A sensitive bio-probe to in situ detect unlabeled single-stranded DNA targets based on optical microfiber taper interferometer coated by a high ordered pore arrays conjugated polymer has been presented. The polymer coating serves as tentacles to catch single-stranded DNA molecules by π-π conjugated interaction and varies the surface refractive index of the optical microfiber. The microfiber taper interferometer translates the refractive index information into wavelength shift of the interference fringe. The sensor exhibits DNA concentration sensitivity of 2.393 nm/log M and the lowest detection ability of 10−10 M or even lower.
© 2015 Optical Society of America
The selective detection of single-stranded DNA in mixed solution (including double-stranded DNA) contributes to the screening of DNA lesions and variation in such as food safety testing, clinical diagnostics, and forensics . Various effects have been exerted in the development of biosensors for the detection of DNA [2–4 ].In most of these sensors, which are based on absorbance , reflectance , fluorescence [7,8 ], chemiluminescence , or bioluminescence , labels are usually necessary. However, label-free techniques are highly desired in analysis of medicine, environmental monitoring, and biochemical researchbecause they remove possible effects on the labels on the functions of target molecules .
As an excellent candidate for sensing, opticalmicrofiber’s miniature size and mechanical flexibility allow convenient integration with current medical tools for sensing in inaccessible locations, and multiple parameters of the its profile can be optimized for different applications. And based on the evanescent wave mechanism of optical fiber, its micro-scale diameter endows it with high refractive index (RI) sensitivity . With its RI sensitivity, optical microfiber could realize the measurement of biological molecules through its surface modification. The authors have previously demonstrated a microfiber Bragg grating biosensor detecting DNA target based on the surface functionalization of single-stranded DNA probe . Li and his associates realized the detection of alpha-fetoprotein by an optical microfiber functionalized by special anti-body .Recently, optical microfiber taper interferometer has attracted considerable attention due to its advantages such as compactness, easy implementation, high fringe visibility and robustness [2, 12, 15 ]. It has been gradually introduced in some measurement of biomoleculars based on its RI sensitivity. For example, based on the RI measurement of optical microfiber taper interferometer, Tian and associates realized the detection of Inmmune globulin G .
In this article, we present a sensitive biosensor to in situ detect unlabeled single-stranded DNA targets based on asilica optical microfibertaper interferometer coated by a high ordered pore arrays conjugated polymer membranes. The membrane serves as tentacles to catch single-stranded DNA molecules by π-π conjugated interaction. The target stacking on the taper surface varies the surface RI of the optical fiber and endows the fiber with DNA sensitivity. The proposed biosensor is with good linearity and high concentration-sensitivity of 2.393 nm/log M, yielding the lower detection limit of ≤ 10−10 M. The ability of these high-sensitive, ultrathin, mechanically compliant, biocompatible device affords minimally invasive in situ single-stranded DNA detecting and has potential in biomedical science and engineering.
2. Experimental details
2.1 Evanescent wave microfiber tapered interferometer
The taper structure fabrication was as previously described in . Briefly, a double cladded single mode optical fiber was tapered down to micron scale-diameterby using a flame-heated drawing technique.The flame with a width of 5 mm scanned across the optical fiber once accompanied by slowly stretching the optical fiber with two linear stages. The moving speeds of the flame and stages mainly determined the geometrical parameters including the diameter of the optical fiber and the length of the transition regions. A taper with a uniform region whose diameter and length were 7.5 μm and 1.4 cm, respectively, was fabricated to use in the DNA detection. The transition region was 0.4 cm in length. This geometry allows the coupling and recombination of modes in the microfiber, thus achieving an interferometer. When the fundamental core mode of the untapered fiber enters the downtaper region, it excites a fundamental mode and a higher order mode in the taper region. Although more than two modes may be excited, mode beating is mainly between the HE11 and HE12 modes because they have the similar azimuthal symmetry and the smallest phase mismatch. Normally, the external RI sensitivity of interferometer can be expressed by Formula (1) .12], so the term in the bracket is also negative. As a result, the transmission dips red shift with increasing RI. On the other side, both the dispersion parameter Γ and the dΔn/dnext are strongly dependent on the optical fiber diameter. By using thinner microfibers, the sensitivity can be greatly enhanced due to the stronger evanescent-field interaction and reduced dispersion factor.
