Towards the Mass Production of Molecularly Imprinted Polymers via Cost-Effective Photopolymerization Synthesis and Colorimetric Detection via Smartphone

Abstract

The need for rapid, on-site contaminant detection is becoming increasingly vital for tackling environmental and public health challenges. This study introduces an efficient method for detecting sulfamethoxazole (SMX), a widely used antibiotic with significant environmental implications. A cost-effective, scalable approach was developed using lab-on-paper devices integrated with molecularly imprinted polymers (MIPs), synthesized through an in situ photopolymerization process that was completed in just 10 min. Using only 2 mL of MIP solution enabled the efficient mass production of 100 disks. Traditional template extraction, which often takes hours or days, was reduced to just 10 min using a multichannel micropipette and absorbent fabric. The MIP-PAD achieved a limit of detection (LOD) of 0.8 µg/mL and a limit of quantification (LOQ) of 2.4 µg/mL, with measurements obtained using a smartphone-based colorimetric detection system. It exhibited excellent repeatability, with a relative standard deviation (RSD) of 3.26% across seven tests, high reusability for up to eight cycles, and recovery rates for real samples ranging from 81.24% to 99.09%. This method provides notable improvements in sensitivity, reproducibility, and environmental sustainability over conventional techniques. The user-friendly platform integrating smartphone-based colorimetric detection is highly practical for real-time applications, offering broad potential for environmental monitoring, food safety, and healthcare.

1. Introduction

The contamination of water with pharmaceuticals, particularly antibiotics, has become an escalating concern for both environmental and public health [1]. These contaminants, often originating from pharmaceutical residues, agricultural runoff, and improper disposal of medications, pose significant risks by inducing antibiotic resistance and disrupting aquatic ecosystems [2]. SMX, a widely used antibiotic in both human and veterinary medicine, is frequently detected in environmental samples, raising alarms about its persistence and ecological impact [3,4].

Given the critical need to monitor and mitigate the presence of SMX in water systems, the development of rapid, efficient, and selective detection methods is paramount. Traditional analytical techniques, such as high-performance liquid chromatography (HPLC) coupled with mass spectrometry [5], are effective, but require costly equipment, extensive sample preparation, and highly trained personnel. These limitations have driven the exploration of alternative approaches that are both cost-effective and accessible.

MIPs have emerged as promising materials for the selective recognition and extraction of target molecules like SMX [6,7,8,9]. MIPs are synthetic polymers with tailor-made recognition sites that mimic the shape and functional groups of specific analytes. However, one of the significant challenges in the MIP process is the effective removal of the template molecule [10,11], which is critical to ensuring the specificity and sensitivity of the MIP for future target recognition.

In this work, we addressed the challenge of template removal by employing a surface-imprinting technique using paper as the support for the MIP [12,13,14,15]. This innovative approach significantly reduced the time required for template extraction—from several hours to just a few minutes—while also minimizing the use of extraction solvents. The template removal process involved the application of 30 µL of methanol and acetic acid mixture using a multichannel micropipette, repeated ten times to ensure thorough extraction. The efficiency of the extraction was closely monitored through a colorimetric method [16,17], which provided visual confirmation of complete template removal. Using this approach, we achieved a more efficient, rapid, and environmentally friendly extraction process.

The surface-imprinting technique combined with photopolymerization [18,19,20] facilitates the creation of MIPs directly on paper, resulting in a sensor that is not only highly selective but also simple and rapid to produce. This method avoids many of the challenges associated with traditional extraction techniques, such as the need for large volumes of solvents and lengthy processing times. Additionally, the use of paper as a substrate offers a low-cost and easily disposable platform, making it ideal for widespread environmental monitoring.

In this study, we present an efficient method for the detection of SMX utilizing surface-imprinted MIPs on paper, synthesized via a straightforward photopolymerization process. This method provides rapid and efficient extraction of SMX from complex matrices coupled with visual detection capabilities, thereby offering a robust and user-friendly tool for environmental monitoring.

2. Materials and Methods

2.1. Chemicals and Reagents

For the synthesis of molecularly imprinted polymers (MIPs), the following reagents were procured: sulfamethoxazole (SMX) was utilized as the template molecule, methacrylic acid (MAA) with 99% purity served as the functional monomer, 2,2′-azobis(isobutyronitrile) (AIBN) was used as the initiator, and ethylene glycol dimethacrylate (EGDMA) with 98% purity acted as the cross-linker. All of these reagents were sourced from Sigma-Aldrich (St Louis, MO, USA). Dimethyl sulfoxide (DMSO) with a purity of ≥99% was obtained from Loba Chemie (Mumbai, Maharashtra, India) and used as the porogenic solvent.

