LP02, LP03 and LP04 modes according to the 10 cross sections in the half taper region at the wavelength of 1340 nm.
The Fig. 3(a), (b) and (c) present the changes of effective refractive indexes of LP01, LP02, LP03 and LP04 modes versus 19 cross sections at the wavelength of 1340 nm, 1470 nm, 1600 nm, respectively; the Fig. 3(d), (e) and (f) show the corresponding effective refractive index differences between the LP01 mode and the three cladding modes. It is obvious that both the modes effective refractive indexes and effective refractive index differences go through large fluctuations with the decrease of fiber diameter, especially the higher order cladding LP04 mode possess the maximal variations, which is sufficient to prove that the electric arc discharge taper cannot be regarded as perturbation, and the traditional coupled mode theory is no longer applicable for the micro-tapers-based ULPFG, the local integration in the taper region should be considered to analyze the mode coupling of the micro-tapers-based ULPFG. Therefore, the phase matching condition is expressed as:
In the length of range from z0 to z0+Λ, in accordance with the Eq. (2), the fundamental mode and cladding modes will be resonant coupled at a certain wavelength position. Due to the period of ULPFG reaches a length of few millimeters, high harmonics of cladding modes would be excited and coupled with fundamental mode [10], N is the number of harmonics. neff,co(z) and neff,cl(z) are the effective refractive index of fundamental mode and cladding modes respectively. According to the transmission spectrum observed by the spectrograph, the mode coupling could be confirmed in rough at the three wavelengths of 1340 nm, 1470 nm, 1600 nm, which is marked in the Fig. 4(a). The dip at 1340 nm may consist of the resonances of 8LP03 mode and 9LP04 mode; the dip at 1470 nm is constructed mainly by the resonance of 6LP02 mode; and the dip at 1600 nm is formatted mainly by the resonance of 5LP02 mode, respectively. Considering the effect of micro-tapers fabrication, the changes of mode effective refractive index are not linear in the taper region as shown in the Fig. 3, so it is possible that high-order harmonics of two similar cladding modes resonate at two similar wavelengths. In addition, due to the limitation of spectral resolution of OSA, if the wavelength positions of the two modes resonances are close each other, the resonance dips will overlap, therefore, it is possible that there are two cladding modes with different orders at the resonance dip at the central wavelength of 1340 nm. The frequency spectrum obtained by Fourier transform is offered in the Fig. 4(b), it is also proved that the transmission spectrum contains multiple mode components. In addition, multi-peaks and dips with lower contrast ratio are mingled in the transmission spectrum, that is because of the intermodal interference behavior possibly resulted by the non-uniformity of the period and the size of the arc tapers caused by the fabrication technique, the spectrum can be purified by improving the fabrication process to avoid the intermodal interference.Fig. 3. The changes of effective refractive indexes of LP01, LP02, LP03 and LP04 modes versus 19 cross sections at the wavelength of 1340 nm (a), 1470 nm (b) and 1600 nm (c), respectively; (d), (e) and (f) The corresponding effective refractive index differences between the LP01 mode and the three cladding modes.
Fig. 4. (a) Transmission spectrum of the cascaded micro-tapers-based ULPFG; (b) The corresponding spatial frequency spectrum.
3. Experimental setup and processes of immunobiologic sensing
The experimental setup of cascaded tapers-based ULPFG integrated bio-sensor is shown in Fig. 5. A super luminescent light emitting diode (SLED) broadband source covering a spectral range of 1250-1640 nm with a spectrum density of -30dBm/nm is utilized to provide the broadband incident light. The light propagating through the ULPFG carried external information is monitored by the optical spectrum analyzer (OSA: Yokogawa aq6370c) with a wavelength resolution of 0.1 nm. The area of ULPFG is encapsulated into a silica capillary with a diameter of 300µm and two micro-T-branch pipes with the diameter of 1.6 mm and length of 37.5 mm, two tiny rubber tubes connected with it serve as the inlet and outlet of liquid, all above components constitute a microfluidic cell, which provides a microfluidic channel for liquid loading. In addition, a syringe pump is used to inject biological liquids from the tube of inlet, and the outlet tube leads to the beaker for collecting waste liquids. All joints of the microfluidic cell are sealed with paraffin to keep samples tightly inside the capillary jacket.
