Practical sensitive fluorescence sensing with microstructured fibres E. P. Schartner*, R. T. White, S. C. Warren-Smith, T. M. Monro Centre of Expertise in Photonics, School of Chemistry & Physics, University of Adelaide, Australia ABSTRACT We demonstrate a new detection limit for fluorescent species in small-core glass microstructured optical fibres. Two detection schemes are explored: forward detection by an optical spectrum analyser; and backward detection by a photodiode. In the second scheme, characterisation of the fluorescence signal during fibre filling allows us to accurately separate quantum-dot fluorescence from intrinsic glass fluorescence. The spectral overlap of these two fluorescence sources is the principal limit to the detection sensitivity. We demonstrate a detection limit of ~200 pM. Keywords: Optical-fibre sensors; fluorescence; optical sensing; microstructured optical fibres.
INTRODUCTION Optical fibres are ideal for environmental sensing applications because of their ability to transmit optical signals to and from the sensing region without the use of free-space optics. By accessing the evanescent field, the fibre itself can be the sensing element and long interaction lengths can be achieved. Microstructured optical fibres (MOFs) are particularly suited to such applications as the material to be detected can occupy the air spaces inside the fibre. By tailoring both the MOF material and the geometry, the light-matter overlap can be increased to values much larger than with conventional fibres. The extremely small transverse structure of MOFs also means that very small samples can be measured. Detection of biomolecules attached to fluorescent labels has recently been demonstrated in small-core MOFs1-3, yielding a detection sensitivity down to 1 nM for antibodies labelled with quantum dots (Qdots)1. The fluorescence-detection approach is attractive because of its simplicity. When one end of the fibre is dipped into the sample, capillary forces draw the liquid into the voids within the fibre. The evanescent field of the pump light excites the fluorescent labels and a portion of the fluorescence is captured by the fibre core and propagates to the fibre tips. Captured fluorescence can be detected at either end of the fibre, although backward detection provides the convenience of single-ended devices and an improved signal-to-pump ratio4. Efficient fluorescence-based MOF sensors require a large evanescent field in the fibre holes, such as in band-gap fibres, liquid-core fibres or small-core fibres. Here we concentrate on small-core fibres, which offer comparatively simple filling requirements. We make use of lead-silicate (Schott F2) glass, from which we can produce suspended-core fibres with core diameters as small as 400 nm5. In this paper, we use our small-core fibres for the detection of fluorescent Qdots and compare two detection schemes. While a spectrum measurement can provide better sensitivity (200 pM has been achieved here), a simple photodiode measurement can provide detection at concentrations as low as 500 pM, provided the system is well characterised. This paves the way for practical realisation of MOFs in biological and chemical sensing applications.
EXPERIMENTAL RESULTS Recent advances in fibre fabrication have enabled the core size of the wagon-wheel (WW) fibres to be decreased significantly5, as shown in Fig. 1(a). The resulting increase of the overlap of the evanescent field with the surrounding medium is expected to correspond to an increase in the sensitivity of the system. The fibre used here has a core size of ~800 nm, which was chosen as a good balance between increased overlap and increased loss from decreased core size. 50jim
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Fig. 1. Comparison of fibre used in Ref. 1 (left) to recently fabricated small-core fibre (right).
The fluorophore chosen was Qdot® 800 ITK™, a commercially available CdSe semiconducter Qdot from Invitrogen. This fluorophore is ideal for characterisation of this system as, unlike typical organic fluorophores, it does not suffer *Corresponding author:
[email protected] 20th International Conference on Optical Fibre Sensors, edited by Julian Jones, Brian Culshaw, Wolfgang Ecke, José Miguel López-Higuera, Reinhardt Willsch, Proc. of SPIE Vol. 7503, 75035X © 2009 SPIE · CCC code: 0277-786X/09/$18 · doi: 10.1117/12.835350 Proc. of SPIE Vol. 7503 75035X-1
from photobleaching. For these trials, the Qdots were unconjugated, although they can be easily conjugated, and protein detection in fibre has previously been demonstrated using Qdots at concentrations down to 1 nM1. Two separate detection methods were employed, based on an optical spectrum analyser and a photodiode for detection in forward and backward directions, respectively (see Fig. 2). Advantages and disadvantages of each approach are discussed below.