For the selectivity study, sulfadiazine (SDZ), sulfanilamide (SPD), and sulfamerazine (SMZ) were purchased from Sigma-Aldrich (St Louis, MO, USA)

In the context of colorimetric SMX detection, additional reagents were also sourced from Sigma-Aldrich (USA). Sodium nitrite (NaNO₂) 99%, N-(1-naphthyl)ethylenediamine dihydrochloride (NED), and sulfamic acid were purchased from Merck, while hydrochloric acid (HCl) was sourced from Sigma-Aldrich (St Louis, MO, USA)

For the MIP extraction process, methanol (≥99.9%) were acquired from Loba chimie (Mumbai, Maharashtra, India). and acetic Acid (≥99%) were obtained fromHoneywell Fluka (Wunstorfer Strasse, Seeltze, Germany).

The water samples were collected from the Oued Malh River in Casablanca Morocco to test the applicability of our MIP-PAD method in real-world environmental conditions.

2.2. Apparatus and Image Processing

The synthesis of the MIP-PAD was conducted using a portable UV lamp that emits at a wavelength of 365 nm, sourced from Alonefire (Guangdong, China, to facilitate the polymerization process. For the subsequent colorimetric sensing of the MIP, we utilized a Samsung Galaxy J5 smartphone equipped with a 13 MP camera featuring a 28 mm, f/1.9 sensor. This setup enabled high-resolution image capture of the colorimetric reactions. To ensure consistency and accuracy in data collection, images were manually captured from a predetermined optimal distance under controlled ambient lighting conditions to minimize variability. Each image was taken in triplicate, allowing for the calculation of standard deviation (SD) and ensuring the reliability of the results. The digital analysis of color intensities was performed using ImageJ software (Java 1.8.0_345), which facilitated the automatic quantification of color intensities across the red, green, and blue (RGB) channels, providing a detailed and precise measurement of the color changes. Additionally, glass microfiber membranes with a porosity of 1.2 μm and a diameter of 47 mm, which served as the supporting material for the MIP-PAD, were acquired from FiltraTECH (Saint Jean de Braye, France). These membranes were chosen for their high porosity and chemical resistance, which are critical for the effective functioning of the MIP-PAD in various analytical applications. Fourier-transform infrared spectroscopy (FT-IR) measurements were performed using a Frontier Perkin spectrometer, recording spectra over a wavelength range of 4000 to 500 cm⁻¹.

2.3. Procedure for the Colorimetric Determination of SMX

For the colorimetric detection of SMX, a stepwise reagent addition process was employed to facilitate a diazotization reaction, resulting in a measurable color change. First, 5 µL of 1% sodium nitrite (NaNO₂) solution was added to the sample to generate nitrous acid, followed by 5 µL of 1 M hydrochloric acid (HCl) to acidify the reaction medium and promote nitrous acid formation. Next, 5 µL of 2% sulfamic acid was introduced to remove excess nitrite, preventing side reactions that could interfere with the detection. Finally, 5 µL of N-(1-naphthyl) ethylenediamine dihydrochloride (NED) was added, which reacted with the diazonium ion to produce an azo dye, leading to a color change. The colorimetric detection followed the method developed in our laboratory [21] and has been previously compared with advanced instrumental techniques, such as UPLC-MS.

2.4. Preparation of the MIP-PAD Platform for SMX

The preparation of the MIP solution began by combining 60 µL of MAA, 360 µL of 200 ppm SMX as the template, and 1.140 mL of DMSO. This mixture was allowed to self-assemble for 1 h at 4 °C to form a pre-polymer complex. Following self-assembly, 50 µL of EGDMA as the cross-linker, 20 mg of AIBN as the initiator, and 300 µL of water were added to complete the MIP solution. The fiberglass disks were cut separately using a perforator, after which the MIP solution was dispensed onto each fiberglass disk, selected for its chemical resistance and high porosity. Approximately 40 disks were arranged in a petri dish to ensure uniform UV exposure during the polymerization process. The disks were exposed to UV light for several minutes, typically achieving complete polymerization within 10 min, which formed a thin, imprinted polymer layer on the surface of the disks (see Scheme 1 and Scheme S1). This efficient method, using just 2 mL of MIP solution to produce about 100 disks in 10 min, enhanced throughput and reduced production costs. The uniform UV exposure ensured consistent performance across all disks, making it suitable for large-scale environmental monitoring. Additionally, a non-imprinted polymer (NIP) was synthesized using the same procedure, but without the SMX template, serving as a control in subsequent experiments. Figure S1 illustrates the polymer formation on the paper surface.

2.5. Template Removal

To initiate the template removal process, we employed a multichannel micropipette to dispense 30 µL of our extraction solvent using a prepared mixture of methanol and acetic acid (9:1) onto each disk. This solvent mixture was selected for its effectiveness in dissolving and removing the imprinted template molecules from the polymer matrix. To ensure thorough and complete extraction, this step was repeated ten times. This repetitive application maximized the removal of any residual template molecules, ensuring that the imprinted sites within the MIPs were left free of the template and ready for subsequent use.

For practical purposes, this method was applied to a batch of 100 disks with a total solvent volume of 30 mL. The efficiency and effectiveness of the extraction were confirmed by the consistent results observed across all disks, demonstrating that the process was thorough and reliable. This careful monitoring and repetitive extraction ensured that the MIPs exhibited optimal specificity and performance for their intended analytical applications.

