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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editor: Gregory W. Faris
  • Vol. 3, Iss. 12 — Dec. 1, 2008
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Antibody immobilization within glass microstructured fibers: a route to sensitive and selective biosensors

Yinlan Ruan, Tze Cheung Foo, Stephen Warren-Smith, Peter Hoffmann, Roger C. Moore, Heike Ebendorff-Heidepriem, and Tanya M. Monro  »View Author Affiliations


Optics Express, Vol. 16, Issue 22, pp. 18514-18523 (2008)
http://dx.doi.org/10.1364/OE.16.018514


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Abstract

Glass microstructured optical fibers have been rendered biologically active for the first time via the immobilization of antibodies within the holes in the fiber cross-section. This has been done by introducing coating layers to the internal surfaces of soft glass fibers. The detection of proteins that bind to these antibodies has been demonstrated experimentally within this system via the use of fluorescence labeling. The approach combines the sensitivity resulting from the long interaction lengths of filled fibers with the selectivity provided by the use of antibodies.

© 2008 Optical Society of America

1. Introduction

The development of effective MOF-based biosensors requires: 1) a sensitive detection mechanism capable of measuring low-levels of biomolecules and 2) a means of selectively identifying specific biomolecules of interest. The first requirement can be realized by taking advantage of the long interaction lengths offered by the interaction between a guided mode in a fiber and the material of interest. It has been possible to detect proteins at the 1 nM level using soft glass MOFs [6

6. Y. Ruan, E. P. Schartner, H. Ebendorff-Heidepriem, P. Hoffmann, and T. M. Monro, “Detection of quantum-dot labelled proteins using soft glass microstructured optical fibers,” Opt. Express 15, 17819–17826 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-26-17819. [CrossRef] [PubMed]

].

The introduction of selectivity to an MOF-based sensor requires the functionalization of the otherwise inert fiber to allow its response to a biological species to be determined via optical measurements. In principle, this can either be done during the fiber fabrication process or via post-processing of the fiber.

One advantage of using polymer MOFs is that the surface can be chemically modified to allow biomolecules to be attached directly [1

1. J. B. Jensen, P. E. Hoiby, G. Emiliyanov, O. Bang, L. H. Pedersen, and A. Bjarklev, “Selective detection of antibodies in microstructured polymer optical fibers,” Opt. Express13, 5883–5889 (2005), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-13-15-5883. [CrossRef] [PubMed]

, 8

8. J. Cheng, C. Wei, K. Hsu, and T. Young, “Direct-write laser micromachining and universal surface modification of PMMA for device development,” Sens. Actuators B , 99, 186–196 (2003). [CrossRef]

, 9

9. G. Emiliyanov, J. B. Jensen, and O. Bang, “Localized biosensing with Topas microstructured polymer optical fiber,” Opt. Lett. 32, 460–462 (2007). [CrossRef] [PubMed]

]. The low glass transition temperature (e.g. ~90 °C for PMMA) makes polymer fibers impractical for use in high temperature environments or for high power light transmission. Although glass MOFs do not allow direct functionalization, their potential benefits for biosensing are great, since they offer access to particular spectral regions such as the UV-Vis (via high purity silica glass) and the mid-IR (via soft glasses such as tellurite, fluorides, and chalcogenides). Compared to polymer MOFs, glass MOFs have other advantages including lower loss, the potential for higher damage thresholds because of their higher glass transition temperatures, better cleaving, and easier integration with conventional fiber technologies. This motivates the investigation of approaches for functionalizing glass-air boundaries within glass MOFs.

For glass fibers, which have relatively high processing and fabrication temperatures, the most promising approach for incorporating biological functionality within the fiber is to deposit surface layers after the fiber has been drawn. This post-processing can be done from the end of the fiber or via side access holes. Side access holes can be produced by techniques such as drilling at the preform stage, or ion beam milling at the fiber stage [10

10. F. M. Cox, R. Lwin, M. C. J. Large, and C. M. B. Cordeiro, “Opening up optical fibres,” Opt. Express15, 11843–11848 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-19-11843. [CrossRef] [PubMed]

,11

11. C. M. B. Cordeiro, C. J. S. de Matos, E. M. dos Santos, A. Bozolan, J. S. K. Ong, T. Facincani, G. Chesini, A. R. Vaz, and C. H. B. Cruz, “Towards practical liquid and gas sensing with photonic crystal fibers: side access to the fibre microstructured and single-mode liquid-core fibre,” Meas. Sci. Technol. 18, 3075–3081 (2007). [CrossRef]

]. While side-access is attractive for distributed detection schemes, only limited lengths have been fabricated in glass fibres [11

11. C. M. B. Cordeiro, C. J. S. de Matos, E. M. dos Santos, A. Bozolan, J. S. K. Ong, T. Facincani, G. Chesini, A. R. Vaz, and C. H. B. Cruz, “Towards practical liquid and gas sensing with photonic crystal fibers: side access to the fibre microstructured and single-mode liquid-core fibre,” Meas. Sci. Technol. 18, 3075–3081 (2007). [CrossRef]

]. For the work presented here, we choose to explore the approach of functionalizing fibers from the fiber ends, which requires the development of techniques for depositing coatings on the internal surfaces of the holes within the cross-section and along the length of the fiber.

