Introduction

Evanescent wave absorption (EWA)-based fiber-optic sensors are of great use for diagnostic applications because of their high sensor performance and simplicity of design, fabrications and use. These sensors have been successfully used for the detection of glucose, DNA, proteins and microorganisms. These sensors are based on the principle that whenever analyte binds to the sensor surface, the refractive index at the core changes which modulates the propagating light (Sai et al. 2010). This change denotes the binding/detection of the analyte and the intensity of modulation is correlated with the concentration of the analyte in the sample. The performance of these sensors depends upon the evanescent field strength which is influenced by the extinction coefficient, concentration and surface area of interaction of the analyte with waveguide, in addition to the wavelength of light and refractive index of the waveguide and surrounding medium (Sai et al. 2009).

Various modified configurations have been developed to improve the performance of fiber-optic sensors, which includes (1) labeled approach using fluorophores, (2) label-free approach using metal nanoparticles or nano-composites, (3) sandwich-type assay, (4) modulation of optical parameters, (5) number of geometric designs, etc. (Leung et al. 2007). These adaptations showed some degree of improvement in sensor performance. However, scarce efforts have been made to improve the linker chemistry at the sensor matrix, which is a vital component of any sensing device. Many researchers have drawn attention to two-dimensional (2D) conventional linkers, e.g., silanes, polyaniline, poly-l-lysine (PLL), etc. or gel-based matrices.

Most 2D linkers show limitations in developing a stable, dense and well-organized sensor matrix. For example, silane molecules tend to polymerize upon exposure to air/moisture during shelf-life, which results in defective and irreproducible adhesion on the glass. This can also happen if the substrates are not dehydrated properly prior to silane layer deposition (Benters et al. 2001). Poly-l-lysine (PLL) polymeric layers tend to lose their adhesive properties due to damage from the molecular geometry of freely rotating long polypeptide chains after their immobilization onto the glass surface (Ji et al. 2004). Furthermore, some of these linear polymeric linkers form entangled chains, which probably result in a decreased number of available functional groups for subsequent bioconjugation reactions (Bosman et al. 1999).

In case of 3D gels or the membrane-coated surfaces of poly-acrylamide (Arenkov et al. 2000) and agarose gel (Khayyami et al. 1998), leaching of bioreceptor molecules results in larger variations in signal intensity, thus limiting their use. In addition, interaction of analytes in such matrices is diffusion rate limited (Hanbury et al. 1997), which has primarily confined their applications for the detection of low molecular weight analytes including vapor molecules (Aneesh and Khijwania 2011). Taking into account the limitations of the 2D linkers and 3D gel matrices, we felt that there was need to improve the existing sensor matrix chemistry or design a new type of sensor matrix. Recently, supramolecular dendritic architectures have been employed in designing and developing the sensor platforms, particularly for electrochemical and fluorescence-based sensors (Shiddiky et al. 2007; Benters et al. 2001). These dendrimeric linkers have offered more advantages over the conventional 2D and 3D linkers as a glue material.

Dendrimers are well-defined, monodispersed, globular macromolecules constructed around a core unit. The exciting properties of dendrimers, such as globular shape, structural uniformity, nanometric size, the existence of dendritic crevices, high surface functionality, hydrophilicity, versatility to design dendrimer of different composition and high mechanical and chemical stability make them an ideal glue material for sensors (Satija et al. 2007, 2011). Hence, designing a dendrimeric sensor matrix might be a successful approach to obtain superior sensor performance. In this paper, we used amine-terminated poly(amidoamine) dendrimer as glue material to develop EWA-based fiber-optic sensor. Further, the dendrimeric sensor matrix was compared with 2D silanized sensor matrix by developing a RI-sensitive sensor using localized surface plasmon resonance (LSPR) of gold nanoparticles.

Materials and methods

Materials

Optical fiber of core diameter 200 μm (numerical aperture 0.37) was obtained from Ceramoptec®. 1,1′ carbonyldiimidazole (CDI), 3-((2-aminoethyl amino)-propyl) trimethoxysilane (APTMS), amine-terminated fourth-generation polyamidoamine (4G, PAMAM-NH2) dendrimer, fluorescein isothiocyanate (FITC) on Celite and tetra chloroaurate (HAuCl4) (99.99%) were purchased from Sigma-Aldrich. 1,4-dioxane was procured from Merck, India. Acetic acid, borax, boric acid and trisodium citrate were obtained from SD Fine Chemicals, India. All the other reagents used were of analytical grade. Deionized (DI) water was used for cleaning and preparation of solutions.

