Macroporous Polymer Cantilever Resonators for Chemical Sensing Applications

A novel, cost‐effective strategy for sensing performance enhancement of cantilever‐shaped, mass‐sensitive sensors is presented. The developed strategy relies on the introduction of macroporosity in the recognition unit. The developed sensors are successfully applied in air humidity monitoring as well as in chemical sensing. As mass‐sensitive transducers, polymer microelectromechanical systems (MEMS) in the form of poly(vinylidene fluoride‐trifluoroethylene) [P(VDF‐TrFE)] cantilever resonators are specifically developed. These resonators are modified with a hierarchically structured macroporous poly(2,3′‐bithiophene) film, acting as a sensing layer. The design of the recognition layer takes advantage of the synergistic combination of inverse opal structuring, surface imprinting, and semi‐covalent imprinting of proteins. The resulting cantilever resonators are first successfully tested for air humidity monitoring in the whole relative humidity range (ca.10 – 90%). When the recognition layer is composed of human serum albumin‐imprinted polymer (HSA‐MIP) with a hierarchical structure, it can also be used as a selective chemosensor in a concentration range from 10 pM to 20 µM. The obtained results demonstrate that the macroporosity of the receptor film significantly enhances the sensor performance.


DOI: 10.1002/admt.202300771
monitoring via an efficient combination of the analytical signal transducer with the recognition unit.Mass-sensitive transducers are one of the most attractive alternatives for sensing applications as they enable the direct determination of chemical compounds regardless of their physiochemical properties and without labeling. [1]Additionally, these transducers are characterized by a short response time and a relatively high sensitivity, making them suitable for real-time analysis. [2]For this, microelectromechanical systems (MEMS) and vibrating cantilevers, in particular, are appealing forms of mass-sensitive transducers.Miniaturization is the current trend to enhance the sensitivity of mass sensors. [3]However, as the reduction of the cantilever dimensions is challenging for fabrication technologies, it drastically increases the resonator cost.One cost-effective alternative for sensitivity enhancement is the increase of surface area available for the binding of analytes.Surface enhancement combined with the simultaneous demand for device miniaturization can be implemented by introducing porosity or nanostructuration into the recognition part of the chemosensor. [4,5]Porous or nanostructured materials provide a high aspect ratio, increasing the number of recognition sites and thus improving the sensitivity of mass sensors.Generating macroporosity by means of colloidal crystals made out of nanobeads as a mold is an attractive strategy providing very precise control of the material porosity. [6][9] The introduction of macroporosity into structures of gold-based mass-sensitive transducers has been already successfully applied to air humidity monitoring. [10]electivity of the determinations using mass-sensitive transducers can be provided via modification of the resonators surface with appropriate receptor materials.Molecularly imprinted polymers (MIPs) are a class of artificial receptor materials, and an attractive alternative to their natural counterparts i.e. antibodies, enzymes, nucleic acids, viruses, whole microorganism cells, and tissues. [11]Procedures for imprinting low molecular weight compounds were widely investigated and extensively described in the literature. [12,13]The application of MIPs as recognition elements of cantilever chemosensors was already explored for the determination of low molecular weight compounds. [14,15]However, the detection of macromolecules and in particular proteins by MIPs remains challenging. [16,17]Previously developed and optimized strategies for generating macroporous MIPs [8,9,18] by electrochemical synthesis combine three material engineering approaches, namely inverse opal structuring, surface imprinting, and semi-covalent imprinting of the proteins. [19]This synergistic strategy enables fast and high-affinity binding of the proteins by the molecular cavities of the MIP with low unspecific binding by the polymer matrix.Importantly, a macroporous structure of the MIP provides an enhancement of sensitivity due to a high surface-to-volume ratio.Previously developed