Recently, the internal surfaces of silica MOFs have been coated with strands of DNA [7

7. 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. Express14, 8224–8231 (2006), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-14-18-8224. [CrossRef] [PubMed]

]. This approach uses poly-L-lysine to immobilize negatively charged molecules such as DNA to a solid support. Poly-L-lysine has positively charged amino-groups that can bind to the negatively charged silica surface through an ionic binding [12

12. P. D. Sawant, G. S. Watson, S. Myhra, and D. V. Nicolau, “Hierarchy of DNA immobilization and hybridization on poly-L-lysine using an atomic force microscopy study,” J. Nanosci. Nanotechnol. 5, 951–957 (2005). [CrossRef] [PubMed]

]. An alternate approach is based on coating the internal surfaces of soft glass MOFs with nm-scale silane layers [13

13. J. Debs, H. Ebendorff-Heidepriem, J. Quinton, and T. M. Monro, “A Fundamental study into the surface functionalisation of soft glass microstructured optical fibres via silane coupling agents,” accepted for publication, J. Lightwave Technol. (2008).

]. A similar process has been used to coat the core of a photonic bandgap fiber with a silane coating [14

14. S. Gosh, A. R. Bhagwat, C. K. Renshaw, S. Goh, A. L. Gaeta, and B. J. Kirby “Low-light-level optical interactions with rubidium vapor in a photonic band-gap fiber,” Phys. Rev. Lett. 97, 023603 (2006). [CrossRef]

].

Here we demonstrate that it is possible to extend this technique to allow the immobilization of antibodies inside soft glass MOFs. Soft glasses have lower glass softening points than silica, enabling preform and jacket tube fabrication via extrusion through complex stainless steel dies [15

15. H. Ebendorff-Heidepriem and T. M. Monro, “Extrusion of complex preforms for microstructured optical fibers,” Opt. Express15, 15086–15092 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-23-15086. [CrossRef] [PubMed]

]. The extrusion technique allows a large flexibility in the preform and jacket tube geometries that can be achieved. This, in turn, has recently enabled the fabrication of fibers with nanoscale core sizes as small as 400 nm diameter (without requiring a postprocessing tapering step), which is of great advantage for sensing applications [16

16. H. Ebendorff-Heidepriem, S. C. Warren-Smith, and T. M. Monro, “Suspended nanowires: fabrication, design, and characterization of fibers with nanoscale cores,” submitted to Nature Photonics.

]. Furthermore, for nanoscale core sizes, the higher refractive indices of soft glasses compared with silica enables higher sensitivity due to enhanced fluorescence capture fraction [17

17. S. Afshar, V. S. C. Warren-Smith, and T. M. Monro, “Enhancement of fluorescence-based sensing using microstructured optical fibers,” Opt. Express 15, 17891–17901 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-26-17891. [CrossRef] [PubMed]

].

The immobilization of antibodies inside soft glass MOFs is based on adapting the established procedure used for the immobilization of antibodies onto glass slides [18

18. S. K. Bhatia, L. C. Shriver-Lake, K. J. Prior, J. H. Georger, J. M. Calvert, R. Bredehorst, and F. S. Ligler, “Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces,” Anal. BioChem , 178, 408–413 (1989). [CrossRef] [PubMed]

] to the internal holes within an MOF, and here we implement this procedure in non-silica glass fibers for the first time. Proof of concept of this approach was demonstrated in Ref. [19

19. T. M. Monro, Y. Ruan, H. Ebendoff-Heideprien, H. Foo, P. Hoffmann, and R. C. Moore, “Antibody immobilization with glass microstructured fibers: a route to sensitive and selective biosensor,” Proc. Internat. Soc. Opt. Engin. (SPIE) 17, 70046Q-1–4 (2008).

]. Here we present a detailed description of the experimental demonstration of the selective detection of immobilized proteins within a glass fiber. In Section 2, the immobilization processes and experimental results on bulk glasses are introduced. The way in which these processes can be applied to soft F2 glass MOF for biosensing is then described in Section 3.