2.2 Materials and reagents
All chemicals and solvents supplied by Aladdin were of analytical grade and were used without further purification. The conjugated polymer used in our work was synthesized via Pd-catalyzed Sonogashira reaction according to the work of Zhang et al . The conjugated polymer was dissolved in the mixed solvent with concentration of 10−4 g/ml including of tetrahydrofuran (THF)/toluene (v) = 1:1, and transferred onto SiO2 optical fiber sensor surfaces followed. The DNA oligonucleotides (ssDNA 5′ > CA TCA ATG TAT CTT ATC ATG TCT GGA <3′) and phosphate buffer saline (PBS, pH 7.4) solution were synthesized and purified by Sangon Inc. (Shanghai, China). The DNA solution was diluted by PBS solution to concentrations of 100 pM, 1 nM, 10 nM, 100 nM, and 1 μM, respectively.
Surface morphology of conjugated polymer on glass surface and polymer coated optical microfiber were observed by field emission scanning electron microscope (SEM) (ULTRA 55, ZEISS, BRUKER).
2.4 Immobilization of conjugated polymer membrane onto optical fiber sensor surface
The functionalization of optical microfiber by conjugated polymermembrane was performed as follows. The optical fiber was cleaned for 30 min in a bath with a piranha solution which consisting of 1 vol of 30% H2O2 and 3 vol of concentrated H2SO4 to generate reactive hydroxyl groups. The cleaned optical fiber was then immersed in the conjugated polymer mixed solvent solution for 30 min. Then, the polymer functionalized optical fiber was pulled out of the solution and dried in vacuum.
2.5 Experimental setup and optical configuration
The experimental setup permitted the sensor to operate in the transmission. During the experiments, the stability of sensor and biotesting was protected by designing and fabricating a microchannel chip. Each individual sensor was fixed in the microchannel (width 200 μm by height 150 μm) with help of UV-sensitive adhesive both sides over the sensing element of centimeter in length (for a total sensing volume of ~50 μL, taking into account the volume taken up by the optical fiber). The single-stranded DNA solutions were injected into the micro-fluidic chip via an electronic-controlled pump, eliminating the potential environmental influence during the bio-sample measurement. The sensing taper interferometer was excited by a broadband source (BBS) emitting light over the 1250-1650 nm range and its interference spectra were monitored by an optical spectrum analyzer (OSA) with minimum wavelength resolution of 0.02 nm.
3. Results and discussions
Figure 1 presents the SEM micrographs of the typical order membranes with irregular pore arrays on SiO2 substrate. The pores interconnected with each other by polymer paths forming a piece of membrane. Different dissolving capacities of polymer in THF and toluene may be responsible for the control of morphological changes in membrane organization . Solvent-mediated transformations between solid phases in a solvent could beproceed by the growth of precipitates of a stable phase as a metastable phase dissolves. In a lot of conditions, these metastable phases may develop more quickly than the stable phase . Due to the different polarities of THF and toluene, the conjugated polymer presented chain aggregations and induced local inhomogeneity in the mixed solvent solution, resulting in phase separation and formation of pore arrays membranes in the transition process of metastable phase precipitates in a saturated solution when drying .
The transmission of taper interferometerwas tested by the experimental setup illustrated in Fig. 2(a) . The sensing region of 0.4 cm in length was connected to the OSA and BBS by normal silica optical fiber. The scheme of cross section structure of sensing region for biomolecular sensing in an encapsulation is shown in the inset. The sensing region demonstratedthe cross section structure and the conjugated polymer membrane coated on the surface of the taper. A numerical simulation was performed by means of a full-vector finite-element method to calculate the mode characteristics as shown in Fig. 2(b). This model has a diameter ratio between the core and the cladding of 6.1 μm/125 μm, a core-cladding index contrast of 0.0125, a silica fiber diameter of 5.2 μm. The material dispersion of the fused silica in the cladding is taken into account . The interference fringe was red-shifted about 4.5 nm after the taper was coated with the conjugated polymer due to the increasing RI of the taper surface . And the calculation shows good agreement with the experimental values. Figure 2(c) testifies that the conjugated polymer membrane has successfully deposited on the taper interferometer surface. It was not free-standing but could be easily transferred on to silica optical fiber surface. The surface hydroxylation of optical fiber made the pores on the membrane smaller and more structured .The regular morphology offers an appropriate pore size for biomolecule detecting as well as a larger contact area. Figure 2(c) also presents that the diameter of sensing region of taper interferometer was~5 μm. And the SEM image of fiber cross section as shown in Fig. 2(d) illustrates the thickness coating is ~200 nm. The small size of taper allows more precise, cleverer in situ detection of biomoleculars to be performed.