capacitive [8,9] and field-effect transistor (FET) [7,8] chemosensors, selective with respect to some proteins, confirmed the outstanding analytical performance of the macroporous MIP receptor material.9] One of the most elegant resonator strategies is based on piezoelectric materials.Currently, the most common piezoelectric materials for MEMS transducers are rigid inorganic crystal materials i.e. quartz, PZT, LiNbO 3 , ZnO, and LiTaO 3 . [20]However, the fabrication process of the resonators using these materials requires very precise cutting with respect to the crystal structure or sophisticated crystallization procedures.Moreover, resonators made of such rigid crystalline materials are very fragile, sensitive to mechanical stress, and prone to breakage.An appealing way around these technological limitations is the use of poly(vinylidene fluoride-trifluoroethylene) [P(VDF-TrFE)] films as a piezoelectric element of the resonator.P(VDF-TrFE) is a ferroelectric polymer, exhibiting interesting piezoelectric and pyroelectric properties. [21]Up-to-date, P(VDF-TrFE) has been widely used for the fabrication of various types of physical sensors and actuators, as well as for energy harvesting devices. [21]In compar-ison to crystalline piezoelectrics, sensors based on P(VDF-TrFE) are lightweight, elastic, stretchable, with low energy consumption, and relatively inexpensive.Importantly, the P(VDF-TrFE) resonators fabrication procedure is simple, cheap, and can be implemented in cleanroom-free environment.The application of P(VDF-TrFE) films as piezoelectric elements of a cantilever resonator was already explored. [22,23]However, up-to-date there is only one report in the literature describing a piezoelectric membrane made of P(VDF-TrFE) and applied as a biosensor for singlestranded DNA oligomers. [24]In parallel, cantilever resonators using nanostructured mesoporous silica were already explored in chemosensing [25] and humidity monitoring applications. [26]t is worth noting that sensitivity enhancement of cantilever chemosensors using nanostructuration generated by colloidal crystals of nanobeads as a mold was not yet studied.
In the present contribution, we propose a versatile masssensitive polymer transducer comprising a millimeter-sized (1.45 × 2.20 mm 2 ) piezoelectric P(VDF-TrFE) cantilever resonator (Figure 1 a).The transducer was applied for air humidity monitoring and selective chemosensing of human serum albumin (HSA) protein as two proof-of-principle applications.In both cases, the bottom side of the cantilever was modified with an appropriate recognition unit.For air humidity monitoring, poly(2,3′-bithiophene) in the form of a non-porous or macroporous inverse opal film was deposited.For HSA chemosensing a molecularly imprinted polymer (MIP) with a hierarchical structure was employed (Figure 1 a) to provide selectivity and surfaceenhanced sensitivity.
The core of the P(VDF-TrFE) cantilever resonators is made out of a 25 μm-thick polyethylene naphtalate (PEN) substrate, and a P(VDF-TrFE) piezoelectric film sandwiched between two Au electrodes (Scheme S1 of Supporting Information).This structure was hermetically coated with a SU8 passivation film ensuring cantilever protection with respect to a broad range of chemical conditions e.g. for recognition unit synthesis and deposition as well as sensor operation. [27]Additionally, the underside Au electrode of the cantilever enabled electrochemical deposition of the recognition unit in the form of macroporous MIP or poly(2,3′bithiophene).
Changes in the resonance frequency of the developed transducer can be triggered by changes in the resonator's total mass and/or by cantilever stiffness.While the mass and stiffness of bare cantilevers stay intact during the experiments, the mass and stiffness of the recognition unit could be affected by, e.g., adsorption of water, polymer swelling, or selective binding of the analyte.Therefore, air humidity could be monitored and selective binding of the HSA protein molecules inside MIP film can be investigated via the cantilever resonance frequency as the analytical signal of the sensors (Figure 1b).The sensing performances of the air humidity sensors with non-porous and macroporous films were compared based on resonance frequency monitoring in real-time.For the MIP chemosensors, imprinted with HSA, the analytical signal with respect to the HSA target analyte and interfering myoglobin protein were studied in vacuum.