2. Antibody immobilization on glass

To detect specific proteins, it is necessary to immobilize antibodies onto the glass surface that forms the fiber core in order to facilitate the selective binding of antigens to antibodies to occur in a region where the overlap with the guided mode of the fiber is strong. However, as mentioned above, antibodies cannot be attached directly to a glass surface. Bhatia et al. [18

18. S. K. Bhatia, L. C. Shriver-Lake, K. J. Prior, J. H. Georger, J. M. Calvert, R. Bredehorst, and F. S. Ligler, “Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces,” Anal. BioChem , 178, 408–413 (1989). [CrossRef] [PubMed]

] immobilized antibodies onto glass slides by first introducing silane and crosslinked layers. In brief, first a silane layer is attached to the glass surface, followed by attachment of a so-called crosslinker onto the silane layer. Finally, the antibodies are attached to the crosslinker [18

18. S. K. Bhatia, L. C. Shriver-Lake, K. J. Prior, J. H. Georger, J. M. Calvert, R. Bredehorst, and F. S. Ligler, “Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces,” Anal. BioChem , 178, 408–413 (1989). [CrossRef] [PubMed]

]. A solution containing a range of antigens that may be labeled with different dyes can then be introduced into the holes of the fiber by dipping the end of the fiber into the solution and allowing it to fill under capillary action. The antigens that correspond to the immobilized antibodies will bind to them, and any unmatched antigen can be flushed away.

2.1 Demonstration of immobilization on glass plates

This immobilization process was first tested on glass plates made from 3 different glass materials: conventional glass slides, F2 and LLF1 glass plates. F2 and LLF1 glasses are commercially available Schott glasses. To obtain a hydrophilic surface for silanization, the glass plates are first cleaned with a mixture of concentrated hydrochloric acid and methanol, and then treated with concentrated sulphuric acid. After cleaning, the oxide surfaces of the glass plates exhibit a relatively low water contact angle (that is, a more hydrophilic surface). After silanization, the contact angle of water with the glass plates is 52°, in agreement with literature [18

18. S. K. Bhatia, L. C. Shriver-Lake, K. J. Prior, J. H. Georger, J. M. Calvert, R. Bredehorst, and F. S. Ligler, “Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces,” Anal. BioChem , 178, 408–413 (1989). [CrossRef] [PubMed]

]. Similar results are obtained for the F2 and LLF1 plates. The silane and crosslinker layers were attached via immersion of the glass plates in the corresponding solutions. To reduce cost, in most of our experiments, a single drop (30 µL) of antibody/antigen solution was placed on the glass surface for incubation to achieve protein immobilization or binding.

To determine whether the antibodies were attached to the glasses, we labeled the antibodies with quantum dots, whose fluorescence was detected with a Typhoon imager. The imager is equipped with lasers at 488, 532, and 630 nm, and has filters to block the excitation light and improve the image contrast. We used two different quantum dots, which separately emit light at 800 nm (Qdot800) and at 705 nm (Qdot705).

Fig. 1. Images of the glass samples with immobilized Q800-labeled antibodies.

The first antibody sample used here is Qdot800 goat F(ab′)2 anti-mouse IgG conjungate. The maximum wavelength of the filters within the imager is 670 nm (30 nm bandwidth), which will block 800 nm emission from the Qdots used for antibody labeling [6

6. Y. Ruan, E. P. Schartner, H. Ebendorff-Heidepriem, P. Hoffmann, and T. M. Monro, “Detection of quantum-dot labelled proteins using soft glass microstructured optical fibers,” Opt. Express 15, 17819–17826 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-26-17819. [CrossRef] [PubMed]

]. Thus no filter was used for fluorescence measurement of the samples with Qdot800 labeled antibodies. A 532 nm laser was chosen as an excitation source. This selection was made as a result of a trade-off between the loss of the glass at the pump wavelength, the source availability, and the pump absorption. Figure 1 shows the images of the three glass plates with each treated via application of a 100 nM 30 µL antibody solution drop. The drop was firstly allowed to remain on the glass surface for one hour before the plate was rinsed using deionized water. The area the antibody drop covered appears dark in color and indicates strong fluorescence from Qdot800, demonstrating that the antibody has attached to the glass surfaces via silane and cross-linked layers. The contrast of the fluorescence signal between the droplet and the background regions is clear. The signal from the conventional slide is stronger than the other glasses, indicating higher attachment efficiency for the slides compared to the F2 and LLF1 glasses.

To further test the immobilization process, we used an antibody labeled with an alternative quantum dot (Qdot705 goat F(ab′)2 anti-mouse IgG conjungate) with an emission wavelength located at 705 nm. When the image of the glass slide immobilized with this conjungate was taken together with those of the F2 and LLF1 samples immobilized with the Qdot 800 labeled antibody (Fig. 1) using the 670 nm filter, only the glass slide presents the antibody drop image with the emissions from Qdot800 on the F2 and LLF1 glasses removed by the filter. This confirmed that all the drop images are produced by fluorescence light emitted by the Qdot-labeled antibodies.

To determine the quantity of immobilized antibody, the image of the drop with the same volume (30 µL) and concentration of the antibody on the glasses has been taken as a reference. Image analysis software (IMAGEQUANT SOLUTIONS) has been used for signal quantification. Figure 2 shows the image of the drop of 100 nM 30 µl Qdot800 goat F(ab′)2 anti-mouse IgG conjungate, which remained on the glass slide when the image was taken. The object and background are defined in Fig. 2 for fluorescence analysis. The total fluorescence of the image is obtained by integrating the signal corresponding to the object and subtracting the background. Table 1 summarizes the ratios of the total fluorescence of the images shown in Fig. 1 relative to that of the standard 100 nM 30 µL antibody drop shown in Fig. 2. Based on the concentration of the drop and their image areas, the density of the immobilized antibody can be extracted. As shown in Table 1, the relative immobilization efficiency was found to be: conventional glass slide > LLF1 glass > F2 glass.

