Wireless and Flexible Optoelectronic System for In Situ Monitoring of Vaginal pH Using a Bioresorbable Fluorescence Sensor

Herein, a miniaturized wireless sensing vaginal ring for the in situ continuous monitoring of vaginal pH and real‐time transmission of the pH data to a smartphone is reported, aimed at the diagnosis and management of bacterial vaginosis, a common condition frequently and adversely affecting women. The sensing vaginal ring consists of a bioresorbable pH fluorescence sensor placed on top of a polydimethylsiloxane ring encapsulating a miniaturized driving/readout optoelectronic circuit, data acquisition system, wireless transceiver, and power supply. The pH sensor consists of a micrometer‐thick porous silica scaffold conformably coated with a nanometer‐thick polymer multilayer stack and is intended to be replaced after 4 days. The sensor fully dissolves in biocompatible by‐products eliminating waste management issues; conversely, the ring embedding the circuit is reusable with new sensors. The pH sensor, as well as the sensing vaginal ring, show excellent performance in the continuous measurement of pH in vaginal fluid and can monitor the pH level over the physio‐pathological range of 3–7.5 with high linearity, accuracy, and reliability, transmitting the data to a smartphone in real time. The proposed technology can be immediately translated to other diseases, among which wound healing, intragastric activity, and cancer progression, where continuous monitoring of pH is required, as well as to other markers/analytes by engineering the polymer stack with suitable receptors, such as aptamers and other molecular probes.


Introduction
The development of flexible and wearable chemical (bio) sensing systems with wireless data readout and transmission use, though have poor reliability. [18][19][20] Further, they only allow single-in-time measures and are not suitable for continuous pH tracking. On the other hand, accurate and continuous measurements of vaginal pH in situ from a few hours to days are of key importance for prevention, early diagnosis, and treatment of BV. Wireless miniaturized systems able to operate in the vagina and transmit data to a smartphone and, then, to a doctor would be game changing.
Among the different biocompatible materials that have been used for the preparation of (bio)sensors for clinical applications, nanostructured porous silicon (PSi) has been increasingly exploited over the latest two decades [21][22][23] thanks to its vast specific surface that allows accumulation of a tremendous number of molecules, [22] effective refractive index modulation that permits straightforward preparation of diverse optical structures, [23] and dissolution in safe byproducts in physiological conditions, which has enabled the in-vivo application of sensing and drug delivery devices based on PSi. [24][25][26] A few works leveraging PSi for pH optical sensing have been also reported, namely, by full infiltration [27] or top coating [28,29] of PSi with a pH-sensitive hydrogel. More recently, we reported on an implantable PSi fluorescence sensor able to monitor pH in vivo, leveraging a micrometer-thick porous silica (PSiO 2 ) scaffold conformably coated with a pH-responsive stack of fluorescent polyelectrolytes. [30] The sensor was able to perform continuous pH measurements for 4 days, then dissolve in additional 4 days. However, pH measurements were carried out with a benchtop setup featuring a discrete laser as the excitation source, a fiberoptic link to guide the laser light to the sensor and collect the sensor fluorescence, a commercial spectrometer to measure the sensor fluorescence spectrum and measure the peak intensity value.
Herein, we report on a wireless and flexible sensing vaginal ring for the continuous in situ monitoring of vaginal pH and real-time transmission of the dataset to a smartphone. The sensing ring leverages a biodegradable PSi-based fluorescence sensor able to monitor pH in the range of 3-7.5 over 4 days of continuous operation. The pH sensor is placed on top of a polydimethylsiloxane (PDMS) vaginal ring encapsulating a miniaturized driving/readout optoelectronic circuit, data acquisition system, wireless transceiver, and battery-power supply. The pH sensor, as well as the sensing vaginal ring, allow measuring pH in vaginal fluid with high linearity over the physio-pathological range of vaginal pH, namely, from 3 to 7.5, showing good accuracy and stability over time and over repeated measurements, despite the extreme acidic environment of vagina and high complexity of vaginal fluid. The pH sensor degrades in biocompatible by-products after its use, eliminating the need of handling the sensor disposal and reducing medical waste; conversely, the vaginal ring encapsulating the optoelectronic system can be reused multiple time with new sensors.