Fabrication and Passivation of P(VDF-TrFE) Cantilever Resonators
The fully-operational bare P(VDF-TrFE) cantilever resonators were fabricated using a previously developed procedure, [22,23] modified for particular requirements of chemosensing applications (Scheme S2a, Supporting Information).To fulfill these requirements, Au was used as the electrode material of the resonator.Thus, the resonators presented high resistance with respect to harsh chemical environment, including strong acids (e.g.HF) and bases (e.g.NaOH) used for further modification of the cantilevers with dedicated recognition units.For successful resonator operation, optimal Au electrode thicknesses were 40 and 150 nm for the bottom and top electrodes, respectively.Relatively high P(VDF-TrFE) film roughness in comparison to the PEN substrate (Figures S1-S3, Supporting Information) imposed that the thickness of the top electrode was higher than the one of the bottom electrode.Additionally, the chosen P(VDF-TrFE) film thickness was 8 μm (Figure S2, Supporting Information).Using P(VDF-TrFE) films, thinner than 8 μm, led to a major loss of the operational resonators during the P(VDF-TrFE) film polarization step.Due to the insufficient rigidity of the thin P(VDF-TrFE) film, designing a free-standing cantilever requires the use of a 25 μm-thick PEN substrate playing the role of a rigid core, allowing also for oscillations at relatively high frequencies.Among various tested polymeric materials for the resonators' passivation, including PDMS, methacrylate, and SU8, only the latter resulted in satisfying sealing properties.SU8 offers substantial chemical resistivity, low swelling in organic solvents, and sufficient elasticity.Thus, a four-step passivation procedure comprising SU8 spin-coating and dip-coating steps was developed (Scheme S2b, Supporting Information).Importantly, the initial SU8 spin-coating (Step 1 in Scheme S2b, Supporting Information) is crucial in the whole passivation procedure, because sole dip-coating (Step 3 in Scheme S2b, Supporting Information) of the bare resonator with a diluted SU8 solution (≈10% solid content) resulted in damages of the P(VDF-TrFE) film.It is worth noting that the adhesion of the evaporated Au electrode used for functionalization (Step 4 at Scheme S2b, Supporting Information) was enhanced via modification of the outer, dip-coated SU8 thin film processing. [28]Indeed, UV crosslinking and post-exposure baking of the dip-coated SU8 thin film were performed after evaporation of a 35 nm-thick Au electrode on the bottom side of the devices.Technically, this UV crosslinking was performed through the thin semi-transparent Au film. [29]