Fig. 2. Fluorescence image of the standard 30 µL100 nM antibody drop on the glass slide. The definition of the object and background for fluorescence analysis is also displayed.

Table 1. Antibody immobilization for different glasses

table-icon
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2.2 Quantification of the binding efficiency of the antibody to the antigen

Fig. 3. Binding between antibody and antigen with only one of them labeled: (a) labeled antibody (Qdot800 goat F(ab′)2 anti-mouse IgG conjungate) bond to unlabeled antigen (Purified mouse IgG); (b) antigen (mouse anti-human Qdot705 conjungate) bond to unlabeled antibody (Goat anti-Fab2 anti-Mouse IgG (H+L)); (c) 30 µL 100 nM antigen solution (mouse anti-human Qdot705 conjungate) as a reference.

In any practical sensor, it is necessary to use specific antibodies to detect specific antigens within the sample. Thus for the second experiment, we reversed the sequence of the antibody and antigen. The unlabeled antibody (goat anti-Fab2 anti-Mouse IgG (H+L)) was first immobilized onto the treated LLF1 glass slide. A large volume (>130 µl) was used to allow immobilization to occur across the whole surface. A high concentration (330 nM) was used to increase the density of the immobilized antibody (and of the bound antigen). Indeed, a thick antibody layer was observed after immobilization (with a 100 nM antibody solution, the layer is not evident to the eye). Following a rinse using the buffer solution, a portion of the layer washed away, indicating weak attachment between the thick antibody layer and the crosslinked film. Applying a 30 µL 100 nM antigen solution (Qdot705 mouse anti-human conjungate) on the surface of the remained thick film for one hour and then washing the antigen solution away, the image of the bound antigen is shown in Fig. 3(b). Figure 3(c) shows the image of the antigen solution drop with the same concentration/volume as that used in Fig. 3(b). Through comparing the fluorescence signals of Fig. 3(b) and 3(c), and calculated antigen number included in the antigen drop in the Fig. 3(c), the calculated density of the attached antigen in Fig. 3(b) is 2.5 fmol/mm2. This corresponds to 68% total density of immobilized antigen relative to the density of immobilized antibody. As expected, a higher binding density results from a higher density of immobilized antibody. This value is also equivalent to that achieved on the silica substrate in Ref. [18

18. S. K. Bhatia, L. C. Shriver-Lake, K. J. Prior, J. H. Georger, J. M. Calvert, R. Bredehorst, and F. S. Ligler, “Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces,” Anal. BioChem , 178, 408–413 (1989). [CrossRef] [PubMed]

], where no blocker was used as well. Since no blocker was used, nonspecific binding of antigen or antibody to the crosslinker layer was not prevented. For the silica substrate, Ref. [18

18. S. K. Bhatia, L. C. Shriver-Lake, K. J. Prior, J. H. Georger, J. M. Calvert, R. Bredehorst, and F. S. Ligler, “Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces,” Anal. BioChem , 178, 408–413 (1989). [CrossRef] [PubMed]

] demonstrated that the nonspecific binding represented 38% of the total binding to antigen and crosslinker [18

18. S. K. Bhatia, L. C. Shriver-Lake, K. J. Prior, J. H. Georger, J. M. Calvert, R. Bredehorst, and F. S. Ligler, “Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces,” Anal. BioChem , 178, 408–413 (1989). [CrossRef] [PubMed]

]. The ratio of nonspecific binding to the specific binding is believed to be mainly dependent on respective reactivity of the antigen to the antibody and to the crosslinker. Thus we can conclude that specific antigen binding has been achieved in the LLF1 glass.

3. Application to fibers for biosensing

After immobilization, the loss of the F2 MOF increased to about 30 dB/m at 532 nm compared to 2 dB/m before processing. The reason for this increased fiber loss is not entirely clear and requires further investigation. Thus only a short fiber could be used for observation of fluorescence. The same setup was used for fluorescence measurement as illustrated in Ref. [6

6. Y. Ruan, E. P. Schartner, H. Ebendorff-Heidepriem, P. Hoffmann, and T. M. Monro, “Detection of quantum-dot labelled proteins using soft glass microstructured optical fibers,” Opt. Express 15, 17819–17826 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-26-17819. [CrossRef] [PubMed]

]. The measured fluorescence signals with different output power from the 19.2 cm long F2 MOF with immobilized antibodies is shown in Fig. 4(b). The background fluorescence from the MOF itself has been subtracted in Fig. 4(b). The strong fluorescence clearly indicates that antibodies have become attached to the core surface. Figure 4(c) shows the fluorescence signal of another F2 MOF with 25 cm length from the same fiber draw as that used in Fig. 4(a) but instead filled with a 100 nM antibody solution. Both fibers have the same core size, and the same experimental conditions were used. Note that the loss of the solution filled F2 MOF was only 2 dB/m. Comparing Fig. 4(b) and 4(c), it can be seen that the F2 MOF with immobilized antibody exhibits a fluorescent signal about 10% of that from the MOF filled with solution.