Fig. 5. Experimental setup diagram of cascaded micro-tapers-based ULPFG integrated immunobiologic sensor.
The realization of immunobiologic sensing is benefited from the fiber surface modification of HGFI, the self-assemble of HGFI could form an amphipathic nano-film with thickness of 10 nm on the surface of optical fiber, which could modify the hydrophobicity of fiber surface into hydrophilicity by hydrophobic force and enhance the adsorption capacity and uniformity for the probe antibody molecules [18]. To demonstrate the effect of self-assembled monolayers of HGFI on fiber surface, the labeled HGFI with fluorescein isothiocyanate (FITC) is employed to immerse the optical fiber overnight, and the control experiment without HGFI is carried out synchronously, their fluorescence micrographs are offered contrastively in the Fig. 6, the FITC is bound uniformly on the surface of optical fiber as shown in the Fig. 6(a), but the fiber without HGFI displays little fluorescence as shown in the Fig. 6(b), which prove the hydrophobin HGFI is capable of coating a uniform nano-film on the surface of optical fiber.
The operational processes of fiber surface modification and immunobiologic sensing are diagramed in the Fig. 7. The HGFI dissolved in distilled water with concentration of 100µg/ml is firstly injected into the microfluidic cell. After a sufficient reaction by self-assembly of the HGFI molecules, a monolayer film is coated on the surface of micro-tapers-based ULPFG. The microfluidic channel is then rinsed repeatedly with deionized water to remove the dissociative HGFI molecules. Next, the probe antibody GAR IgG (produced by Bioss) dissolved in phosphate-buffered saline (PBS) solution (pH 7.4) with concentration of 4 µg/ml is pumped into the ULPFG integrated microfluidic channel for 40 min, due to the hydrophilic group of HGFI is exposed on the outer surface, the GAR molecules are immobilized on it by electrostatic incorporation. After washing by deionized water, the 5% Bovine Serum Albumin (BSA) blocking buffer is injected into the microfluidic channel for 1 hour to block the nonspecific binding sites of HGFI, which can avoid the nonspecific binding between the target antigen and the nano-film of HGFI. Through the above modification processes, the immobilized GAR is ready to be used for immunobiologic recognition. After the antigen R IgG (produced by Santa Cruz Biotechnology) matched with antibody GAR is injected into the microfluidic channel, the specific interaction between the immobilized antibody and antigen molecules performs quickly at the surface of ULPFG. This series of biomolecular reactions in the near field of ULPFG will cause the changes of optical parameters of evanescent field, the responses are monitored and recorded in the section 4.
Fig. 7. The processes diagram of HGFI self-assembly, antibody immobilization, and immunobiologic detection.
4. Results and discussion
Figure 8 shows the responses of ULPFG transmission spectrum versus different observation times during the biological reaction processes of optical fiber surface modification and immunological recognition. In order to obtain a high adsorptive optical fiber surface, the self-assemble process of HGFI on the surface of ULPFG maintain 15 min at first, due to the hydrophobic force, large number of HGFI molecules gather near the fiber surface, the external refractive index in the near field of the ULPFG increase accordingly, which result in the transmission spectrum gradually move toward the longer wavelength region. In our experiment, the resonant dip near 1550 nm is selected for analysis of self-assemble process of HGFI, it is found that the selected resonance dip exhibits a red wavelength shift of about 3.3 nm within 15 min, however, the wavelength shifts tend to flat after reacting over 10 min, which implies that the self-assemble process of HGFI molecules on the surface of ULPFG nearly becomes saturated.