Electrochemical Synthesis with Simultaneous Deposition of Macroporous Poly(2,3′-bithiophene) or Hierarchical MIP Films
The receptor material in the form of macroporous poly(2,3′bithiophene) or MIP with a hierarchical structure imprinted with HSA was synthesized and simultaneously electrodeposited on the Au electrode of the cantilever resonator, according to a previously developed five-step procedure (Scheme 1). [9]A non-porous or macroporous poly(2,3′-bithiophene) (NIP) film was deposited on a bare Au electrode or an Au electrode modified with HSA-free colloidal crystal, respectively.
After Langmuir-Blodgett (LB) deposition of a colloidal crystal (Step 1 in Scheme 1), the procedure for receptor unit deposition was modified by the introduction of an Au underlayer electrodeposition.This modification was necessary to improve colloidal crystal stability and to promote its adhesion to the evaporated Au electrode of the cantilever during the five-step procedure for MIP deposition.Indeed, evaporated Au electrodes have a relatively low roughness.Poly(2,3′-bithiophene) films adhere well to rough Au surfaces, [30] so the increase of the Au roughness via initial electrodeposition of Au improves adhesion of the macroporous MIP recognition part to the cantilever transducer and ensures the integrity of the chemosensor devices.The thickness of the Au underlayer was controlled by monitoring the number of current oscillation peaks (minima and maxima) of the chronoamperometric curves recorded during potentiostatic electroreductive deposition of Au on the Au electrode of the cantilevers (Figure S4, Supporting Information).To optimize the thickness of the Au underlayer, its electrodeposition was terminated at the first chronoamperometric curve maxima (Figure S5, Supporting Information) to obtain one half-layer of macropores.
In the most important step (Step 4 in Scheme 1) the HSA template structure was imprinted into the polymer matrix, during the electrochemical synthesis of poly(2,3′-bithiophene) inside the colloidal crystal mold.On the surface of the mold, HSA template molecules with attached functional monomers were immobilized.During electrodeposition of poly(2,3′-bithiophene), the voids between the beads are filled with polymer, and simultaneously the bithiophene moieties of the functional monomers were co-polymerized with 2,3′-bithiophene cross-linker.Conditions of the potentiostatic deposition of poly(2,3′-bithiophene) were carefully optimized previously, leading to stable macroporous polymer structures in the form of inverse opals. [9]The deposition of poly(2,3′-bithiophene) inside the voids between the beads of the colloidal crystal was conducted from the bottom (electrode surface) to the top of the colloidal crystal.The active surface of electrodeposition is changing periodically during deposition, leading to current oscillations in the chronoamperometric curves.This allows for precise control of the polymer film thickness. [6]Current oscillations are quite well pronounced in the chronoamperometric curves for the macroporous MIP or NIP synthesis (Figure 2a,b) in comparison to electrodeposition of poly(2,3′-bithiophene) on a bare Au electrode (Figure 2c).This suggests that the colloidal crystal quality was relatively high.
Apart from current oscillations observed during electrodeposition, another method for tuning the thickness of the macroporous poly(2,3′-bithiophene) film can be implemented via the control of the total charge passing through the working electrode during electrodeposition.For this purpose, the correlation between the number of macropore layers of the poly(2,3′bithiophene) film and total charge density (Q D ) passed through the working electrode was investigated.The expected thickness of macroporous poly(2,3′-bithiophene) films was 6 and 10 macropore layers for +210 mC•cm −2 and +326 mC•cm −2 , respectively.Macroporous film thicknesses were estimated based on control electrodeposition experiments conducted on Au-plated glass slides modified with the same colloidal crystals of 660 nm silica beads (Figure S6, Supporting Information).For further air humidity monitoring and chemosensing experiments, cantilevers with 6 and 10 macropore layers of MIP or NIP films were fabricated.These film thicknesses were chosen to compare the analytical performance of HSA chemosensors and also for technical reasons.Chemosensors with MIP films comprising less than 6 macroporous layers show only a modest improvement in sensitivity.On the other hand, the fabrication of cantilevers comprising a sensing film with more than 10 macroporous layers is technically more demanding.
As expected, the resulting macroporous films present an inverse opal morphology.In both cases of MIP and NIP, the spherical shape and diameter of macropores were preserved (Figure 3).Moreover, all macropores (insets in Figure 3) are well-interconnected, allowing for easy permeation of the solution through the MIP film, as well as easy access to imprinted cavities localized exclusively on the inner pore surface.However, the macropore ordering is not exactly the expected hexagonal packing.Apparently, the inhomogeneous and rough surface of the cantilevers causes defects in the colloidal crystal mold during LB transfer.However, the quality of macropore packing of the MIP or NIP films composed of 6 and 10 macropores is very similar.The observed defects in the macroporous film, including pore deformations, vacancies or pore dislocations do not substantially affect the global connectivity and the diffusion through the sensing film. [31]Therefore, these defects in the macroporous structure do not impact the sensor's performance.
An increase in the specific surface area of the macroporous films can be achieved by following two strategies-namely, an increase in the number of macroporous layers or a decrease in macropore size for a given total layer thickness.The number of pore layers inside the sensing film is limited by the LB technology used for colloidal crystal deposition.The maximum number of bead layers in a colloidal crystal and consequently the maximum number of pore layers in the sensing film are approximately 30 and 25, respectively.Macroporous MIP films with various pore diameters can also be fabricated.The possible pore size is limited by the LB fabrication of the colloidal crystals and varies from approximately 100 to 1200 nm.However, in the case of HSA chemosensing, the size of the connecting channels between the pores should be significantly bigger than the size of the HSA analyte molecules (7.5 × 6.5 × 4.0) in order to allow unrestricted penetration of the protein into all the pore layers.As the dimension of the connecting windows between neighboring pores typically equals approximately 10% of the pore size, the diameter of the beads should exceed 200 nm.On the other hand, larger beads typically result in better organized layers.As it can be demonstrated by geometric considerations that the bead diameter has no direct influence on the active surface area for a given num-ber of pore layers, we have therefore chosen to preferentially use larger beads for all the experiments.