In the deionized water without DNA, the taper sensor shows transmission peak centered by 1498.69 nm. When moved into the solution with 10−10 M DNA, a sharp red-shift of 7.24 nm in transmission peak was recorded, as shown in Fig. 3(a) . With DNA concentration increasing from 10−10 M to 10−6 M, the sensor showed regular red-shift in transmission wavelength (Fig. 3(b)). And dependence of transmission wavelength on the DNA concentrations is almost linearly, which a linear fitting of the data gives
It is clear that the concentration-dependence coefficient is 2.393 nm/logM. The detection limit is much lower than the former reports of 5*10−7 M based on the reflective microfiber Bragg grating , ~10−7M (100 ng/μL) based onphotonic crystal fiber long-period gratings , and 2*10−6 M based on fiber optic surface plasmon resonance biosensing  in the literature. After detecting the conjugated polymer membrane coating on the taper interferometer surface could be removed by a piranha solution in 30 min and recoated by the conjugated polymer after 30 min-immersing in the polymer solution. After that, the biosensor could be employed in a new DNA measurement process. This easily realizes the repeatability of the tapered microfiber. The linearity and repeatability offer a very convenient way to in situ measure the concentration of DNA, especially in the ultra-diluted concentration DNA detection.
To reveal why theas-prepared biosensor has high sensitivity to DNA concentration, the schematic of conjugated polymer tentacles get the single-stranded DNA on the sensor surface is shown in Fig. 4 . In the DNA solution the single-stranded DNA was in the dissociated state, and the concentration was uniform throughout the solution. After the conjugated polymer membrane coating on the taper surface, the single-stranded DNA attached onto the conjugated polymertentacle by the force of π-π stacking, generally. And the conjugated polymer membrane acts as the tentacles and catches the single-stranded DNA molecules. A similar interaction has happened between the single-stranded DNA and graphene oxide, basing on which the single-stranded DNA adsorbed onto the graphene oxide rapidly [22, 23 ]. Resulting, the DNA stacked on the surface of the biosensor, improving the RI increasement further. And the regular pore arrays on the conjugated polymer tentacle increase the contacting area between membrane and single-stranded DNA, and enhance the surface catching ability on the other hand. This surface aggregation aggrandizes the surface DNA concentration on the taper, resulting of the enhanced RI variation on the taper surface.
In all, the conjugated polymer membrane coating improves the DNA concentration sensitivity of the biosensor as tentacles: (i) increasing the contacting area between membrane and single-stranded DNA by the regular pore arrays; (ii) catching the single-stranded DNA on the sensor surface by π-π stacking. And the coating of conjugated polymer membrane enhances the evanescent field of the biosensor causing the optical fiber more sensitive to the change of surrounding RI. The stacking of DNA on the membrane surface increases the surface RI of the optical fiber.
A high-sensitivity DNA biosensor based on optical microfiber taper interferometer coated with conjugated polymer has been proposed, designed and demonstrated: The conjugated polymer membrane acting as tentacles, catches single-stranded DNA molecules and enlarges the surface RI increasementof the tapered optical fiber. And the taper interferometer gets the tiny RI change information and changed it into optical signals. With the combination of tentacle-like coating and the optical fiber taper interferometer structure, the biosensor demonstrates improved DNA concentration sensitivity of 2.393 nm/log M and good linearity, yielding the lower detection limit of ≤ 10−10 M. This high sensitivity and biocompatibility enable the biosensor in precision in situ DNA detection, even in ultra-diluted DNA solution. Above all, based on our work, coating the appropriate sensitive membrane to capture the target molecules on the surface of high RI sensitive optical taper, could convert the concentration information of target molecular to the RI variation, and further to light signals by the taper. This way provides a novel and convenient platform to detect the concentration of biomolecules and is potential for broad utility in biomedical analytical science and engineering.
This work was supported by the National Science Fund for Distinguished Young Scholars of China (No. 61225023), the National Natural Science Foundation of China (No. 51403077), the Research Fund for the Doctoral Program of Higher Education (No. 20114401110006), the Guangdong Natural Science Foundation (No. S2013030013302), the Planned Science and Technology Project of Guangzhou (No. 2012J5100028), and Fundamental Research Funds for the Central Universities (No. 21614317).
References and links
5. P. Lucas, M. A. Solis, D. L. Coq, C. Juncker, M. R. Riley, J. Collier, D. E. Boesewetter, C. Boussard-Pledel, and B. Bureau, “Infrared biosensors using hydrophobic chalcogenide fibers sensitized with live cells,” Sens. Actuat. Biol. Chem. 119(2), 355–362 (2006).
6. M. Watanabe and K. Kajikawa, “An optical fiber biosensor based on anomalous reflection of gold,” Sens. Actuat. Biol. Chem. 89(1–2), 126–130 (2003).