Fabrication of U-shaped fiber-optic probes

U-shaped fiber-optic probes were fabricated as described earlier by our group (Sai et al. 2009). Briefly, the decladded portion (2 cm) of the optical fiber was bent into a U shape with the help of butane candle flame to obtain a probe with a bending diameter of ~1.5 mm. The bend diameter was measured using an optical microscope.

Functionalization of fiber-optic probes

First, the U-shaped probes were cleaned in methanol–HCl solution (1:1) followed by dipping in concentrated H2SO4 and thorough rinsing in DI water. To generate additional silanol groups, cleaned probes were treated with sulfochromic acid for 10 min followed by washing with DI water twice and drying under nitrogen. These cleaned probes were stored in DI water until functionalization.

Amino-silanization of fiber-optic probes

Silanization of fiber-optic probes was carried out as per Sai et al. (2009). Briefly, cleaned probes were heated at 110 °C for 2 h to remove surface adsorbed water. Thereafter, fiber probes were dipped in APTMS silane solution (1 % v/v in ethanol:acetic acid (5:2) mixture) for 10 min in argon ambience. The excess amount of silane on probes was removed by rinsing in ethanol. This was followed by condensation at 115 °C in argon ambience for at least 90 min.

Dendrimer immobilization on glass substrates

The dendrimer was immobilized on fiber-optic probes in two steps as described earlier (Pathak et al. 2004) with slight modification. Cleaned fiber probes were kept in CDI solution (80 mM in dioxane) for 24 h at room temperature. These CDI-activated probes were washed twice in dioxane followed by incubation in 4G PAMAM-NH2 dendrimer solution (20 nM in ethanol) for 24 h. Thereafter, probes were washed twice in ethanol to remove any loosely bound dendrimer molecules followed by nitrogen drying.

Change in surface chemistry on glass, i.e., CDI and dendrimer binding, was studied using contact angle measurements. Since it is almost impossible to measure contact angle on the curved fiber surface, functionalized glass slides (CDI and dendrimer coated) were used for this study. The contact angle was measured with a Digidrop (model-DS) instrument with water as the test liquid at room temperature. The measurements were taken on at least three different samples at three different locations on each sample. Further, the presence of dendrimeric primary amine groups was evaluated using an amine reactive fluorescent dye, i.e., FITC. Dendrimer-coated fiber probes were subjected to 50 μM FITC solution (prepared in 25 mM borate buffer, pH 8.3) and real-time absorbance was measured at λmax = 500 nm. After experimentation, the binding of FITC to fiber probes was verified by observing fluorescence from the probes under a fluorescence optical microscope (ZEISS Axioskope-2 MAT).

Development of gold nanoparticles-anchored fiber-optic sensor

GNPs of 40 ± 5 nm diameter were synthesized using citrate reduction method (Turkevich et al. 1951). The wavelength at peak absorbance was 530 nm. GNP binding on both types of functionalized probes was carried out real time using an optical setup. When the absorbance at 535 nm (A535nm) reached ~2.5 absorbance units, further GNP binding was interrupted by flushing the gold colloidal solution with DI water. Thereafter, these GNP-coated probes were used for RI sensitivity measurements. A certain weight percent of sucrose solution (0.5 mL) was introduced into the flow cell and absorbance spectrum was recorded. This procedure was repeated for higher RI values by replacing the liquid in the flow cell with a higher concentration of sucrose solution. RI sensitivity was obtained by measuring the absorbance changes at 535 nm wavelength in the presence of sucrose solutions of different RI between 1.33 and 1.37.

Results and discussion

Amine-terminated dendrimers were covalently immobilized on fiber probes using CDI cross-linker. CDI linker molecule binds with hydroxyl group at the probe surface leaving one imidazole moiety. This CDI-activated probe forms a carbamate-like structure with amine-terminated dendrimer molecules and releases the second imidazole molecules. In this fashion, PAMAM-NH2 dendrimers were covalently bonded to the fiber probe surface.

The contact angle of CDI-immobilized glass slides was found to be 18°, which increased to ~47° after dendrimer immobilization. This increase in contact angle was due to large number of short chain alkane moieties present in dendrimer molecules. However, the contact angle of the dendrimerized glass surface was found to be lower than that of the silanized surface (~70°) (Wang et al. 2005; Blinka et al. 2010). It can be attributed to a large number of amine groups of PAMAM dendrimer (64 groups in 4G), as compared to 2D silane molecules, which might have provided a more hydrophilic sensor surface.