To predict the density of the immobilized antibody on the core surface, and also the potential of the immobilized fiber for sensing, the fluorescence capture within the immobilized MOFs is compared with solution-filled MOFs has been calculated using the models recently developed by our group [17

17. S. Afshar, V. S. C. Warren-Smith, and T. M. Monro, “Enhancement of fluorescence-based sensing using microstructured optical fibers,” Opt. Express 15, 17891–17901 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-26-17891. [CrossRef] [PubMed]

,20

20. S. C. Warren-Smith, S. Afshar, and T. M. Monro, “Highly-efficient fluorescence sensing using microstructured optical fibres; side access and thin-layer configurations,” Proc. Internat. Soc. Opt. Engin. (SPIE) 17, 70041X-1–4 (2008).

]. In order to simplify these calculations, a step-index structure is used for these calculations. For the antibody solution filled fibers, the model assumes that a length of MOF is fully filled with the solution. For the immobilized fibers, a single molecular layer is assumed to be immobilized on the core surface. Thus the distance from the Qdots to the surface of the fiber core is regarded as layer thickness, and is approximately 10 nm according to the data provided by Invitrogen [21]. The extinction coefficient of the Qdots is 2×108 M-1m-1 [22]. The loss of the solution-filled MOF at the excitation wavelength of 790 nm was measured as 1 dB/m using a standard cut-back measurement. The fluorescence measurements are made at the opposite end of the fiber from which the pump light is launched. The calculated ratio of the captured fluorescence in the immobilized fiber (with a loss of 30 dB/m) to that in the solution-filled MOF is shown in Fig. 5(a). This quantity is described as the ratio of the fluorescence capture fraction (FCF) in Fig. 5. As a comparison, the ratio of the captured fluorescence for an immobilized fiber with a loss of 2 dB/m is also displayed in Fig. 5(b). This corresponds to assuming that no loss is introduced in the immobilization processes. It can be seen from both figures that the fluorescence signal of the immobilized F2 MOF is strongly dependent on the surface density with which the antibody attaches to the surface of the core.

Fig. 4. Fluorescence intensity captured by the forward propagating mode of F2 MOFs using Qdot 800 labeled antibodies for the case of: (a) the SEM image of the MOF used here; (b) immobilized antibodies on the internal surfaces; (c) holes with a 100 nM solution of antibodies.

Using this data, the antibody surface density corresponding to the fluorescence capture ratio of 10% (as defined above) as measured in our experiment can be estimated to be 1.42×10-3 fmol/mm2 as illustrated by a grey dot in Fig. 5(a). If no additional loss is introduced to the fiber during the immobilization process, the relative fluorescence signal of fibre with immobilized antibodies could in principal be increased to 36% as shown in Fig. 5(b) (grey dot). Additionally, the lower loss of the immobilized fiber should also enable much longer fiber to be used for further fluorescence enhancement. Figure 5(c) shows length dependence of the fluorescence capture fraction of the low loss immobilized fiber. It can be seen from these results that the fluorescence signal increases with increasing fiber length, with the highest value occurring for a fiber length of approximately 2 m. For longer lengths the fluorescence decreases due to attenuation of the fluorescence signal along the fiber length. For the surface density of 1.42×10-3 fmol/mm2 achieved in our experiments to date, the maximum fluorescence of the immobilized fibers can be enhanced by a factor of 4 using a 2 m length of the MOF (compared with 19.2 cm length used in these experiments).

Fig. 5. Comparison of the FCF in the immobilized MOF and in the solution filled MOF. The grey dots in the figures correspond to the points with the surface density of 1.42×10-3 fmol/mm2. (a) FCF ratio between two fibers with loss of immobilized fiber of 30 dB/m; (b) FCF ratio with assumed loss of the immobilized fiber as 2 dB/m; (c) length dependence of the FCF with the immobilized fiber loss of 2 dB/m.

Note that the fiber hole radius (approximately 4 µm) is smaller than the minimum depletion layer thickness of 9 µm measured for a F2 glass plate in Table 1. This suggests that it should be possible to attach all the antibodies within the solution-filled F2 MOF to the internal hole surface, which would amount to an attachment density of 0.21 fmol/mm2 based on the size of the F2 MOF used above and the solution concentration of 100 nM. This value represents the maximum antibody density that can be immobilized for this fiber. This maximum is 150 times greater than the attached antibody density that we inferred from our experimental results (Fig. 4(a), 1.42×10-3 fmol/mm2). This difference indicates that there is significant scope for substantially improving the magnitude of the fluorescence signal by optimizing the coating processes for improved antibody density.