Results and Discussion
Concept of the sensing vaginal ring is sketched in Figure 1. We envisaged a sensing vaginal ring that looks like and is worn like a common contraceptive ring, yet is equipped with a fluorescence sensor to measure vaginal pH over time and with a wireless circuit to read and transmit in situ pH levels to a smartphone for data storage and visualization, allowing clinicians to monitor vaginal pH of a patient in real time.
The fluorescence pH sensor is fabricated as sketched in Figure 2a. A nanostructured PSi scaffold was prepared with a 100-nm-thick low porosity (61.5%) layer on top and a 4.5-µm-thick high porosity (76.5%) layer at its bottom. The as-prepared PSi scaffold was detached from the native silicon substrate to achieve a free-standing PSi membrane (Figure 2a (1)). [32] The porous membrane was then thermally oxidized at 1000 °C for 5 min to convert PSi to PSiO 2 and enhance surface hydrophilicity and chemical stability in a water-based environment, [31] further providing the scaffold surface with a net negative charge (Figure 2a (2)). [22,23] The PSiO 2 membrane was transfer-printed upside-down onto a flexible 2-mm-thick PDMS slab, with the low porosity layer in contact with the PDMS slab to inhibit oligomer diffusion inside the pores (Figure 2a (3)). [33] PDMS was chosen as the supporting material due to its wellknown biocompatibility, flexibility, and transparency properties. [34] Eventually, the inner surface of the PSiO 2 membrane was conformably coated via electrostatic layer-by-layer (LbL) technique with a nanometre-thick pH-responsive multilayer stack of positive and negative polyelectrolytes labelled with a  green-emitting fluorophore (Fr) (inset in Figure 2a (3)). Assessment of each preparation step was performed monitoring the changes in the effective optical thickness (EOT) of the PSiO 2 scaffold by Fast Fourier Transformation (FFT) of the reflectance spectra ( Figure S1, Supporting Information). [23,30,35] A picture of a PSiO 2 scaffold coated with the fluorescent polyelectrolyte stack on a PDMS slab, i.e., the pH sensor, is shown in Figure 2b. Figure 2c,d shows top-view and cross-section scanning electron microscopy (SEM) images of the as-prepared PSi scaffold, highlighting the morphology of low and high porosity layers with vertical columnar pores with size of 6.9 ± 2.7 and 35.9 ± 13.6 nm ( Figure S2, Supporting Information), respectively. Fr-labelled poly(allylamine hydrochloride) (PAH:Fr) and poly(methacrylic acid) (PMAA:Fr) with fluorescence peaked at ≈530 nm ( Figure S3, Supporting Information) were selected among other weak polyelectrolytes due to their well-known biocompatibility. [36,37] The multilayer stack was assembled in tris(hydroxymethyl)aminomethane (TRIS) buffer at pH 8 to fully ionize both PAH:Fr and PMAA:Fr (pKa PAH:Fr = 9.5 and pKa PMAA:Fr = 6.2). The thickness of the multilayer stack increases linearly with number of polyelectrolytes in the stack, achieving a maximum value of 6 nm with six layers, namely, (PAH:Fr/PMAA:Fr) 3 ( Figure S4, Supporting Information). Figure S5 (Supporting Information) shows bright-field and fluorescence-mode optical microscope images of the cross-section of a PSiO 2 scaffold coated with a multilayer stack of PAH:Fr and PMAA:Fr. A uniform photoluminescence (PL) emission peaked at 530 nm confirms the homogeneous coating of the PSiO 2 scaffold with the fluorescent polyelectrolyte stack over the entire thickness.