Dynamic Characterization of the P(VDF-TrFE) Cantilever Resonators with Macroporous MIP and NIP Films
The designed P(VDF-TrFE) resonators operate properly despite the rather harsh chemical treatment used for macroporous MIP or NIP film deposition.Moreover, the devices entirely preserved their structural integrity.Indeed, out-of-plane vibrations of the resonators in the range from 0 to 200 kHz were characterized in the air using laser Doppler vibrometry in order to identify their mode shapes (Figures S7-S15, Supporting Information) and associated resonance frequencies and quality factors (Table S3, Supporting Information).Importantly, the resonance spectra were  characterized by relatively high reproducibility for resonance frequency values of particular oscillation modes, resonant peak shapes, and quality factor values.Furthermore, the experimental results match quite well with the theoretical calculations, as indicated in Table S3 (Supporting Information).The butterflylike resonance mode (Figure 4a,b) was chosen for further sensing measurements, because of the well-observable resonance peak in the resonance velocity spectra (Figure 4b) at a relatively high frequency (≈53.51 ± 0.50 kHz, n = 10) as well as a very high homogeneity of the resonance frequency (peak maximum) over the cantilever area.The value of the quality factor was ≈17.1 ± 0.50 (n = 10) in air.

Monitoring of Air Humidity Using Cantilever P(VDF-TrFE) Resonators with Macroporous and Non-Porous Poly(2,3′-Bithiophene) Films
The possibility of continuous online monitoring of air humidity using P(VDF-TrFE) cantilever resonators modified with a polymeric film was first explored.The P(VDF-TrFE) resonators with a macroporous or non-porous poly(2,3′-bithiophene) film were used for this application.For both cases, the mass of the deposited poly(2,3′-bithiophene) film was supposed to be equal because it was controlled by the charge density passed through the working electrode during poly(2,3′-bithiophene) electrodeposition.The poly(2,3′-bithiophene) electrodeposition was stopped after Q D = +326 mC cm −2 has passed through the working electrode.Measurements of the resonance frequency of the piezoelectrically actuated cantilever resonators were carried out continuously using laser Doppler vibrometry focusing in the middle of the cantilever tip.Optical vibrometry in comparison to other frequency read-out methods (i.e.piezoelectric method) offers low noise levels and extraordinary sensitivity.Moreover, optical vibrometry prevents undesired coupling effects, reducing the resolution of the measurements.Dynamic curves of the time dependence of the resonance frequency changes were recorded for the cantilevers with macroporous (Figure 5a) and non-porous (Figure 5b) poly(2,3′-bithiophene) films.An increase in the relative air humidity (RH) from 10% to 90% induced a decrease in the resonance frequency.This phenomenon is triggered by two independent and synergistic factors.First, it could be explained by an increase in the polymer mass, caused by the adsorption of water molecules by the polymer.Second, this frequency drop could be triggered by polymer swelling and the resulting decrease in polymer stiffness.Variations of the resonance frequency for the cantilever with macroporous poly(2,3′-bithiophene) films are much more pronounced and clearly visible for the levels of humidity applied, whereas the non-porous cantilever provides a much weaker response.The calibration curves of relative frequency changes versus relative humidity were plotted for cantilevers with macroporous (curve 1 in Figure 5c) and non-porous (curve 2 in Figure 5c) films.Changes in the resonance frequency were linearly proportional to humidity in the whole tested range (≈10 -≈90%) for both films.The slopes of the calibration curves were −298 ppm %RH −1 and −46 ppm %RH −1 for sensors with macroporous and non-porous poly(2,3′-bithiophene), respectively.[34][35][36][37] Additionally, the sensitivity of the sensor with a macroporous film was ≈6.5 times higher than that of the sensor with a non-porous film.The calculated specific surface area of the inverse opal polymer film composed of ten layers of spherical pores is 12.1 times higher compared to a continuous polymer film of the same projected area.This clearly illustrates that the introduction of macroporosity into the polymer structure is beneficial for improving the sensitivity of MEMS-based humidity sensors.

