4.2. MIP and Waveguides
The simplest photonic structure to use in biosensing is a waveguide. In a waveguide, the light is physically confined by the different refractive index between the waveguide material and the surrounding and each event occurring at the waveguide interface produces a variation in the local refractive index, and consequently, a variation in the light spectrum, as described in paragraph 3. Different materials, both organics and inorganics, have been used for waveguides fabrication and various are the measurement configurations reported (see Table 2
). Among the different optical configurations, the following are coupled to MIPs and herein discussed: “free-standing filaments” that are obtained by polymerizing MIP structures in a filamentous shape, after the polymerization the MIP filament is peeled off from the substrate and used as free-standing. The integrated optical waveguide (IOW) uses the longitudinal total internal reflection of a light beam through a planar waveguide. The interferometer, Young interferometer or Fabry-Peròt interferometer represent different configurations that take advantage of interfering waves. Diffraction gratings are optical components with a periodic structure that splits and diffracts light into several beams travelling in different directions. Optical fibers are cylindrical dielectric waveguides, surrounded by a cladding layer that transmits light along the longitudinal axis by total internal reflection. Finally, lossy mode resonance structures combine the guided mode in the core of the optical fiber and the lossy mode in semiconductor metal oxide layer.
A first attempt at coupling the MIP and waveguide is reported in 2001. Yan and Kapua fabricated an MIP microstructure on a silicon wafer using micromolding in capillaries: through a polydimethylsiloxane (PDMS) stamp, they built MIP micromonoliths [87
]. In this configuration, the MIP was itself the waveguide, and the specific interaction of the analyte 2,4-dichlorophenoxyacetic (2,4-D) was guaranteed by the imprinted cavities. Following this idea, Brazier and coworkers patterned polymer waveguides onto a silicon substrate, imprinting polyurethane filaments with anthracene [88
]. Although they did not reach high performances, they laid the groundwork for the future works. Two years later, the same authors published a paper with a detailed study of the optical properties revealing that MIP parameters have to be optimized, although the fabrication technique based on micromolding capillaries allowed the achievement of the preparation of MIP arrays on a single chip, enabling the simultaneous analysis of multiple analytes [89
A different technique was employed by Walker and coworkers [90
]. A submicron molecularly imprinted sol-gel film was deposited onto a waveguide made by a SiO2
layer on microscope glass to specifically detect the explosive 2,4,6-trinitrotoluene (TNT). When the analyte binds to the MIP film, it is converted into a colored anionic form facilitated by an amine group remaining in the binding site after cleavage of the template. The selectivity is, therefore, assured by the specific binding to the imprinted sites and by the chemistry required to form the colored product. A LoD of 5 ppb was achieved.
A sol-gel technique was also used by Edminton and coworkers to deposit an MIP layer onto a Si3
waveguide for TNT detection [91
]. The sensor was based on an interferometric configuration, where two waveguides (the reference and the sensing one, respectively) were crossed by laser light. The two waveguides produced an interference useful for the detection of wavelength shift. The configuration allowed three orders of magnitude to be gained with respect to the work of Walker et al., and there was a good selectivity for TNT compared to structural analogues. The MIP layer was about 10 times thinner than that proposed by Walker and colleagues, which undoubtedly represents a great advantage (Table 2
Barrios and coworkers [92
] determined the basic optical properties (refractive index, optical attenuation and optical response to the recognition of the antibiotic enrofloxacin) of their MIP film, depositing it onto a glass slide in a grating shape or as a bulk film. The optical parameters were then used to simulate the geometry of a Si3
waveguide in order to have the highest performance. To achieve this result, two effects produced by the analyte binding to the MIP were considered: a variation in the film refractive index and a variation in the film thickness. To take into account the first effect, they hypothesized an infinite film thickness, while for the second one, they imposed a 300 nm MIP thickness value. The parameters that better match fabrication costs with sensor performances resulted in a waveguide width of ~1 μm and a waveguide thickness between 60 and 80 nm (in quasi-transverse-magnetic (TM) operational mode).
A polymeric integrated Young interferometer functionalized with an MIP film for melamine detection was instead proposed by Aikio and coworkers [93
]. An input waveguide branched into four waveguides, forming two parallel Young interferometers integrated into one sensor chip. Both of the interferometers contained a reference and a measurement waveguide. An MIP or control nonimprinted polymer (NIP) were deposited by spin-coating technique: the MIP interferometer was meant to measure the specific binding of analyte, while the NIP interferometer measured the nonspecific one. The phase change, due to the binding of melamine to MIP film, was four times higher with respect to the corresponding NIP film, and a saturation above 0.5 g/L was recorded.
