Modular Polymer Biosensors by Solvent Immersion Imprint Lithography

Abstract

We recently demonstrated Solvent Immersion Imprint Lithography (SIIL), a rapid benchtop microsystem prototyping technique, including polymer functionalization, imprinting and bonding. Here, we focus on the realization of planar polymer sensors using SIIL through simple solvent immersion without imprinting. We describe SIIL's impregnation characteristics, including an inherent mechanism that not only achieves practical doping concentrations, but their unexpected 2-fold enhancement compared to the immersion solution. Subsequently, we developed and characterized optical sensors for detecting molecular O₂. To this end, a substantially high dynamic range is reported, including its control through the immersion duration, a manifestation of SIIL's modularity. Overall, SIIL exhibits the potential of improving the operating characteristics of polymer sensors, while significantly accelerating their prototyping, as it requires a few seconds of processing and no need for substrates or dedicated instrumentation. These are critical for O₂ sensing as probed by way of example here, as well as any polymer permeable reactant.

Graphical abstract

Solvent Immersion Imprint Lithography (SIIL) enables complete microsystem prototyping in less than 1 minute with no instrumentation requirements. SIIL can also enable planar polymer biosensors through impregnation by simple solvent immersion. An unexpected solvent transport mechanism enables practical impregnation concentrations and functional O₂ sensors of controlled sensitivity and dynamic range. Overall, SIIL-made sensors necessitate no substrates or instrumentation, with typical processing times of less than 30 seconds.

Introduction

Polymer sensors have received considerable attention in recent years due to their prototyping simplicity, cost-effectiveness and compatibility with patient monitoring [1, 2]. Since their emergence more than two decades ago, numerous sensing architectures have emerged [3, 4], including novel material systems such as conjugated polymers [5], nanocolloids [6] and graphene [7]. Such advances have successfully addressed a wide variety of applications in areas such as security, health and the environment [8-10]. Generally, such modalities sense the presence or activity of an analyte either electrically or optically, with the latter being substantially more compatible with microscopic and high-throughput applications such as optofluidics [11-14] and microfluidics [15-17].

Similarly, the optical sensing of oxygen – a widespread electron acceptor in nature – has gained substantial attention [18]. Such sensors are employed in the form of thin films (i.e. planar sensors), either as ‘patches’ on containers, or directly integrated with optical fibers. Application of such planar oxygen sensors typically involve monitoring respiration kinetics in bioreactors, disease diagnostics, fuel tank stability analysis and food packaging quality control. Additionally, oxygen sensing has been successfully implemented in microfluidics for identifying the impact of oxygen tension on mammalian cells, as well as fundamental metabolic studies in microbial communities that extend even down to the single cell level [19-21]. In both cases of planar and microfluidic integrated oxygen sensors, their prototyping typically involves the spin coating of a polymer solution mixed with an oxygen sensing chromophore [15-17, 22], resulting in a thin sensing film deposited on a substrate. This includes microfluidics designed to image the microfluidic's spatial structure [16]. While the approaches based on spin-coating thin-films accurately control the sensor thickness and doping uniformity, the use of a substrate is far from ideal in applications within liquid or chemically challenging environments that may undermine the film stability, including its delamination. Alternative approaches involve the use of super-critical CO₂ mediated impregnation in bulk polymer slabs, thereby eliminating the need of a substrate [23], as well as sol-gel strategies [24].

While the previously reported approaches have successfully addressed a plethora of sensing challenges and needs, they usually require dedicated instrumentation and expertise, as well as long and multi-step processing, especially in the context of optofluidic sensors. This is particularly pertinent in addressing the inherent photobleaching of phosphorescent probes, which necessitates frequent sensing module replacement, thus decreasing cost-effectiveness. To overcome this shortcoming, we have recently demonstrated a substantially faster and simpler alternative process, termed Solvent Immersion Imprint Lithography (SIIL) [25]. SIIL is beneficial in polymer processing since it employs commonly available solvents for reducing the polymer glass transition temperature. This enables the reduction of polymer inter-chain interactions and mobility enhancement without the need for dedicated instruments operating under high temperature conditions. Thus, following solvent immersion – a single processing step lasting less than 1 minute – it is possible to prototype a complete polymer microsystem, including imprinting, bonding and 3D functionalization (or impregnation). Such advantages are pertinent to both optofluidics and microfluidics in life and energy sciences [26].