Chemosensing of HSA Using P(VDF-TrFE) Cantilever Resonators with Macroporous MIP Films
In the next step, the analytical performance of the cantilever chemosensors functionalized with macroporous MIP and NIP First, the resonance frequency of the cantilevers with HSAextracted MIP films was measured after incubation with PBS (pH 7.4).The cantilevers with macroporous NIP films were also treated with the same extraction and conditioning procedure as cantilevers with MIP films in order to avoid apparent resonance frequency shifts not related to HSA binding.To estimate the reproducibility of the resonance frequency of the chemosensors, they were consecutively incubated three times with PBS (pH 7.4), dried and their resonance frequency was then measured.Standard deviations of the relative resonance frequency of the cantilevers with MIP films composed of ≈6 and ≈10 layers were 0.06% and 0.32%, respectively.Standard deviations of the relative resonance frequency of the cantilevers with NIP films composed of ≈6 and ≈10 layers were 0.33% and 0.44%, respectively.Then, the resonance frequencies of the cantilever chemosensors were measured after incubation with HSA solution in PBS (pH 7.4).Initial conditions for binding of the HSA analyte were selected to obtain saturation of all molecular cavities present inside MIP films.For this purpose, a concentration of 20 μm HSA and 2 h incubation time were selected.The decrease of the resonance frequency for cantilevers with MIP films was much higher than for cantilevers with NIP films (Figure 6a), for MIP and NIP films composed of either ≈6 or ≈10 macropore layers.The apparent positive resonance frequency shift of the cantilever with a NIP film composed of 6 macropore layers is within the relatively high standard deviation.This result confirms the presence of molecular cavities in the MIP films for the specific detection of HSA molecules by MIP recognition units of the chemosensors.Calculation of the ratio between the relative resonance frequency shift after HSA binding and the MIP film thickness enables direct comparison of chemosensors with 6 and 10 macroporous recognition layers.The ratio was equal to 1.96 × 10 −3 and 1.85 × 10 −3 [%•mC −1 •cm 2 ] for cantilevers with a MIP composed of 6 and 10 macroporous layers, respectively.This clearly indicated that the resonance frequency change triggered by HSA binding is proportional to the thickness of the macroporous MIP film.
To verify the obtained chemosensing results, we estimated the theoretical total mass of the cantilevers with macroporous MIP or NIP films and the maximum mass load of HSA proteins inside the molecular cavities of the MIP film.These calculations were based on the dimensions of the cantilever resonators, the dimensions of macroporous films, and the dimensions of the HSA molecule.The total mass of the bare unmodified cantilever resonator was calculated as the sum of the masses of each resonator functional element (Table S5, Supporting Information).The mass of non-porous construction elements was estimated using Equation S1 (Supporting Information).The mass of the macroporous Au underlayer, and the mass of macroporous MIP/NIP polythiophene films, were calculated by using Equation S2 (Supporting Information), a modified equation for the fraction of space occupied by spheres in a hexagonal close-packed structure.The estimated mass of the unmodified cantilever and the cantilever with ≈6 and ≈10 macropore layer films were 187, 190, and 192 μg, respectively.The estimated maximum mass of HSA proteins adsorbed in the ≈6 macropore layer MIP film, and 10 macropore layer MIP film were ≈0.4 and ≈0.7 μg, respectively.The theoretical maximal mass of HSA adsorbed inside the MIP film was estimated using Equation S3 (Supporting Information).This approximation is based on the assumption that the total surface of the macropores present in the MIP film is occupied by HSA molecules.Thus, the maximum relative shift of the reso- nance frequency should be on the order of ≈−0.2% and ≈−0.4% for cantilevers with a 6 and 10-macropore layer thick MIP, respectively.Measured values of resonance frequency shifts (−0.631% and −0.939%) exceed the maximum theoretical values, however, they are in the same order of magnitude.On the other hand, this rough theoretical approximation does not include changes of mechanical properties of the MIP films induced by HSA binding by the MIP film.
Then, the selectivity of the MIP chemosensors toward the target HSA analyte was tested via a measurement of the resonance frequency changes recorded after incubation of the HSAextracted MIP films with 20 μM myoglobin in a PBS (pH 7.4) solution.The resonance frequency changes measured for both chemosensors with 6 and 10 macroporous layers were negligibly low in comparison with the responses of the MIP chemosensors when incubated with an HSA analyte (Figure 6a).Therefore, the analytical response of the developed MIP cantilever chemosensors was selective for the target analyte, indicating that the molecular cavities present in MIP films enable specific protein binding.
The calibration curve was plotted for different cantilevers with MIP films composed of 6 macroporous layers.The frequency changes were measured sequentially for 10 pm, 1 nm, 100 nm, and 20 μm HSA (Figure 6b).The obtained calibration curves show a linear dependence of the relative resonance frequency with the logarithm of the HSA concentration.A similar logarithmic dependence was reported for other millimetersized gravimetric cantilever biosensors for proteins [38,39] or bacteria spores. [40]The observed logarithmic concentration dependence is typical for receptor-ligand interaction.The linear regression equation describing the calibration plot of the cantilever chemosensor with the HSA-templated MIP film (Figure 6b ].This value is slightly higher than the sensitivities of cantilever biosensors selective for Staphylococcal enterotoxin B (SEB) [39] and -methylacyl-CoAracemase (AMACR) proteins [38] (Table S6, Supporting Information).However, with these biosensors, the biochemical recognition measurements were performed in liquid media.Thus, the resonator oscillations could be damped by the liquid medium, resulting in a decrease in the biosensor's sensitivity.The limit of detection (LOD) at 3 of the developed MIP chemosensors was ≈6 pm (≈431 pg mL −1 ), which is five and eight orders of magnitude below HSA concentrations in urine (0.45-4.51 μm) and blood (0.53-0.75 mm), respectively.Therefore, the developed cantilever chemosensor could be eventually applied in determinations of HSA in urine as well as in blood.The LOD value is three orders of magnitude higher than the LOD of millimetersized cantilever biosensors selective with respect to SEB in apple juice (100 fg•mL −1 ). [39]It is also two orders of magnitude higher than the LOD (13 fm) of previously developed chemosensor using hierarchical MIP imprinted with HSA as recognition unit and FET as a transducer. [7]The LOD of the developed chemosensors could be improved via the implementation of three strategies for excluding interference in analyte-receptor affinity.The first strategy is an increase in mass loading with the analyte.For this, increasing the number of macropore layers would result in a higher number of analyte binding sites.The second strategy relies on the improvement of the mass sensitivity of the resonator transducer via resonance frequency increase or reduction of the chemosensor's effective mass.The third strategy consists of the reduction of analytical signal noise via an enhancement of the cantilever resonator Q factor.Nevertheless, the present results clearly show the capacity of polymer cantilevers functionalized with macroporous MIP films to selectively detect analytes at low concentrations.