A different waveguide configuration is represented by an optical fiber. In a fiber, the inside radiation pathway is determined by the total internal reflection, and consequently, fibers act as a waveguide.
The simplest way to functionalize an optical fiber with MIP is dipping the fiber tip into partially polymerized solution. This approach was firstly reported by Jenkins and coworkers in 2001 [94
]. They directly dip coated the distal end of a glass fiber into a prepolymerized solution for the specific detection of organophosphate pesticides and insecticides, with detection limits less than 10 parts per trillion (ppt), with an extended linear dynamic range (ppt to ppm) and response times of less than 15 min. Another work was reported by Queiros and coworkers [95
] where a glass fiber tip was dip coated in the MIP mixture and then polymerized for the specific recognition of Microcystin-LR, an hepatotoxin present in drinking-water. The comparison between MIP and NIP membrane revealed that MIP showed better performances than NIP and also a higher stability to temperature. In a similar way, Xiong and coworkers dipped an optical fiber into an MIP solution designed for Bisphenol A recognition, finally inserting the prepared optical fiber into a transparent capillary for the measurements [96
], taking advantage of the evanescent wave field produced on the fiber core surface to excite the bisphenol A fluorescence. The LoD was 1.7 × 10−9
; selectivity and stability were proved.
Similar to the approach followed by Walker and coworkers [90
], Nguyen et al. immobilized a fluorescent MIP to the distal end of an optical fiber for cocaine detection [97
]. The carboxylic groups on the fluorophore inside the MIP cavity and the amine group on the analyte formed a complex that enhanced the fluorescence signal at the analyte binding. The system response to cocaine was in the concentration range 25–500 μM. Instead, Ton and coworkers [98
] coated a polystyrene optical fiber with a fluorescent microMIP, containing N-(2-(6-4-methylpiperazin-1-yl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl-ethyl)acrylamide [99
], and demonstrated the sensor ability to detect the herbicide (2,4-D). The evanescent wave passing through the fiber excited the MIP. The analyte binding to the MIP produced a strong fluorescence enhancement; the fluorescence intensity was proportional to the analyte concentration in the nanomolar range, with a Limit of Quantification (LoQ) of 2.5 nM. Moreover, the authors proposed a further advancement [98
]: the evanescent wave passing through the polystyrene optical fiber was exploited to photopolymerize the fluorescent MIP directly on the fiber, and the performance of the in situ fabricated MIP-sensor was compared to the coated sensor, showing similar performances.
Taking advantage of the lossy mode resonance, Usha and Gupta [100
] built a nanocomposite of ZnO/MoS2
on a silica core fiber optic immobilizing an MIP selectively designed for p-cresol, a highly toxic phenolic compound detected in urine. The nanocomposite was designed to enhance the refractive index of the system resulting in a low detection limit for the analyte; indeed, the reached LoD was of 28 nM.
A more complicated configuration was adopted by Carrasco et al. [101
]: an optical fiber was etched creating microwells of approximately 3.1 μm in depth, and MIP (or NIP) fluorescently-encoded microspheres were deposited inside the wells and imaged through epifluorescence microscopy. The specific detection of the analyte enrofloxacin, a fluoroquinolone antibiotic widely used for both human and veterinary applications, was proved in a competitive assay: a fluorescent analogue of the antibiotic competes for active sites within the imprinted microspheres, highlighting the high specificity of MIP. Interfering substances were tested to assess cross-reactivity, which was negligible. Moreover, tested with sheep serum samples (diluted 6-fold), the results suggested that matrix effects were negligible. The excellent long-term stability of these materials was demonstrated for over five years of experiments.
In a recent and extensive review presented by Gauglitz [52
] on optical detection systems based on fibers and waveguides, MIPs are acknowledged as a valid alternative to antibodies in complex matrices and real samples, combining good specificity, short time of responses and selectivity.
MIPs coupled to waveguides.