Fig. 8. The joint resonance wavelength shifts as functions of observation time in the processes of HGFI, antibody GAR IgG, BSA blocking, and antigen R IgG.
The hydrophilic group of HGFI nano-film is exposed to the external environment, which is often used for antibody coating in the field of immunodetection. The probe antibody GAR molecules are absorbed nonspecifically on the nano-film of HGFI by electrostatic adsorption. The resonant dip at 1380 nm is selected to analyze the adsorption process of GAR, as the Fig. 8 shows the wavelength shift 2.3 nm in total within 40 min, and it still tends to red shift at the observation time of 40 min, that is, the absorption of antibodies did not reach saturation. Therefore, the 5% BSA blocking buffer is infilled in the microfluidic cell to block adequately the binding sites of HGFI nano-film within 1 hour, it is obvious that a wavelength shift of 1.84 nm generated after the GAR binding, the illusion of “adsorption saturation” on the surface of HGFI nano-film can avoid the absorption of antigen on it, thus reducing the error of immunodetection.
The specific matching between the binding antibody GAR and antigen R is investigated by injecting R/PBS solution with concentration of 1µg/ml (refractive index RI=1.3350) in the ULPFG-based immune-sensing region. Due to the blocking of BSA, the antigen R molecules cannot be absorbed by the HGFI nano-film indirectly, but rather be attracted by the immobilized GAR molecules. Large number of antigen R molecules cluster on the surface of ULPFG, the boundary conditions of micro-tapers are modified by the three molecular layers, which result in the changes of optical parameters in the evanescent field near the ULPFG surface, and the effective refractive index difference increases with the increase of specific adsorption between antigen and antibody molecules. Eventually the transmission spectrum come out a large shift towards to the longer wavelength direction. The Fig. 8 displays that the resonant dip at the wavelength about 1600 nm appear an obvious red shift of 4.6 nm within 90 min. Two other groups of specific antigen R detection with different concentration of 0.5µg/ml (RI=1.3346) and 0.1µg/ml (RI=1.3344) respectively are studied, as the Fig. 8 displays that the maximum wavelength shifts of transmission spectrum achieve 2 nm and 0.56 nm respectively. Base on the three groups responses of antigen R detection, the sensing sensitivity for the specific antigen R could be deduced as 4.5 nm/(µg/ml) (6733 nm/RIU), which is much higher than the pre-report [22] (3135 nm/RIU), report [23] (180 nm/RIU), and report [24] (722.3 nm/RIU). and considering that the resolution of the OSA is set at 0.1 nm, the detection limit of antigen R could be estimated to be 0.02µg/ml, which is 10 times lower than the report [25] (0.2 mg/L). therefore, the ULPFG-based sensor is a competitive tool in the field of immune sensor with the high sensing sensitivity and low detection limit.
In addition, in order to evaluate the stability and error of the global transmission spectrum to the immune-sensing, resonant dip A, B and C at the wavelength of 1379.3 nm, 1433.8 nm and 1596.3 nm respectively are extracted to analyze the laws of wavelength shift, and then calculating the wavelength shift errors of these three dips by standard deviation, which are shown in the Fig. 9(a), the three dips have wavelength shifts of 4.2 nm, 4 nm and 4.6 nm respectively, and the Fig. 9(b) presents the standard deviation of average spectrum response is 8.8% merely, which means that there is an error of about 0.4 nm in the maximum wavelength shifts of the transmission spectrum. The index proves that the difference of global spectral responses in the wavelength range from 1300 nm to 1620 nm is very small, and the ULPFG-based sensor has good stability and accuracy for the sensing of antigen R. Furthermore, the response time for antigen R detection is investigated, according to the average spectral response, when the wavelength shifts increase from 0 nm to 0.1 times of the maximum, the corresponding time is defined as the response time generally. The average wavelength shift of maximum for the antigen R with 1µg/ml is 4.26667 nm, the response time of the corresponding wavelength shift to 0.1 times the maximum value is 35 minutes, which possess of great value in clinical laboratory.