Fig. 2. System for fluorophore sensing using forward (a) and backward (b) fluorescence capture with detection by an optical spectrum analyser (OSA; a) and photodiode (b). The OSA is an Anritsu model AQ6315E.
Optical Spectrum Analyser Results Figure 3 show the results obtained using forward detection by the OSA. Observe that the 0.2 nM (200 pM) concentration Qdot solution can be clearly detected, which represents a significant improvement on previous reports1. This result was obtained using relatively a short length (30 cm) of fibre and input powers in the range 1–5 mW.
Fig. 3. a) OSA results for low concentration Qdot solutions in decane. b) Data + low-pass filtered signal to show trend in fluorescence signals
The primary limitation encountered was background fluorescence from the F2 glass, which is believed to originate from metal impurities in the glass. This has previously been encountered1 with F2 glass, and several changes from previous results have been implemented to reduce the impact of this unwanted fluorescence. Firstly, the glass composition was changed from commercially available F2 rods to bulk glass, which was observed to significantly reduce glass fluorescence. The second change is the reduced core size of the fabricated fibre; as the core size decreases, the power fraction located within the glass decreases. As the glass fluorescence is related to the power within the glass, reduction of the pump power acts to decrease the recorded glass fluorescence. The advantage of the OSA detection method is that the results can be analysed in more detail than with a photodiode because the entire spectrum can be viewed. This is negated somewhat by the sensitivity of the OSA, as low concentrations are close to its noise level and differentiation between background and Qdot signals becomes difficult. Photodiode Results With the sensing concept verified, we set out to make the sensing configuration more practical for field-based applications. The OSA was replaced with a high-gain photodiode capable of measuring pW power levels. The experimental arrangement is shown in Figure 2(b). To ensure that the pump signal was completely filtered, a combination of a dichroic mirror (HR@514 nm; HT@>700 nm), a 550-nm long-pass filter and a prism was used. The alignment was adjusted such that minimal green pump light reached the photodiode. Coupling into the fibre was optimised, and the free end dipped into the solution under investigation. The fibre was periodically re-aligned using the fluorescence signal detected from the photodiode. Fibre length was 30±1cm. Fig. 4(a) shows the fluorescence power observed as the fibre is filled with a solution of n-decane. As there are no Qdots present, this signal is due solely to the glass fluorescence. Of particular note is the significant decrease in the fluorescence recorded as the fibre fills. This is attributed to two processes: reduced confinement of pump light to the core; and reduced fluorescence capture efficiency6 of glass fluorescence. Both contributions result from a reduction in index contrast between the core and the holes (as they are filled). These processes are discussed in detail in the next Section.
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Fluorescence curves for three concentrations of Qdots are shown in Figs 4(b)–(d). For all cases the Qdot concentration is low enough that the refractive index of the solution can be assumed to be equal to that of decane. Therefore, the contribution of the glass fluorescence can be assumed to be equivalent to results shown in Fig. 4(a) and thus the Qdot fluorescence is taken to be the difference between the total fluorescence and the glass fluorescence. The results in Fig. 4 indicate that Qdot concentrations as low as ~0.5 nM can be measured by this approach. Below this concentration, the difference between the total and the glass fluorescence is too low for reliable results.
Fig. 4. Fluorescence power recorded during filling with various concentrations of Qdots in decane. Blue circles show the measured total fluorescence (blue lines are a fit to this data). The green dashed curves show the glass-fluorescence contributions (decane-only curve). Red curves are the fluorescence attributed to the Qdots (the difference between the total fluorescence and the glass fluorescence).
A significant advantage of the photodiode-based system over the OSA-based system is its increased sensitivity. This enables variations of the pump intensity to be exploited that cannot be effectively used with the OSA due to the proximity of the current results to the noise floor of the detector. The results in Fig. 5 demonstrate that the Qdot fluorescence saturates significantly more than glass fluorescence. As the detection limit is determined by the power of the Qdot signal relative to that of glass, the use of lower pump powers is expected to further reduce the detection limit. In addition to this the photodiode also provides a more rapid detection time, limited primarily by the filling time as unlike the OSA scan times are virtually instantaneous.
Fig. 5. Dependence of 1-nM Qdot fluorescence (red squares) and glass fluorescence (blue circles) on incident pump power, showing the saturation of the Qdot fluorescence. The powers of the two curves are scaled independently.