We leveraged the high specific surface of the PSiO 2 scaffold, which is ≈400 times larger than that of a flat substrate, to accommodate a massive quantity of Fr molecules per unit area and amplify, in turn, the fluorescence intensity of the multilayer stack. The PL intensity increases linearly with the number of polyelectrolyte layers in the stack, with a slope of 6862 ± 315 counts layer −1 (Figure 2e  intensity is ≈470-fold larger than that achieved from a flat SiO 2 substrate coated with the same fluorescent multilayer stack regardless of the number of layers (control, black dots, and inset in Figure 2f), consistently with the surface increase factor of the porous scaffold. No differences were observed on the PL intensity of PSiO 2 scaffolds coated with same multilayer stacks either sitting on the native Si substrate or transfer-printed on the PDMS slab, thus indicating that the assembling process of the fluorescent stack is not affected by the substrate hosting the PSiO 2 scaffold (compare red and green dots in Figure 2f).
The PSiO 2 scaffold coated with a multilayer stack of (PAH:Fr/ PMAA:Fr) 2 +(PAH:Fr) 1 was used for vaginal pH sensing, based on our former studies on this subject. [30] The pH sensor was tested in PBS buffer at 37 °C at different pH values ranging from 3 to 7.5 to mimic physio-pathological conditions of vagina. Figure 3a shows PL spectra of the pH sensor versus pH values. A monotonic reduction of the PL intensity as the pH level increases from 3 to 7.5 is apparent. Taking the peak value (at ≈530 nm) of the PL emission as the output signal of the sensor, high linearity and reliability are achieved throughout the whole pH range tested over several sensing cycles and different samples (Figure 3b). The sensitivity value extrapolated from bestfitting of experimental data is −7470. 8   on PDMS slabs. Similar sensing performance were found for pH sensors left on the native Si substrate, thus corroborating the robustness of pH sensors transfer-printed on PDMS slabs ( Figure S6, Supporting Information).
The linearity range of the proposed pH sensor is twice that of state-of-the-art fluorescent sensors based on pH-sensitive dyes, which typically feature a sigmoidal calibration curve with a limited linear range of ≈2 pH points. [38][39][40] This is due to the unique operation principle of our pH sensor that relies on the change of ionization degree and, in turn, of cohesion force of polyelectrolytes in the multilayer stack with pH, which causes the swelling (shrinking) of the polymer stack as the pH decreases (increases). As the polyelectrolyte stack swells upon reduction of the pH level, the distance of nearest neighbors Fr molecules in the stack increases, reducing the self-quenching and increasing PL intensity of the polymer multilayer; vice versa for increasing pH. To confirm shrinking/swelling of the multilayer stack as the sensing mechanism, PL intensities of PAH:Fr and PMAA:Fr solutions were investigated at different pH values. A significantly smaller variation of the PL intensity with pH in the range of 3-7.5 was measured for both the polyelectrolyte solutions, compared to that of the pH sensor ( Figure S7, Supporting Information), namely, sensitivity to pH is reduced by a factor of 93% and 98% for PAH:Fr and PMAA:Fr solutions, respectively. Figure 3c shows the time-resolved response of the pH sensor measured in phosphate-buffered saline (PBS) buffer (in a flow cell) upon step changes of the pH value from 7.5 to 3, and back. The PL intensity (at ≈530 nm) quickly changes for each tested pH value achieving a new steady state value in less than 20 min for unit pH changes. Excellent reproducibility (CV% ≈2%) and good resolution (0.2 pH points) are achieved, and no significant drift or hysteresis are appreciable over the whole pH range tested.
We next carried out a set of experiments in vaginal fluid simulant (VF) at different pH values to mimic real-setting operation of the pH sensor. [41] The sensor performed well in VF despite the complexity of the fluid containing proteins, glucose, glycerol, and several salts, with no difference in terms of linearity, sensitivity, and dynamic range with respect to the performance achieved in PBS buffer (Figure 3d,e). The slight downward shift of the calibration curve in VF compared to PBS is explainable with the increased absorption/scattering of the polyelectrolyte fluorescence emission in VF due to the presence of proteins, salts, and other components. For comparison, pH sensors left on the native Si substrate showed similar performance in VF (compare Figure 3e and Figure S6b, Supporting Information).