MIPs coupled to waveguides.
|Waveguide Material||Optical Configuration||MIP Thickness||Analyte||LoD or LoQ||Reference|
|Polymer||Free-standing filaments||20 µm||2,4-Dichlorophenoxy acetic (2,4-D)||LoQ: 0.021 µmol/g|||
|Polymer||Free-standing filaments||<20 µm||Anthracene||LoQ: 1.6 µmol/g|||
|Polymer||Free-standing filaments||100 µm||Anthracene||-|||
|SiO2/TiO2||IOW||∼0.3–1.0 µm||2,4,6-Trinitrotoluene (TNT)||LoD: 5 ppb|||
|Si3N4/SiO2||Interferometry||20–120 nm||TNT||LoD: 2.4 ppt|||
|Si3N4/SiO2||Diffraction grating||322 nm||Enrofloxacin||LoQ: <50 µM|||
|Polymer||Young interferometer||-||Melanine||LoQ: <0.1 g/L|||
|Glass||Fabry-Pérot interferometer||19 µm||Microcystin-LR||LoQ: >1.8 μg/L|||
|Glass||Fiber microarrays||3.1 µm||Enrofloxacin||LoD: 40 nM|||
|Glass||Fiber||200 µm||Organophosphates||LoD: <10 ppt|||
|Glass||Fiber||<5 µm||Bisphenol A||LoD: 1.7 ng/mL|||
|Glass||Fiber||-||Cocaine||LoQ: <25 μM|||
|Polymer||Fiber coupled to spectrofluorimeter||few µm to nm||2,4-D||LoQ: 2.5 nM|||
|ZnO/MoS2||Lossy mode resonance||1.2 µm||p-Cresol||LoD: 28 nM|||
4.3. MIP and Photonic Crystals
An alternative approach for developing optical biosensors is based on Photonic Crystals (PC) structures. These systems consist of two or more materials with different refractive indices, which are periodically arranged in space and manipulate light in the scale of optical wavelengths. From a historical point of view, the PC, where proposed in the pioneering work of Yablonovitch [102
] and John [103
], aimed to create materials that can manipulate photons in a way that resembles a semiconductor crystal and influences the properties of electrons. Indeed, the shifts in the reflection peak of these 3D photonic structures and the high surface to volume ratio makes them interesting for sensing [104
]. The detection systems for PCs ranges from fluorescence [105
], to enzymatic reactions [106
], to label-free, just by observing the reflection peak change of the structures [107
]. Moreover, both inorganic and hydrogel-based 3D photonic structures have been reported.
PC configurations range from 1D structures, based on multilayers Bragg stack films, to 3D ones, such as opal systems (both direct or inverse). The most exploited configurations are the opals due to their low realization cost, flexibility, lightweight and power free employment. Moreover, these systems are suitable for the analysis of both liquids and gasses.
The inverse opal configuration is, to date, the most chosen for the development of MIP PC sensors [108
] (see Table 3
). Different opal 3D structures have been reviewed: the main configuration is based on silica nanoparticles covered with MIPs deposited onto a solid substrate “film” (Table 3
), while another configuration is based on MIP photonic hydrogel particles based on an inverse opal structure and called “hydrogel”, and the last configuration relates to silica microspheres decorated with MIP “microspheres”. Dai and coworkers [109
] combined the features of an inverse opal colloidal crystal and an MIP hydrogel, composed of acrylamide and EGDMA, to detect 2-butoxyethanol (2BE), a pollutant associated with hydraulic fracturing contamination, with an LoD of 3.4 ppb. Reflection measurements allowed the correlation of the Bragg’s peak position shifts of MIP PC with the 2BE concentrations from 1 ppb to 100 ppm. A similar approach was developed for the recognition of pesticides, in particular for the detection of parathion. Zhang and colleagues used a gold doped inverse opal PC and an MIP hydrogel (MAA, NIPAM, EGDMA) [110
]. A shift in wavelength (Δλ
) of the reflection peak position of about 10 nm was reported for a concentration of parathion of about 10 ng/mL.
More recently, Huang et al. [111
] developed a portable label-free inverse opal photonic sensor, constituted of MIP hydrogel particles, addressed to pesticides, such as methanephosphonic acid (MPA), by means of colorimetric monitoring. Authors proved that a concentration of MPA of 10−6
M induced a large red shift of the Bragg peak that can be seen to the naked eye, with a good recovery over 5 cycles.
The sensor reported by Hou and coworkers [112
] consists of an inverse colorimetric PC sensor (see Figure 3
) based on hydrophilic–hydrophobic patterned MIP-PC for the detection of tetracycline. The main advantage of this strategy was to realize MIP structures of millimetric dimension on the hydrophobic PC substrate. Upon analyte binding, the MIP-PC sensor underwent a colorimetric transition from cyan to red (optical shift > 200 nm), clearly visible to the naked eye. Moreover, varying the diameter of the MIP dots permits the variation of the detection range of the sensor. Overall, the sensitivity of this MIP-PC sensor was superior by one order of magnitude with respect to that of PC based on MIP films [113
]; the LoD was 2 nM.