Conclusion
We have successfully tested an original surface enhancement strategy for the sensing performance improvement of cantilever mass-sensitive sensors.This strategy constitutes an attractive and cost-effective alternative to current trends aiming at expensive and sophisticated miniaturization technologies.We have developed novel and versatile mass-sensitive transducers that were applied for air humidity monitoring, as well as for selective protein detection.P(VDF-TrFE) films were applied as piezoelectric elements of the resonators offering relative simplicity of resonator fabrication at low cost, and enabling integration of their actuation.The fabrication strategy for the P(VDF-TrFE) cantilever resonators with macroporous MIP and NIP films was successfully applied to design devices that are fully resistant to a range of chemical conditions.As a first test of the beneficial effects introduced by the macroporosity, cantilevers with macroporous poly(2,3′-bithiophene) were applied for online air humidity monitoring.Then, cantilevers with MIP films with a hierarchical structure were successfully used for HSA chemosensing.The mass-sensitivity of the developed P(VDF-TrFE) cantilever resonators allows a clear observation of the resonance frequency shift triggered by air humidity changes and by selective binding of HSA target analyte inside the MIP film.Importantly, the macroporosity of the receptor film provides in both cases a significant enhancement of the sensor sensitivity.Therefore, the developed P(VDF-TrFE) resonators in the form of micro-and nanostructured cantilevers constitute a versatile and label-free platform for mass-sensitive transduction for chemosensing.The combination of the cantilevers with other receptor materials opens up interesting perspectives for the development of a range of masssensitive chemosensors with improved selectivity and sensitivity.The developed polymer cantilever resonators are therefore an attractive alternative to resonators based on inorganic rigid crystalline piezoelectric materials, like the well-known quartz crystal microbalance (QCM).

Experimental Section
Sections describing materials and instruments (Table S1, Supporting Information) used within all experiments were presented in Supporting Information.
Fabrication and Passivation of P(VDF-TrFE) Cantilever Resonators: The bare cantilever resonators were fabricated via a four-step procedure (Scheme S2a, Supporting Information) and then passivated using SU8 epoxy-based photoresist (Scheme S2b, Supporting Information).
First, on the surface of a poly(ethylene naphtalate) (PEN) substrate, a gold electrode was thermally evaporated.Afterward, a P(VDF-TrFE) film was spin-coated and thermally annealed.After the evaporation of the second gold electrode, the shape of the cantilever was formed by means of xurography, a numerical vinyl-cutting approach.Then, the bare cantilever resonators were coated with SU8 passivation film.For this purpose, the top side of the device was spin-coated with a thin SU8 film.Subsequently, a thin glass slide was bonded onto the top part of the resonators in order to fix the cantilever clamped base.Then, a second thin sealing film of SU8 was formed via dip-coating of the cantilever part of the resonator.Ultimately, a thin semi-transparent Au electrode was thermally evaporated on the bottom side of the device.All experimental details are precisely described in the Supporting Information.
Synthesis and Deposition of a Macroporous Molecularly Imprinted Poly(2,3′-Bithiophene) Film in the Form of Inverse Opals: MIP films with a hierarchical structure imprinted with HSA were synthesized and deposited on the Au electrodes of the cantilever resonators according to a previously developed procedure (Scheme 1). [9]irst, silica nanobeads (≈660 nm), surface modified with 3aminopropyl moieties, were synthesized and used for the Langmuir-Blodgett (LB) deposition of colloidal crystals on the Au electrodes of the cantilever resonators.Then, a one-half-layer-thick Au underlayer was electrodeposited.Afterward, the HSA template for imprinting was immobilized on the surface of the colloidal crystal with glutaraldehyde as agent coupling proteins with the amine moieties present on colloidal crystal surface.Subsequently, the HSA molecules were derivatized with bithiophene-based functional monomers using amide chemistry.Synthesis with simultaneous deposition of the MIP and non-imprinted polymer (NIP) films in the form of inverse opals was implemented via electrooxidative polymerization of 2,3′-bithiophene in propylene carbonate solution performed at a constant potential (1.25 V vs Ag pseudo-reference electrode).After polymerization, the colloidal crystal was dissolved with 5% HF acid and the HSA template was extracted from the MIP films with a concentrated 30% NaOH solution.Then, the cantilever clamped base was fixed a second time to obtain higher reproducibility in resonance frequency measurements.Ultimately, the P(VDF-TrFE) film of the cantilever was polarized, and all oscillation modes of the resonators were characterized using laser vibrometry.All experimental details of each procedure were precisely described in the Supporting Information.
Monitoring of the Relative Air Humidity Using a Sensor with a Macroporous Poly(2,3′-Bithiophene) Film: A cantilever with a macroporous or non-porous poly(2,3-bithiophene) film was mounted and piezoelectrically actuated inside a humidity chamber (VGIMEMS from Surface Measurements Systems).The resonance frequency of the cantilever butterfly-like resonance mode was monitored continuously via subsequent recording and analysis of the resonance spectra using an MSA 500 laser vibrometer from Polytec focused at the center of the cantilever tip.
Testing of the Analytical Performance of the MIP Chemosensor: Before measuring the resonance frequency, the cantilevers with a MIP or NIP film were dried for at least 2 h in a vacuum (≈100 mTorr), then mounted inside a vacuum chamber and kept for at least 12 h under reduced pressure (≈300 mbar) for resonance frequency signal stabilization.
In order to perform binding of the HSA analyte, the resonators with HSA extracted MIP or NIP film were immersed into HSA solution of a given concentration in phosphate buffer saline (PBS) (pH 7.4) for 2 h.Afterward, the resonators were washed with water for 15 min.
After the HSA binding experiment, the HSA molecules were extracted from the MIP or NIP film with a 3% NaOH solution for 2 h.Then, the device was washed with water for 2 h.
For efficient permeation of the solution into the pores of the MIP or NIP film during HSA binding, washing, and HSA extraction, the solution was degassed continuously by reduction of the gas pressure inside the vessel.
All resonance frequency measurements of the butterfly-like resonance mode of the cantilever chemosensors were performed optically using an MSA 500 Doppler-effect laser vibrometer from Polytec.