4. Conclusion

We have successfully demonstrated the adaptation of protein immobilization and binding processes in soft F2 and LLF1 glasses, and achieved good immobilization and binding efficiency. We successfully applied these processes into microstructured fibers made of the F2 glass material. Immobilized proteins have been detected within soft glass MOFs for the first time using fluorescence labeling techniques. This paves the way to sensitive and selective biosensors through binding process. This approach lends itself to the measurement of multiple biomolecules via the immobilization of multiple antibodies. The primary task for building on this work is to optimize the coating procedures to reduce the fiber loss and increase the density of the attached antibody after processing, which will enable high sensitivity of detection shown by our modeling.

At the same time, the fiber design can be systematically optimized for enhanced fluorescence capture by increasing the mode field fraction in the fiber holes and/or increased field intensity in the glass-air interface [17

17. S. Afshar, V. S. C. Warren-Smith, and T. M. Monro, “Enhancement of fluorescence-based sensing using microstructured optical fibers,” Opt. Express 15, 17891–17901 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-26-17891. [CrossRef] [PubMed]

,20

20. S. C. Warren-Smith, S. Afshar, and T. M. Monro, “Highly-efficient fluorescence sensing using microstructured optical fibres; side access and thin-layer configurations,” Proc. Internat. Soc. Opt. Engin. (SPIE) 17, 70041X-1–4 (2008).

]. Note that when a glass-air interface within a fiber cross-section is located at a point of high intensity within a guided mode, a localized region of high intensity is created on the low-index side, and this effect is particularly striking in high index glasses. Such localized regions can be used to enhance the efficiency of excitation and capture of fluorescent photons for sensing [20

20. S. C. Warren-Smith, S. Afshar, and T. M. Monro, “Highly-efficient fluorescence sensing using microstructured optical fibres; side access and thin-layer configurations,” Proc. Internat. Soc. Opt. Engin. (SPIE) 17, 70041X-1–4 (2008).

]. Thus in the future, improvements in the density of antibodies immobilized on the fiber core surface combined with the deployment of new small-core high-index glass-based MOFs, promises the development of highly sensitive selective biosensors that have ability to compete with existing commercial technologies, such as ELISA [23

23. J. R. Crowther, ELISA: Theory and Practice (Humana, 1995).

]. While ELISA is widely used both in research and industry, it lacks the ability to perform real-time in-situ measurements. By adapting this approach to allow the detection of backscattered fluorescence from the input (launch) end of the MOFs, as demonstrated in Ref. [24

24. S. Afshar, Y. Ruan, and T. M. Monro, “Enhanced fluorescence sensing using microstructured optical fibers: a comparison of forward and backward collection modes,” Opt. Lett. 33, 1743–1745 (2008).

] for a solution-filled biosensor, this approach promises to lead to the development of biosensors that can move beyond these limitations.

Acknowledgments

We acknowledge funding from DSTO Australia and the Australian Research Council Discovery project DP0665486 for this project, and to S. Afshar and E. Schartner for useful discussions. T. Monro acknowledges the support of an ARC Federation Fellowship.

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Y. Zhu, H. Du, and R. Bise, “Design of solid-core microstructured optical fiber with steering-wheel air cladding for optimal evanescent-field sensing,” Opt. Express14, 3541–3546 (2006), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-14-8-3541. [CrossRef] [PubMed]

5.

T. Ritari, J. Tuominen, H. Ludvigsen, J. C. Petersen, T. Sorensen, T. P. Hansen, and H. R. Simonsen, “Gas sensing using air-guiding photonic bandgap fibers,” Opt. Express12, 4080–4087 (2004), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-12-17-4080. [CrossRef] [PubMed]

6.

Y. Ruan, E. P. Schartner, H. Ebendorff-Heidepriem, P. Hoffmann, and T. M. Monro, “Detection of quantum-dot labelled proteins using soft glass microstructured optical fibers,” Opt. Express 15, 17819–17826 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-26-17819. [CrossRef] [PubMed]

7.

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. Express14, 8224–8231 (2006), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-14-18-8224. [CrossRef] [PubMed]

8.

J. Cheng, C. Wei, K. Hsu, and T. Young, “Direct-write laser micromachining and universal surface modification of PMMA for device development,” Sens. Actuators B , 99, 186–196 (2003). [CrossRef]

9.

G. Emiliyanov, J. B. Jensen, and O. Bang, “Localized biosensing with Topas microstructured polymer optical fiber,” Opt. Lett. 32, 460–462 (2007). [CrossRef] [PubMed]

10.

F. M. Cox, R. Lwin, M. C. J. Large, and C. M. B. Cordeiro, “Opening up optical fibres,” Opt. Express15, 11843–11848 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-19-11843. [CrossRef] [PubMed]

11.

C. M. B. Cordeiro, C. J. S. de Matos, E. M. dos Santos, A. Bozolan, J. S. K. Ong, T. Facincani, G. Chesini, A. R. Vaz, and C. H. B. Cruz, “Towards practical liquid and gas sensing with photonic crystal fibers: side access to the fibre microstructured and single-mode liquid-core fibre,” Meas. Sci. Technol. 18, 3075–3081 (2007). [CrossRef]

12.