8. L. Polavarapu, J. Pérez-Juste, Q. Xu, and L. M. Liz-Marzán, “Optical sensing of biological, chemical and ionic species through aggregation of plasmonicnanoparticles,” J. Mater. Chem. C Mater. Opt. Electron. Devices 2(36), 7460–7747 (2014). [CrossRef]
9. M. Magrisso, O. Etzion, G. Pilch, A. Novodvoretz, G. Perez-Avraham, F. Schlaeffer, and R. Marks, “Fiber-optic biosensor to assess circulating phagocyte activity by chemiluminescence,” Biosens. Bioelectron. 21(7), 1210–1218 (2006). [CrossRef] [PubMed]
10. S. M. Gautier, L. J. Blum, and P. R. Coulet, “Fibre-optic biosensor based on luminescence and immobilized enzymes: microdetermination of sorbitol, ethanol and oxaloacetate,” J. Biolumin Esc. Chemiluminesce. 5(1), 57–63 (1990). [CrossRef]
11. S. Wang, X. Shan, U. Patel, X. Huang, J. Lu, J. Li, and N. Tao, “Label-free imaging, detection, and mass measurement of single viruses by surface plasmon resonance,” Proc. Natl. Acad. Sci. U.S.A. 107(37), 16028–16032 (2010). [CrossRef] [PubMed]
12. L. P. Sun, J. Li, Y. Tan, S. Gao, L. Jin, and B. O. Guan, “Bending effect on modal interference in a fiber taper and sensitivity enhancement for refractive index measurement,” Opt. Express 21(22), 26714–26720 (2013). [CrossRef] [PubMed]
13. D. Sun, T. Guo, Y. Ran, Y. Huang, and B.-O. Guan, “In-situ DNA hybridization detection with a reflective microfiber grating biosensor,” Biosens. Bioelectron. 61, 541–546 (2014). [CrossRef] [PubMed]
14. K. Li, G. Liu, Y. Wu, P. Hao, W. Zhou, and Z. Zhang, “Gold nanoparticle amplified optical microfiber evanescent wave absorption biosensor for cancer biomarker detection in serum,” Talanta 120, 419–424 (2014). [CrossRef] [PubMed]
15. Z. Tian, S. S.-H. Yam, and H.-P. Loock, “Refractive index sensor based on an abrupt taper Michelson interferometer in a single-mode fiber,” Opt. Lett. 33(10), 1105–1107 (2008). [CrossRef] [PubMed]
16. W. Zhang, C. Hu, Y. Huang, M. Zhang, H. Liang, and X. Chen, “Incorporation of light-emitting polymer into large cage-type mesoporous silica: toward new luminescent nanocomposites,” Acta Chim. 70(23), 2425–2432 (2012).
17. L. Chao, F. Chen, K. F. Jensen, and T. A. Hatton, “Two-dimensional solvent-mediated phase transformation in lipid membranes induced by sphingomyelinase,” Langmuir 27(16), 10050–10060 (2011). [CrossRef] [PubMed]
18. A. Putnis and C. V. J. Putnis, “The mechanism of reequilibration of solids in the presence of a fluid phase,” Solid State Chem. 180(5), 1783–1786 (2007). [CrossRef]
19. D. J. Sirbuly, N. O. Fischer, S. C. Huang, A. B. Artyukhin, J. B. Tok, O. Bakajin, and A. Noy, “Biofunctional subwavelength optical waveguides for biodetection,” ACS Nano 2(2), 255–262 (2008). [CrossRef] [PubMed]
20. L. Rindorf, J. B. Jensen, M. Dufva, L. H. Pedersen, P. E. Høiby, and O. Bang, “Photonic crystal fiber long-period gratings for biochemical sensing,” Opt. Express 14(18), 8224–8231 (2006). [CrossRef] [PubMed]
21. J. Pollet, F. Delport, K. P. F. Janssen, K. Jans, G. Maes, H. Pfeiffer, M. Wevers, and J. Lammertyn, “Fiber optic SPR biosensing of DNA hybridization and DNA-protein interactions,” Biosens. Bioelectron. 25(4), 864–869 (2009). [CrossRef] [PubMed]
22. Z. Tang, H. Wu, J. R. Cort, G. W. Buchko, Y. Zhang, Y. Shao, I. A. Aksay, J. Liu, and Y. Lin, “Constraint of DNA on functionalized graphene improves its biostability and specificity,” Small 6(11), 1205–1209 (2010). [CrossRef] [PubMed]