Here, we analyze polymer impregnation pertaining to planar sensing modules, one aspect of SIIL that relies only on solvent immersion without imprinting or bonding (Fig. 1a). This simple impregnation step gives rise to uniformly coated polymer slaps at depths controlled simply through the immersion duration. In the following sections, we analyze the mechanism of impregnation during solvent immersion through imaging and spectroscopy. Subsequently, we investigate the optical detection of molecular O₂ in SIIL-modified polymer slabs, and by using advanced photophysical characterizations we gain deeper insight into SIIL's modularity for controlling the sensor's dynamic range and sensitivity.

Figure 1.

(a) A schematic illustration of the SIIL mediated impregnation of the upper surfaces of a polymer slab, thereby giving rise to planar polymer sensors. (b) The dependence of the impregnation depth as a function of the solvent immersion time (t_i). The insets plot the absorption spectrum of the polymer sensor for different immersion solution concentrations and an immersion time of 30 sec (left) and displays two cross-sectional views of the SIIL sensors using confocal microscopy (right) for 10 sec and 60 sec immersion durations.

Experimental/Methodology

Sensor Fabrication

To form the sensing slabs, 1.2 mm thick polystyrene (PS) slabs were cut to approximate areas of 1 cm² (GoodFellow Cambridge Limited, UK). The slabs were then immersed in an acetone solution of the platinum porphyrin compound Pt(II) meso-Tetra(pentafluorophenyl)porphine (PtT975, Frontier Scientific, USA) at specific concentrations. The immersion duration (t_i) was controlled with a digital timer. Following immersion, the PS slabs were removed from the solvent and their edge was rapidly brought in contact with a Kimwipe for approximately 2 sec to remove the excess solvent from their surface. Subsequently, the slabs were placed between two polydimethylsiloxane (PDMS) slabs to dry under low pressure (exerted by a 500 g weight). The evaporation step was found to be as short as 30 seconds for the abovementioned polymer and solvent combination. The PDMS slabs were not structured, and were employed to avoid random morphology modifications of polymer's surface during solvent evaporation. During this process, both the upper and lower surfaces of the polymer slab are impregnated. This had no effect on the sensor characterization, which was performed using a focused excitation and collection from a single surface. We have also achieved a single-surface impregnation simply by coating one of the slab surfaces with epoxy that was straightforward to manually remove due to its poor adhesion from the polymer.

Impregnation Characterization

To characterize the impregnation mechanism, SIIL modified polymer slabs were placed on an inverted microscope (Leica DMI6000) coupled with a spinning disk confocal system (Yokogawa CSU10). A 20x objective was used and a z-scan was performed at a 2 μm step size. Additional confocal imaging was performed using the OptiGrid module on a Leica DMI6 inverted microscope, at a step size of 1.2 μm using a similar 20x objective. With both methods, the impregnation was characterized by intensity thresholding. Absorption spectroscopy was performed using a UV-VIS spectrometer (UV-1700-PharmaSpec, Shimazdu). For this, the same immersed samples were used and the absorption spectra were collected from 190 nm – 800 nm in steps of 1 nm. The imaging and spectroscopic measurements were combined to determine the doping concentration within the polymer matrix.