Figure 1 .
Figure 1.a) Structure of the P(VDF-TrFE) resonator in the form of a vibrating cantilever with a macroporous HSA-imprinted MIP film, and b) the analytical signal of the chemosensors: the measured shift of the resonance frequency of the cantilever resonator with an HSA-extracted MIP film before (curve 1 and curve 1′) and after selective binding of the HSA analyte in a 100 nm HSA solution in PBS (pH 7.4) (curve 2 and curve 2′).

Scheme 1 .
Scheme 1. Synthesis and deposition of a macroporous poly(2,3′-bithiopene) MIP film imprinted with HSA on an Au electrode at the surface of the P(VDF-TrFE) resonator.

Figure 2 .
Figure 2. Changes of current during potentiostatic deposition of poly(2,3′-bithiophene) on (a,b) Au electrodes at the surface of P(VDF-TrFE) resonators modified with 20 layers of 660 nm diameter nanoparticles forming a colloidal crystal modified with functional monomers derivatized HSA and (c) a bare Au electrode.The propylene carbonate solution contained 0.5 m of the 2,3′-bithiophene cross-linking monomer and 1.0 m of LiClO 4 supporting electrolyte.The charge density passed through the working electrode during the deposition of poly(2,3′-bithiophene) films was Q D = +210 mC cm −2 for curve a, and Q D = +326 mC cm −2 for curves b and c.

Figure 3 .
Figure 3. Top view SEM images of (a,b) macroporous MIP films composed of a) ≈6 and b) ≈10 macroporous layers synthesized with a deposition charge density of Q D = +210 mC cm −2 and Q D = +326 mC cm −2 , respectively.

Figure 4 .
Figure 4. a) Visualization of the butterfly-like resonance mode of the cantilever resonator that was chosen for monitoring the resonance frequency changes triggered by changes of the air humidity or by selective binding of the HSA analyte inside the MIP film, and b) velocity resonance spectra of the butterfly-like resonance mode of the cantilever resonator with a macroporous MIP or NIP film (quality factor was Q ≈ 17).

Figure 5 .
Figure 5.Time dependence of the resonance frequency change of the P(VDF-TrFE) resonator a) with a macroporous poly(2,3′-bithiophene) film composed of ≈10 macroporous layers (Q D = +326 mC cm −2 ) and b) with a non-porous poly(2,3′-bithiophene) film during changes of the relative air humidity (RH) and c) corresponding change of relative resonance frequency as a function of the relative air humidity (RH).The vertical arrows with %RH value indicate the moment of humidity change.

Figure 6 .
Figure 6.a) Comparison of the relative resonance frequency changes after incubation of the chemosensors with MIP and NIP films composed of ≈6 (Q D = +210 mC•cm −2 ) and ≈10 macroporous layers (Q D = +326 mC•cm −2 ) in PBS (pH 7.4) containing 20 μm HSA or 20 μm myoglobin, and b) calibration curve for the chemosensor with MIP films composed of ≈6 macroporous layers (Q D = +210 mC•cm −2 ) after incubation with 10 pm, 1 nm, 100 nm, and 20 μm HSA solutions in PBS (pH 7.4).The blue vertical dashed line represents the LOD at 3.All resonance frequencies were measured in a vacuum.