Stability and long-term operation of the pH sensor in physiological (pH 4) and pathological (pH 7.5) vaginal conditions were investigated by monitoring the PL intensity at ≈530 nm over 100 h of continuous operation in PBS buffer at 37 °C (Figure 3f,g). The pH of the buffer solutions was maintained constant over the full-time span, apart from specific time intervals starting at t = 0, 50, and 100 h during which the pH value of the solution at pH 4 was varied in the range 3-7.5, and back. An excellent stability was observed over >4 days of continuous operation both at pH 4 and 7.5, with a worst-case variation of 2.75% over the whole experiment (Figure 3f). Calibration curves of the sensor recorded at t = 0, 50, and 100 h from the beginning of the experiment are given in Figure 3g. The sensor response to pH is reliable over time, keeping high linearity and good sensitivity (maximum variation ≈14%) after 100 h of operation ( Figure S8, Supporting Information). These results clearly support operation of the sensor for at least 4 days for physio-pathological vaginal screening, such as BV. The sensor then fully dissolved in additional 4 days with safe byproducts, as we reported previously. [30] We then carried out a series of experiments to investigate the performance of the pH sensor upon repeated bending and twisting cycles. Specifically, we tested the sensor as prepared and after 1, 10, and 100 bending cycles at different curvature radii, namely, 1, 2, and 2.5 cm, the latter being the radius of a commercial vaginal ring; then, we tested it upon 100 twisting cycles. The pH sensor features high robustness to bending and twisting in terms of mechanical stability, adhesion, and sensing performance (Figures 3h,i; Figure S9, Supporting Information). The sensor calibration curve remains linear over the whole pH range after 100 bending and twisting cycles, with a maximum sensitivity variation of ≈12% at bending radius of 1 cm, which is consistent with the sensor reproducibility obtained over multiple measurements (Figure 3e).
A key issue for real-setting in situ measurement of vaginal pH is the coupling of the pH sensor with a miniaturized, biocompatible, wireless optoelectronic circuit able to interrogate the sensor and transmit the sensor data to a smartphone or notebook. The use of a fiber-optic link connecting the pH sensor to an external light-source for fluorescence excitation and to a spectrometer for acquisition of the PL spectra and retrieval, in turn, of the vaginal pH value is indeed not applicable in real settings.
We designed a sensing vaginal ring integrating both the pH sensor and the driving/readout circuit (Figure 4a, top). Among several intravaginal inserts, e.g., tampon, "T"-shaped Intra Uterine Device (IUD), and vaginal ring, this latter has several advantages, among which: high conformability and easy to use, which enable application of the ring in the vagina likewise a tampon, whereas IUD requires a trained healthcare professional; in situ operation up to 4 weeks, which is compatible with the lifetime of our pH sensor (>4 days), whereas a tampon has to be replaced after 8 h maximum; use during sexual intercourse, differently from a tampon that has to be removed beforehand. [42] The sensing vaginal ring consists of a thin PDMS layer featuring: i) the pH sensor on the outer surface of the PDMS layer, in contact with the vaginal fluid; ii) a 125-µm-thick polyethylene terephthalate (PET) foil containing the driving/readout optoelectronic circuit and batteries, fully embedded within the PDMS layer. The sketch is shown in Figure 4a. A light-emitting diode (LED, emission peak at 455 nm) and a photodiode (PD, maximum sensitivity at 560 nm) were placed underneath the pH sensor to ensure high coupling efficiency both for excitation and collection of the sensor fluorescence at different pH levels in the range of 3-7.5 (Figure 4b,c), as demonstrated by preliminary experiments in PBS and VF with the stand-alone optoelectronic circuit ( Figure S10, Supporting Information). The PD was provided on top with an adhesive filter film (cutoff ≈520 nm) acting as a rejection filter for the LED light (Figure 4b,c). Optical properties of the LED-PD pair, filter film, and pH sensor emission integrated in the vaginal ring are given in Figure 4c. The pH sensor is effectively excited with the LED through the encapsulating PDMS layer, which is known to have high transmittivity in the visible range ( Figure S11, Supporting Information); then, the fluorescence emission of the sensor is collected with the PD through the PDMS layer that acts as a waveguide. The PD photocurrent is eventually converted into a pH-dependent voltage signal using a transimpedance amplifier ( Figure S12, left, Supporting Information).