DISCUSSION AND THEORETICAL RESULTS To gain a better understanding of our sensor configuration, we have performed a number of numerical calculations. Here we define a fluorescence-sensing figure of merit (FOM), which is the fraction of modal power in the sensing region multiplied by the fluorescence capture efficiency. This FOM is proportional to the total collected fluorescence of the fibre, and can be applied to either the core (background fluorescence) or the cladding (Qdots), in the limit of lowconcentration fluorophores, low fibre loss, and in the absence of pump depletion and fluorophore saturation. It can be derived by taking the low concentration limit of previously determined fluorescence capture theory6, noting that here we
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also consider the contribution of higher order modes. For simplicity we have used a step-index model with analytic vectorial solutions7, where we assume that the cladding is equivalent to the holes of a wagon wheel fibre.
Figure 6. (a) Fraction of fundamental mode power in the core and cladding of a step-index fibre. (b) Fluorescence-sensing figure of merit (FOM) for the background glass fluorescence for different cladding materials. (c) Comparison of the FOM for cladding versus core-excited fluorescence; that is, the ratio of quantum-dot fluorescence to background fluorescence. Cladding materials considered are air (n = 1.00), water (n = 1.33), and decane (n = 1.41).
Fig. 6(a) shows the fraction of fundamental mode power present in the core and cladding regions. Note that it is quite difficult to use a solid-core index-guiding fibre to reduce the amount of power that can excite background glass fluorescence. For example, the core diameter needs to be the order of 400 nm in order for more than 50% of the light to be outside the core. Alternatively, Fig. 6(b) considers the FOM for the core fluorescence (background) only. Here we see that filling the fibre with a liquid is an effective way to reduce the background fluorescence level. This results from the decreased index contrast between the core and cladding, which reduces the numerical aperture of the fibre and thus the fluorescence capture efficiency. These results explain the drop in fluorescence observed in Fig. 4(a), which occurred as the fibre was filled with decane. Unfortunately, reducing the index contrast also reduces the capture efficiency for evanescently excited fluorophores (Qdots). To determine the fibre parameters that can be used to improve the Qdot signal with respect to the background signal, we determined the ratio of their respective FOMs, which is shown in Fig. 6(c). Here we see that refractive index of the solvent has very little effect but that an advantage can be gained by reducing the core diameter, which is mostly due to increased power in the sensing (cladding) region. In summary, while a high index contrast is generally favourable due to an increase in absolute fluorescence signal, it is by using small-core fibres with high evanescent fields that the contrast between analyte and glass-background signals can be improved.
CONCLUSIONS A reduced detection limit of 200 pM for fibre-based fluorescent-molecule detection has been demonstrated using smallcore fibres. An alternative system for more practical field measurements has also been demonstrated down to 500 pM using a photodiode for detection. These limits should be able to be further decreased through the use of improved detection hardware (cooled CCD or PMT), or alternatively reduced core sizes and lower pump powers giving a higher contrast between the glass and fluorophore signals. Detection time can also be improved by increasing the hole size or utilising pressure to increase the capillary fill rates that limit the current detection time.
REFERENCES 1. Y. L. Ruan, E. P. Schartner, H. Ebendorff-Heidepriem, P. Hoffmann, T. M. Monro, "Detection of quantum-dot labeled proteins using soft glass microstructured optical fibers," Opt. Express 15, 17819–17826, 2007. 2. Y. Ruan, T.C. Foo, S. Warren-Smith, P. Hoffmann, R.C. Moore, H. Ebendorff-Heidepriem, T.M. Monro, "Antibody immobilization within glass microstructured fibers: a route to sensitive and selective biosensors," Opt. Express 16, 18514-18523, 2008. 3. 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. 4. S. Afshar V., S. C. Warren-Smith, and T. M. Monro, "Enhanced fluorescence sensing using microstructured optical fibrers; a comparison of forward and backward collection modes," Opt. Lett. 33, 1473–1475, 2008. 5. H. Ebendorff-Heidepriem, S. C. Warren-Smith, and T. M. Monro, "Suspended nanowires: Fabrication, design and characterization of fibers with nanoscale cores," Opt. Express 17, 2646–2657, 2009. 6. S. Afshar V., S. C. Warren-Smith, and T. M. Monro, "Enhancement of fluorescence-based sensing using microstructured optical fibres," Opt. Express 15, 17891–17901, 2007. 7. K. Okamoto, Fundamentals of Optical Waveguides (Academic Press, San Diego, 2000).
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