Sensor Characterization

The planar polymer sensors were placed in a sealed chamber, equipped with a silica window for optical access, as well as one inlet and one outlet. The inlet of the chamber was connected with gas impermeable Tygon tubes, comprising of a single long component (approx. 3 meters) to enable adequate mixing in line with two additional Tygon tubes through a V connector. The latter two tubes were connected to N₂ and O₂ gas cylinders through two separate – manually controlled – mass flow controllers (Alicat Scientific MC-55LPM-D/5M, equipped with 8-pin mini-DIN connectors). Prior to characterization, the chamber and sensing films were purged with pure N₂ for approximately 40 min. Subsequently, different O₂-N₂ mixtures were introduced and the sensor was characterized in an inverted optical microscope (Leica DMI6000) coupled with a fluorescence lifetime imaging setup (LI2CAM-P, Lambert Instruments, Netherlands). For oxygen sensing, an LED with an emission spectrum centered at 400 nm modulated at 5 kHz was employed.

Impregnation with SIIL

In SIIL processing, a polymer slab is immersed in an organic solvent for a controlled amount of time (t_i) [25]. Provided that the polymer-solvent system is properly chosen, during the immersion step solvent molecules penetrate the polymer matrix at a rate that is roughly inversely proportional to the cohesive-energy difference between the solvent and the polymer (Hildebrand solubility parameter) [27, 28]. Two outcomes of solvent penetration are essential to SIIL processing, namely [25]: (1) the gel formation in the upper layers of the polymer slab that enables imprinting/bonding, and (2) the co-transport of solutes dissolved in the solvent that gives rise to polymer impregnation (Fig. 1a).

Both the imprinting and impregnation depths can be controlled via the solvent penetration rate or the immersion duration time (t_i). We have previously explored in detail the effects of both the type of solvent and the immersion duration time (t_i) on imprinting and bonding [25]; here, our specific focus is to elucidate the impregnation process and its impact on the sensing performance of molecular O₂ [28]. Polystyrene (PS) was chosen as the polymer matrix and acetone as the solvent. The Hildebrand parameters (δ) for acetone and PS are 20.4 (J·cm^–3)^1/2 and 18.7 (J·cm^–3)^1/2, respectively. Such a solubility parameter difference allows for the regulation of the impregnation depth with immersion time (Fig. 1b and inset). In contrast, smaller (larger) differences in the Hildebrand parameters would be characteristic of much faster (practically no) mixing between the solvent and the polymer matrix, leading eventually to complete polymer dissolution. O₂ sensing was enabled by the platinum porphyrin compound Pt(II) meso-Tetra(pentafluorophenyl)porphine, dissolved in acetone under variable concentrations ranging from 0.25 mg/ml up to 2 mg/ml (see Experimental/Methodology section for more details).

Impregnation Mechanism

We initially investigated the underlying impregnation process to gain mechanistic insight into the possibilities and limitations in SILL-mediated polymer doping. To this end, PS slabs were immersed for 20 sec in an acetone-chromophore solution at various concentrations, followed by a brief drying step at room temperature between two pieces non-structured (PDMS). The impregnation depth (d) of the sensing moieties was subsequently analyzed by confocal microscopy to determine its dependence on the immersion duration (t_i) (see Experimental/Methodology section). Absorption spectroscopy was subsequently performed on the same polymer slabs (Fig. 1b – inset) in order to quantify their absorbance (A). In this way, the dopant concentration (c) in the polymer was determined through the Beer-Lambert law: $\mathrm{c}=\frac{\mathrm{A}}{2\cdot \epsilon \cdot \mathrm{d}\left({\mathrm{t}}_{\mathrm{i}}\right)}$, where ε = 8.61·10⁻⁴ M⁻¹ cm⁻¹ is the molar extinction coefficient for the porphyrin complex [29]. The factor of 2 in the denominator stems from the fact that A was measured for both surfaces of the polymer slabs since both were impregnated during SIIL at similar impregnation depths (data not shown). The results, shown in Fig. 2a, indicate that the concentration of dopant in the polymer matrix increases linearly with the dopant concentration in the immersion solution. This was confirmed by the high Pearson's coefficient of the linear fitting (r = 0.99), with a slope of approximately 2. This slope value indicates essentially that the embedded chromophore concentration in the polymer matrix is two-fold enhanced compared to that of the immersion solution.

Figure 2.