P. D. Sawant, G. S. Watson, S. Myhra, and D. V. Nicolau, “Hierarchy of DNA immobilization and hybridization on poly-L-lysine using an atomic force microscopy study,” J. Nanosci. Nanotechnol. 5, 951–957 (2005). [CrossRef] [PubMed]

13.

J. Debs, H. Ebendorff-Heidepriem, J. Quinton, and T. M. Monro, “A Fundamental study into the surface functionalisation of soft glass microstructured optical fibres via silane coupling agents,” accepted for publication, J. Lightwave Technol. (2008).

14.

S. Gosh, A. R. Bhagwat, C. K. Renshaw, S. Goh, A. L. Gaeta, and B. J. Kirby “Low-light-level optical interactions with rubidium vapor in a photonic band-gap fiber,” Phys. Rev. Lett. 97, 023603 (2006). [CrossRef]

15.

H. Ebendorff-Heidepriem and T. M. Monro, “Extrusion of complex preforms for microstructured optical fibers,” Opt. Express15, 15086–15092 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-23-15086. [CrossRef] [PubMed]

16.

H. Ebendorff-Heidepriem, S. C. Warren-Smith, and T. M. Monro, “Suspended nanowires: fabrication, design, and characterization of fibers with nanoscale cores,” submitted to Nature Photonics.

17.

S. Afshar, V. S. C. Warren-Smith, and T. M. Monro, “Enhancement of fluorescence-based sensing using microstructured optical fibers,” Opt. Express 15, 17891–17901 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-26-17891. [CrossRef] [PubMed]

18.

S. K. Bhatia, L. C. Shriver-Lake, K. J. Prior, J. H. Georger, J. M. Calvert, R. Bredehorst, and F. S. Ligler, “Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces,” Anal. BioChem , 178, 408–413 (1989). [CrossRef] [PubMed]

19.

T. M. Monro, Y. Ruan, H. Ebendoff-Heideprien, H. Foo, P. Hoffmann, and R. C. Moore, “Antibody immobilization with glass microstructured fibers: a route to sensitive and selective biosensor,” Proc. Internat. Soc. Opt. Engin. (SPIE) 17, 70046Q-1–4 (2008).

20.

S. C. Warren-Smith, S. Afshar, and T. M. Monro, “Highly-efficient fluorescence sensing using microstructured optical fibres; side access and thin-layer configurations,” Proc. Internat. Soc. Opt. Engin. (SPIE) 17, 70041X-1–4 (2008).

21.

Http://www.invitrogen.com/site/us/en/home/brands/Product-Brand/Qdot/Technology-Overview.html.

22.

Http://probes.invitrogen.com/products/qdot/.

23.

J. R. Crowther, ELISA: Theory and Practice (Humana, 1995).

24.

S. Afshar, Y. Ruan, and T. M. Monro, “Enhanced fluorescence sensing using microstructured optical fibers: a comparison of forward and backward collection modes,” Opt. Lett. 33, 1743–1745 (2008).

OCIS Codes
(060.2370) Fiber optics and optical communications : Fiber optics sensors
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence
(300.1030) Spectroscopy : Absorption
(300.2140) Spectroscopy : Emission

ToC Category:
Fiber Optics and Optical Communications

History
Original Manuscript: September 9, 2008
Revised Manuscript: October 21, 2008
Manuscript Accepted: October 21, 2008
Published: October 24, 2008

Virtual Issues
Vol. 3, Iss. 12 Virtual Journal for Biomedical Optics

Citation
Yinlan Ruan, Tze Cheung Foo, Stephen Warren-Smith, Peter Hoffmann, Roger C. Moore, Heike Ebendorff-Heidepriem, and Tanya M. Monro, "Antibody immobilization within glass microstructured fibers: a route to sensitive and selective biosensors," Opt. Express 16, 18514-18523 (2008)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-16-22-18514