Dendrimer binding was further confirmed by qualitative determination of amine groups on fiber probe surface using FITC dye. This fluorescent molecule is known to bind with amine groups. Real-time binding of FITC on dendrimerized probes showed a characteristic absorption peak of FITC at 500 nm (Fig. 1a). Thereafter, FITC-bound dendrimerized probe was observed under a fluorescence optical microscope (ZEISS Axioskope-2 MAT) at 10× magnification. Figure 1b shows the fluorescence microscopic image of FITC-bound dendrimer-coated fiber probe. Bright and homogeneous fluorescence was observed on fiber probes confirming the binding of FITC with the dendrimeric amine groups on fiber probe.

Fig. 1
figure 1

a Absorbance spectrum obtained from dendrimer-coated U-shaped fiber probe due to FITC binding; b Microscopic image of dendrimer-coated fiber-optic probe after treatment with FITC

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Gold nanoparticles were immobilized on functionalized fiber probes to develop LSPR-based RI-sensitive sensor. GNP immobilization was monitored real time using a spectrophotometer. When the absorbance change (λmax = 535 nm) reached to ~2.5 Unit, further GNP binding was stopped by flushing the gold colloidal solution with DI water. These GNP-coated probes were used for RI sensitivity measurements. Similarly, GNP binding and RI sensitivity measurements were performed on silanized fiber probes.

Figure 2 shows the real-time changes in absorbance at λmax = 535 nm due to change in RI surrounding the GNP immobilized fiber probes. Dendrimerized probes showed higher change in absorbance than that of silanized probes at different RI values. RI sensitivity of GNP anchored dendrimerized sensor matrix was found 32 ± 1 which is 1.4-fold enhanced than that of GNP on silanized probe (23 ± 2). Also, the RI resolution of sensor was improved by 1.35-fold when the dendrimeric architecture was used in sensor matrix. This improved sensor performance of dendrimer-based matrix may be attributed to homogeneous pendent group (i.e., amine) distribution and globular shape of dendrimer.

Fig. 2
figure 2

a Absorbance spectra obtained from GNP-coated silanized and dendrimerized fiber-optic probe due to 12 (10−3 RIU) change. b Real-time change in absorbance at 535 nm due to change in RI (i.e., sucrose solution of different %w/w concentration) surrounding the GNP-coated silanized and dendrimerized probes

Full size image

The globular shape of dendrimer might have increased the surface area of fiber probe for GNP binding as compared to 2D silane molecules as shown schematically in Fig. 3. Assuming the probe length of 2 cm and fiber core diameter of 200 μm and 100 % linker coverage, two dimensional silanized surface provides 1.26 × 107 μm2 surface area for GNP binding. When the same fiber probes were coated with globular shaped 4G PAMAM dendrimer (diameter 4 nm), the probe surface area was calculated to be 7.89 × 107 μm2 (assuming half of globular surface is available for GNP binding). This increased sensor surface area might have provided better control on GNP distribution and interspacing among the nanoparticles along with better interaction of nanoparticles with the surrounding media. In addition, the presence of dense and uniform amine groups on dendrimerized fiber probe surface might have helped in homogeneous distribution of GNP as compared to GNP distribution on silanized probe.

Fig. 3
figure 3

Schematic representation of silane and dendrimer linkers on sensor platform

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Conclusion

Dendrimeric nanoparticles were successfully used as “nano-glue” material to develop an LSPR-based RI-sensitive sensor. Dendrimer immobilization protocol was simple which involved dipping, washing and drying steps at normal ambience. Dendrimer binding was confirmed using contact angle measurement and FITC binding. Fluorescence microscopic images of FITC bound on dendrimer-coated fiber probes revealed homogeneous bright signal which suggests the uniform distribution of amine groups on fiber probe surface. Dendrimer-coated fiber-optic probes were used to coat gold nanoparticles to develop an LSPR-based sensor. These probes showed 1.4-fold enhanced RI sensitivity than amino-silanized probes. This enhancement was attributed to globular shape and homogeneous pendant group distribution of dendrimer, which provided better interaction of analyte with immobilized gold nanoparticles. Overall, dendrimeric glue material improved the fiber-optic sensor performance and can be used for biosensor applications.