(a) The chromophore doping concentration within the polymer matrix as a function of the solution concentration; the experimental data are denoted by the blue circles, the red line indicates a linear fit and the blue line the 1:1 relationship between the chromophore concentration in the polymer and in the solution. The inset plots the enhancement factor of the polymer doping concentration as a function of the immersion time; the blue line denotes the dose-response curve. (b) A schematic illustration of the immersion and evaporation mediated transport of the chromophore solution (red circles) in the polymer slab (grey), as well as the resulting enhanced doping concentration in the polymer matrix.

To further investigate this practical enhancement mechanism, we varied the immersion time (t_i) for all different solution concentrations (0.25 mg/ml - 2 mg/ml). By taking into consideration the associated variation of the impregnation depth (Fig. 1b), we confirmed the linear correlation between the polymer doping vs. the solution concentration for all immersion durations. The slope of such linear fits (i.e. the enhancement factor) is approximately 2 for all immersion durations, with the exception of immersion durations t_i < 10 sec. This gives rise to the ‘dose-response’ curve illustrated in the inset of Fig. 2a. Such a complex dependence of the doping enhancement on t_i is indicative of the underlying polymer-solvent interactions and the resulting diffusion-mediated kinetics of the solvent penetration in the polymer [27, 28]. During solvent immersion of the polymer slab (Fig. 2b), the solvent diffuses within the polymer (to typical depths in excess of 10 μm depending on the immersion time) simultaneously carrying the chromophores as a result of the strong interactions between the dopant molecules and acetone (via π-OH, C-F^...O and H^...O bonds). Furthermore, a second solvent transport process is involved in the SIIL planar sensor development. This occurs during the drying step, when the solvent escapes the polymer slab through evaporation (Fig. 2b). This in essence reverses the solvent transportation with respect to solvent immersion. During solvent evaporation however, the chromophores are also carried towards the surface of the polymer slab through the strong solvent-chromophore interactions, overcoming the weaker chromophore-polymer interactions (via π-π bonds between the chromophore's and polystyrene's benzene rings). This 2-way transport mechanism is the likely origin of the observed enhanced chromophore concentration at layers closer to the polymer surface in comparison to their concentration in solution (Fig. 2).

Oxygen Response

To analyze the response to gaseous O₂, polymer sensors were fabricated as previously described by simple solvent immersion and drying in-between two flat PDMS stamps. The SIIL sensors were subsequently introduced in a sealed chamber, including a silica window for optical access. Therein, N₂ and O₂ gases were introduced and mixed at different ratios and the sensor response was analyzed by using a FLIM imaging set-up capable of capturing both intensity and lifetime images. A typical intensity and lifetime Stern-Volmer quenching plot is illustrated in Fig. 3a for a SIIL sensor obtained by immersing PS in an acetone solution of the sensing dye (1 mg/ml concentration). The graph plots the I_o/I and τ_o/τ namely the intensity (I) and lifetime (τ) sensor response under different O₂ concentrations with respect to the values under zero O₂ concentration (I_o, τ_o). The immersion duration was 30 sec, resulting in an impregnation depth of approximately 15±2 μm (cf. Fig. 1b). Fig. 3a illustrates that below a 20% O₂ concentration, the lifetime and intensity quenching plots essentially overlap. However, a substantial deviation is observed above a concentration of 20% O₂, indicating the involvement of both dynamic and static quenching mechanisms upon the interaction of the sensing dye with molecular oxygen [30]. This is better visualized in Fig. 3b that shows the sensor's lifetime and intensity responses in alternating exposures to pure O₂ and N₂. It is worth noting that the implication of both quenching mechanisms does not limit the sensor's dynamic range. On the contrary, static quenching enhances the intensity-based detection range – especially above a 20% O₂ concentration (Fig. 3a) – without hindering the repeatability, as also evidenced by time-lapse FLIM experiments in Fig. 3b.

Figure 3.