Fig. 9. (a) The resonance wavelength shifts of Dip A, Dip B and Dip C for the antigen R IgG detection within 90 min; (b) Evaluation of the stability errors of the mean response for antigen R detection.
The specificity is a necessary evaluation for biosensors. To evaluate the specificity of the proposed cascaded tapers-based ULPFG integrated microfluidic cell, three control groups experiments is conducted for nonspecific antigen detection. The mismatched secondary antibody Goat-Anti-Mouse (GAM) IgG (produced by TransGen Biotech), Mouse Anti-transforming-growth-factor-beta 1 (TGFB1) and Mouse IgG solution with the same concentration of 1µg/ml was injected into the ULPFG-based microfluidic cell individually, which has been immobilized the GAR IgG molecules by self-assemble of HGFI in advance. The spectral responses of nonspecific biological detection are obtained by monitoring respective temporal dependence of resonance wavelength shift with similar structures, as shown in Fig. 10. It could be seen that compared with the detection results of antigen R, there are all only slight fluctuation in the wavelength shifts for the three target analytes (antibody GAM, Mouse Anti-TGFB1 and Mouse IgG) during the same detection time of 90 mins, which are great contrasts with the response of matched antigen R detection. That is because of no biological reaction exists between the molecules of nonspecific targets and binding GAR IgG, the refractive index and molecular surface density change little, result in the little variation of transmission spectrum. At the same time, the effectiveness of GAR IgG adsorption and BSA blocking on the HGFI nano-film were also proved. These responses of control group demonstrate the ULPFG-based immunobiologic Sensor dependent on the self-assemble of HGFI possesses good specificity, which is suitable for biological detection areas.
Fig. 10. Temporal-dependent resonance wavelength shifts for nonspecific immune detection for (a) Antibody GAM, (b) Mouse Anti-TGFB1, and (c) Mouse IgG at the Dip A, Dip B and Dip C, respectively.
5. Conclusion
The immunobiologic sensing employing the self-assemble of hydrophobin HGFI has been investigated based on cascaded arc discharged tapers integrated ULPFG. The coupling modes have been theoretically analyzed in the wavelength range from 1300 nm to 1620 nm by the local coupled mode theory and integration of phase matching conditions, which shown a great correlation with the fiber size in the taper area. The responses of transmission spectrum to the ULPFG surface modification, immobilization of antibody GAR, detection of matched antigen R and mismatched antibody GAM, Mouse Anti-TGFB1 and Mouse IgG with the binging GAR were monitored and recorded experimentally. Which authenticated the sensor performing higher sensing sensitivity of 4.5 nm/(µg/ml), faster response time about of 35 min, lower stability error of 8.8%, and excellent immuno-specificity for the matched antigen R detection. In summary, the proposed ULPFG-based immunobiologic sensor features excellent advantages such as good stability, low error, high specificity, simple fabrication, and label-free, which is anticipated to be used as a powerful tool in biomedical detection field.
Funding
National Natural Science Foundation of China (11704283, 11804250, 11904180, 11904262, 61875091); Natural Science Foundation of Tianjin City (18JCQNJC71300); Tianjin Municipal Education Commission (2018KJ146); Opening Foundation of Tianjin Key Laboratory of Optoelectronic Detection Technology and Systems (2019LODTS004).
Acknowledgments
This work was jointly supported by the National Natural Science Foundation of China under Grants 11804250, 11904262, 61875091, 11904180 and 11704283, Tianjin Natural Science Foundation under Grant 18JCQNJC71300, Tianjin Education Commission Scientific Research Project under Grant 2018KJ146, and the opening foundation of Tianjin Key Laboratory of Optoelectronic Detection Technology and Systems No. 2019LODTS004. The authors thank the above Foundations for help identifying collaborators for this work.
Disclosures
The authors declare that there are no conflicts of interest related to this article.
Data availability
No data were generated or analyzed in the presented research.
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