The sensing vaginal ring featured a diameter of 5.4 cm, thickness of ≈5 mm, width of 14 mm in the region containing the electronic circuit and 4 mm otherwise, comparable to commercial vaginal rings. [49] Pictures of the sensing vaginal ring are shown in Figure 4d. The size of the area containing the electronic circuit is larger due to off-the-shelf components used in this work, but it can be strongly reduced using an application specific integrated circuit (ASIC). Figure 4e shows the calibration curve of the sensing ring, namely, output voltage versus pH level. The output voltage linearly increases (decreases) as the pH level decreases (increases) over the range 3-7.5, consistently with the PL intensity of the pH sensor. High reliability (maximum CV% = 1.5%) and sensitivity (−1.65 ± 0.11 mV pH −1 ) are achieved in VF. Remarkably, the sensing vaginal ring showed pH sensing performance consistent with that achieved with the stand-alone electronic circuit, when tested in VF at pH values in the range of 3-7.5 ( Figure S10, Supporting Information).
To mimic in situ operation of the sensing vaginal ring, during which the pH sensor is in contact with the vaginal tissue, synthetic skin flaps were conditioned overnight in VF at pH 3 and 7.5 and coupled with the ring on the top of the pH sensor (Figure 4f inset). In this configuration, the ring provided reliable voltage values over consecutive measurements at pH 3 and 7.5 (maximum CV% ≈1%) that were fully consistent with those achieved with VF solutions (Figure 4f).
Eventually, wireless transmission of the pH-dependent voltage signal of the sensing vaginal ring to a commercial smartphone was proven connecting the optoelectronic circuit to a microcontroller (µC), also featuring an analog-to-digital converter (ADC) for signal digitalization and a wireless transceiver for signal transmission (Figure 4g; Figure S12, right, Supporting Information). The digital signal was successfully transmitted to a smartphone and the pH level measured with the sensor was displayed in real time using a home-made application (Figure 4h). Sensitivity and offset values of the calibration curve (output voltage versus pH level, Figure 4e) were used to convert the transmitted voltage value into the current pH level measured in real time. A good agreement between real and transmitted pH values is apparent in Figure 4h, which shows the data provided in real time to the user on the smartphone. This is further confirmed by the measured pH versus real pH calibration curve shown in Figure 4i and achieved over 3 different pH sensors with the wireless sensing vaginal ring, for which a slope of 1.01 indicated an excellent agreement between measured and real pH values.

Conclusions
In this work, we report on a wireless sensing vaginal ring for the continuous monitoring of vaginal pH in situ and the realtime transmission of the pH level to a smartphone. The sensing vaginal ring consists of a fluorescence bioresorbable pH sensor placed on top of a polydimethylsiloxane ring encapsulating a miniaturized driving/readout optoelectronic circuit, data acquisition system, wireless transceiver, and battery-power supply. The pH sensor leverages a micrometer-thick nanostructured porous silica membrane conformably coated via LbL assembling with a nanometre-thick multilayer stack of fluorescent polyelectrolytes to monitor the pH level in vaginal fluid with high linearity and reliability over the range of 3-7.5 for 4 days of continuous operation. We demonstrate full operation of the sensing vaginal ring in relevant environment, i.e., vaginal fluid, and wireless transmission of the pH data to a smartphone in real time without degradation of the stand-alone sensor performance. After 4 days of operation, the sensor fully degrades into biocompatible by-products, eliminating waste handling issues in an environmental-friendly approach; conversely, the ring embedding the optoelectronic circuit can be reused multiple times with new sensors.
The wireless sensing vaginal ring of this work paves the way toward the development of miniaturized biocompatible wireless systems for in situ monitoring of different analytes of clinical interest, besides pH, in vagina as well as in other body organs, leveraging the use of a porous silica membrane to amplify the fluorescence of polyelectrolytes labeled with both specific fluorophores and suitable (bio)receptors.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.