(a) Typical Stern-Volmer quenching plots for both the intensity (upper) and lifetime (lower) for a polymer film impregnated with the oxygen sensing dye by immersion in an acetone dye solution at a concentration of 1 mg/ml for 30 sec. (b) Time-lapse FLIM imaging of both the intensity and lifetime, where the gas flown over the sensor was manually controlled from 100% N₂ to 100% O₂; error bars denote the standard deviation of the intensity or lifetime over the FLIM image. (c) A phasor histogram plot of the same film for three oxygen concentrations, viz. 0%, 15% and 40%; the x- and y- axes denote the g_i,j(ω) = m_i,j · cos(φ_i,j) and s_i,j(ω) = m_i,j · sin(φ_i,j) components, where [i,j] are the pixel indices, and m and φ are the modulation and the phase of the emission with respect to the excitation and ω = 2·π·ν, where ν was the 5 kHz modulation frequency employed in the FLIM analysis.

Additionally, the Stern-Volmer plot exhibits a downwards non-linearity for both the intensity and lifetime curves at high O₂ concentrations (> 80%). This is a typical response from chromophores embedded in a solid matrix, manifesting the heterogeneous matrix microenvironment. The latter gives rise to combined responses from complexes that exhibit variable accessibility to oxygen [31, 32]. Such microheterogeneity is further suggested by the phasor plot of the FLIM images, illustrated in Fig. 3c for three O₂ concentrations [33]. All three measurements deviate from the phasor plot's universal circle, indicating the involvement of multi-exponential decays. Such micro-heterogeneity is anticipated during SIIL processing, similar to almost all other methods of prototyping polymer sensors (e.g. spin-coating).

SIIL Modularity

In addition to its simplicity, SIIL exhibits enhanced modularity in sensor design through the control of the immersion solution's chromophore concentration, as well as the immersion duration time. Regarding the former, the intensity quenching plot for variable doping densities at a 30 sec immersion time is illustrated in Fig. 4a. Up to approximately 40% O₂, the quenching curves overlap to a high degree indicating that all chromophores in the matrix respond in a similar fashion within this range. Above this level, however, a minor divergence between each concentration emerges, with the highest dynamic range demonstrated by the lowest immersion solution concentration.

Figure 4.

Intensity calibration plots for planar polymer sensors by simple solution immersion without imprinting. Solutions of different chromophore concentrations were employed but at the same immersion time (t_i = 30 sec) (a), as well as different immersion times at 1 mg/ml chromophore concentration in solution (b).

On the contrary, by varying the immersion duration time a considerable modulation of the sensor response is enabled. This is plotted in Fig. 4b for a PS sensor realized by immersion at a concentration solution of 1 mg/ml. The different immersion durations enable a substantially divergent response even above the 10% O₂ concentration border. The origin of this behavior emanates from the different thicknesses of the sensing layer due to the different immersion duration times, and thus the different diffusional barriers to molecular O₂ they represent (Fig. 1b). O₂ transport within the polymer is not instantaneous but rather undergoes Fickian diffusion with a constant of 4·10⁻⁸ cm² · s⁻¹ [25]. Thus, by simply controlling the immersion duration time, the sensing dynamic range can be increased more than twofold, in essence enabling one of the highest dynamic ranges ever reported [15, 22].

Conclusions

In conclusion, we reported the realization of planar polymer sensors using SIIL. The impregnation mechanism was first analyzed using microscopy and spectroscopy, unmasking a 2-fold enhanced doping concentration in the polymer matrix with respect to the corresponding one in the immersion solution. Such enhancement enables practical doping levels even under immersion in dilute solutions. We demonstrated such sensors, exhibiting a high dynamic range that could be further controlled through the immersion duration time, thus revealing SIIL's modularity. Overall, SIIL significantly improves planar polymer sensor prototyping by enabling extremely short processing duration times, while at the same time eliminating the need for substrates or dedicated instrumentation. These are critical not only in sensing – including scintillator applications – but also in chemical synthesis within polymer reactors, as well as complete optofluidic microsystems as we have previously reported [25].