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References

  1. J. B. Jensen, P. E. Hoiby, G. Emiliyanov, O. Bang, L. H. Pedersen, and A. Bjarklev, "Selective detection of antibodies in microstructured polymer optical fibers," Opt. Express 13, 5883-5889 (2005), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-13-15-5883. [CrossRef] [PubMed]
  2. L. Rindorf, P. E. Hoiby, J. B. Jensen, L. H. Pedersen, O. Bang, and O. Geschke, "Towards biochips using microstructured optical fiber sensors," Anal. Bioanal. Chem. 385, 1370-5 (2006). [CrossRef] [PubMed]
  3. C. M. B. Cordeiro, M. A. R. Franco, G. Chesini, E. C. S. Barretto, R. Lwin, C.H.B. Cruz, and M.C.J. Large, "Microstructured-core optical fibre for evanescent sensing applications," Opt. Express 14, 13056-13066 (2006), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-14-26-13056. [CrossRef] [PubMed]
  4. Y. Zhu, H. Du, and R. Bise, "Design of solid-core microstructured optical fiber with steering-wheel air cladding for optimal evanescent-field sensing," Opt. Express 14, 3541-3546 (2006), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-14-8-3541. [CrossRef] [PubMed]
  5. T. Ritari, J. Tuominen, H. Ludvigsen, J. C. Petersen, T. Sorensen, T. P. Hansen, and H. R. Simonsen, "Gas sensing using air-guiding photonic bandgap fibers," Opt. Express 12, 4080-4087 (2004), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-12-17-4080. [CrossRef] [PubMed]
  6. Y. Ruan, E. P. Schartner, H. Ebendorff-Heidepriem, P. Hoffmann, and T. M. Monro, "Detection of quantum-dot labelled proteins using soft glass microstructured optical fibers," Opt. Express 15, 17819-17826 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-26-17819. [CrossRef] [PubMed]
  7. 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, 8224-8231 (2006), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-14-18-8224. [CrossRef] [PubMed]
  8. J. Cheng, C. Wei, K. Hsu, and T. Young, "Direct-write laser micromachining and universal surface modification of PMMA for device development," Sens. Actuators B,  99, 186-196 (2003). [CrossRef]
  9. G. Emiliyanov, J. B. Jensen, and O. Bang, "Localized biosensing with Topas microstructured polymer optical fiber," Opt. Lett. 32, 460-462 (2007). [CrossRef] [PubMed]
  10. F. M. Cox, R. Lwin, M. C. J. Large, and C. M. B. Cordeiro, "Opening up optical fibres," Opt. Express 15, 11843-11848 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-19-11843. [CrossRef] [PubMed]
  11. C. M. B. Cordeiro, C. J. S. de Matos, E. M. dos Santos, A. Bozolan, J. S. K. Ong, T. Facincani, G. Chesini, A. R. Vaz, and C. H. B. Cruz, "Towards practical liquid and gas sensing with photonic crystal fibers: side access to the fibre microstructured and single-mode liquid-core fibre," Meas. Sci. Technol. 18, 3075-3081 (2007). [CrossRef]
  12. P. D. Sawant, G. S. Watson, S. Myhra, and D. V. Nicolau, "Hierarchy of DNA immobilization and hybridization on poly-L-lysine using an atomic force microscopy study," J. Nanosci. Nanotechnol. 5, 951-957 (2005). [CrossRef] [PubMed]
  13. J. Debs, H. Ebendorff-Heidepriem, J. Quinton, and T. M. Monro, "A Fundamental study into the surface functionalisation of soft glass microstructured optical fibres via silane coupling agents," accepted for publication, J. Lightwave Technol. (2008).
  14. S. Gosh, A. R. Bhagwat, C. K. Renshaw, S. Goh, A. L. Gaeta, and B. J. Kirby "Low-light-level optical interactions with rubidium vapor in a photonic band-gap fiber," Phys. Rev. Lett. 97, 023603 (2006). [CrossRef]
  15. H. Ebendorff-Heidepriem and T. M. Monro, "Extrusion of complex preforms for microstructured optical fibers," Opt. Express 15, 15086-15092 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-23-15086. [CrossRef] [PubMed]
  16. H. Ebendorff-Heidepriem, S. C. Warren-Smith, and T. M. Monro, "Suspended nanowires: fabrication, design, and characterization of fibers with nanoscale cores," submitted toNature Photonics.
  17. S. AfsharV. S. C. Warren-Smith, and T. M. Monro, "Enhancement of fluorescence-based sensing using microstructured optical fibers," Opt. Express 15, 17891-17901 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-26-17891. [CrossRef] [PubMed]
  18. S. K. Bhatia, L. C. Shriver-Lake, K. J. Prior, J. H. Georger, J. M. Calvert, R. Bredehorst, and F. S. Ligler, "Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces," Anal. BioChem,  178, 408-413 (1989). [CrossRef] [PubMed]
  19. T. M. Monro, Y. Ruan, H. Ebendoff-Heideprien, H. Foo, P. Hoffmann, and R. C. Moore, "Antibody immobilization with glass microstructured fibers: a route to sensitive and selective biosensor," Proc. Internat. Soc. Opt. Engin. (SPIE) 17, 70046Q-1-4 (2008).
  20. S. C. Warren-Smith, S. Afshar, and T. M. Monro, "Highly-efficient fluorescence sensing using microstructured optical fibres; side access and thin-layer configurations," Proc. Internat. Soc. Opt. Engin. (SPIE) 17, 70041X-1-4 (2008).
  21. Http://www.invitrogen.com/site/us/en/home/brands/Product-Brand/Qdot/Technology-Overview.html.
  22. Http://probes.invitrogen.com/products/qdot/.
  23. J. R. Crowther, ELISA: Theory and Practice (Humana, 1995).
  24. S. Afshar, Y. Ruan, and T. M. Monro, "Enhanced fluorescence sensing using microstructured optical fibers: a comparison of forward and backward collection modes," Opt. Lett. 33, 1743-1745 (2008).

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