Sai and coworkers [114
] reported on an MIP-PC sensor with improved sensitivity for chloramphenicol (Cm). The system exploited the combination of the features of opals (colloidal crystals) and peculiar wettability of different enrichment processes of arrays of MIP spheres embedded in the PDMS matrix. The authors reported a sensitivity of about 1.5 nM for Cm with a linear response in the range of 1 to 10,000 nM. More recently, Chen et al. [115
] developed a smart sensor for the rapid and label-free detection of benzocaine. In fact, authors combined an inverse PC structure with an MIP hydrogel showing high sensitivity and specificity, a fast response time, a regeneration ability and a satisfactory accuracy with respect to high-performance liquid chromatography measurements, with an LoD of about 0.1 mM.
Finally, MIP-PCs have been successfully employed as low cost and optical readout systems in the field of biomolecules. In fact, Kadhem et al. [116
] using an inverse hydrogel structure with polyacrylamide as the matrix were able to realize an optical sensor sensitive to 5–100 ppb of testosterone. More recently, MIP-PCs have been used to create protein sensors; in fact, Dabrowski et al. [117
], taking advantage of the properties of a semicovalently imprinted inverse opal polythiophene film, reached an LoD of about 13 fM in the detection of human serum albumin, while Chen et al. [118
] used surface imprinted silica colloids for hemoglobin bovine (Hb) recognition, demonstrating a sensitivity of 24.8 × 10−7
4.4. MIP and Whispering Gallery Modes Resonators
Another interesting photonic structure suitable to devise sensors is represented by whispering gallery modes (WGM) resonators. Specificity is given to the WGM resonator by surface functionalization; however, the functional layer is required to be in the range 10–100 nm, in order to remain in the evanescent penetration depth and should be homogeneous in order to preserve the high quality of the transducer. The first applications of WGM resonators in biosensors are reported in 2002–2003 [119
]. Since then, many works have been published in this field [44
], but only recently, the coupling between the WGM resonator and MIPs has been reported. Just three works related to microring resonators have been published, but all highlight the high sensitivity reached using these systems; for a comparison, see Table 4
Chen and coworkers [122
] developed a biosensor based on a microring resonator functionalized with an MIP for the specific recognition of testosterone. Sensor chips employing grating couplers were manufactured on silicon-on-insulator (SOI) wafers with a 220 nm-thick top silicon layer and 2 µm-thick buried oxide layer. The radius of the ring was 32 µm, and the whole sensor was coated by SiO2
with a 2 μm-thick upper cladding layer, while the microring resonator was exposed to the analyzed sample by removing the upper cladding layer in the sensing window. The picture of the optical system used is reported in Figure 4
a. The reached LoD was 48.7 pg/mL.
A further example was reported by Xie et al. [123
]. A cascaded-microring resonators system was designed for the recognition of progesterone (Figure 4
b). The specificity was guaranteed by an MIP, layer which allowed progesterone recognition with high specificity and an LoD of 83.5 fg/mL.
The last reported example applies to the detection of TNT [124
]. The microring resonator array chip consisted of a waveguide connected to a fiber-coupled coherent light source that was split into 4 parallel branches with microrings, as shown in Figure 4
c. The transmission of each branch could be measured independently. The possibility to measure four rings at the same time allowed for a simultaneous comparison of significant signals; the authors, in fact, compared signals coming from the first ring functionalized with MIP specific for TNT, with the second one functionalized with a NIP, whereby the other rings were deputed to monitor temperature fluctuations. The sensitivity to TNT was about one order of magnitude higher in comparison to other structural analogues.
Due to the high sensitivity of the WGM resonators and the high specificity of MIP functionalization, it is reasonable to think that in the coming years, there will be an increase in the number of examples combining these two systems. The possibility to build thin MIP film or nanoMIP allows for better remaining inside the evanescent tail, maximizing in this way the interaction with the analyte. There is an only one example that reports the MIP functionalization of a microsphere resonator [125
]: in this work, a silica sol-gel film was imprinted with a small fluorescent dye (fluorescein isothiocyanate), dipping the microsphere into the solution. The coating technique, the time aging and the extraction procedure were studied finding the best parameters to preserve high quality factor of the resonator. A manual coating for 45–50 s followed by an aging for three days resulted in the best coating procedure, while an oxygen plasma treatment appeared as the best template extraction method. Nevertheless, due to the fragile nature of this kind of WGM resonators, it is more realistic to rely on an increase in MIP functionalization of rings, toroids or other photonic structures more easily built on a photonic chip.