2.6. Binding Experiments

To evaluate the binding capacity of the synthesized MIP-PAD and NIP-PAD, we conducted a detailed adsorption test. The MIP-PADs and NIP-PADs were placed on an absorbent tissue, and a solution containing 60 μL of SMX in concentrations ranging from 1 to 25 ppm was prepared. The suspension was applied to the PADs in three separate drops, each with a volume of 20 µL, using a micropipette to ensure precise and consistent application. After the adsorption process, SMX detection was performed using a colorimetric assay. Specifically, 5 µL of each reagent was introduced—1% NaNO₂, 1 M HCl, 2% sulfamic acid, and 1% NED—which are used to develop a colorimetric response indicative of SMX presence. The color intensity was measured using RGB (red, green, blue) channels to quantify the amount of SMX adsorbed by the MIP-PADs and NIP-PADs.

The equilibrium adsorption capacity (Q, mg/g) of the MIPs and NIPs was calculated using the following equation:

$$\mathrm{Q}\mathrm{e}={\displaystyle \frac{(\mathrm{C}\mathrm{i}-\mathrm{C}\mathrm{e})\times \mathrm{V}}{\mathrm{m}}}$$

where Qe represents the equilibrium adsorption capacity (mg/g), Ci and Ce denote the initial and equilibrium concentrations of SMX (mg/L), respectively, V is the volume of the adsorption solution (L), and m is the mass of the imprinted polymer (g).

The imprinting factor (IF), which assesses the specific recognition capabilities of the synthesized MIPs, was determined using:

$$\mathrm{I}\mathrm{F}={\displaystyle \frac{\mathrm{Q}\left(\mathrm{M}\mathrm{I}\mathrm{P}\right)}{\mathrm{Q}\left(\mathrm{N}\mathrm{I}\mathrm{P}\right)}}$$

Here, Q(MIP) and Q(NIP) represent the adsorption capacities of the MIP and NIP, respectively. The adsorption isotherm was obtained by varying the initial concentration of SMX from 1 to 25 mg/L, providing insights into the binding affinity and capacity of the MIP-PAD in comparison to the NIP-PAD. This comprehensive approach enables a thorough understanding of the MIP-PAD’s selective adsorption properties and its effectiveness in detecting SMX at various concentrations.

3. Results and Discussion

3.1. FT-IR Characterization

FT-IR characterization was conducted to verify the incorporation and functionality of the components within the support fiberglass (PAD), MIP-PAD, and NIP-PAD. This analysis confirmed the effective integration of the MIP within the PAD structure. As shown in Figure 1, in both the MIP-PAD and NIP-PAD spectra, characteristic peaks associated with poly(methacrylic acid) were observed, specifically at 1726 cm⁻¹ (attributable to the C=O stretching vibration) and 1450 cm⁻¹ (corresponding to C-H bending), indicating successful polymer synthesis. Additionally, a peak at 1000 cm⁻¹ in the PAD spectrum, attributed to the Si-O-Si bond, confirmed the presence of the microfiber glass membrane’s characteristic functional group. The coexistence of both MIP and PAD functional groups within the MIP-PAD spectrum further validated the successful integration of the molecularly imprinted polymer into the PAD platform, ensuring selectivity and stability in the device.

3.2. Real-Time Colorimetric Monitoring of Template Extraction Efficiency in MIP-PAD

During the extraction process, we employed a colorimetric method to continuously monitor the efficiency of template removal, as outlined in Scheme 2. To quantify these changes, images of the MIP-PAD were captured at regular intervals using a smartphone camera, and the color intensity was analyzed using ImageJ software (Java 1.8.0_345). This allowed for the conversion of RGB values into numerical intensity data, enabling precise monitoring of the extraction process. This visual detection approach relied on tracking changes in color intensity, which served as an indicator of the presence or absence of the template molecules within the MIP. As the extraction solvent with the MIP-PAD, the diminishing color intensity signaled the gradual removal of the template from the polymer matrix. This straightforward method allowed us to verify that the solvent had efficiently dissolved and removed the template, ensuring the MIP’s recognition sites were fully cleared and ready for future analytical applications. Figure 2 shows that after ten cycles of solvent application which required less than 15 min, the color intensity had stabilized, confirming complete template extraction. The process was time-efficient and minimized solvent consumption, making it both practical and environmentally friendly. This real-time observation ensured the MIPs were properly conditioned for precise molecular recognition, highlighting the method’s utility for rapid and scalable environmental and industrial monitoring applications.

3.3. MIPs Performance

The isothermal adsorption results for both the MIP-PAD and the NIP-PAD demonstrated a marked difference in performance, with the MIP-PAD showing significantly higher adsorption capacities across an SMX concentration range of 1 to 25 μg mL⁻¹, as shown in Figure 3. This superior performance of the MIP-PAD is attributed to its specifically designed recognition sites that selectively bind SMX, which is evident from the progressively increasing green intensity corresponding to higher SMX concentrations. This increase in color intensity confirms the effective binding of SMX to the MIP-PAD, illustrating its high affinity for the target molecule. In contrast, the NIP-PAD, lacking these specific recognition sites, exhibited lower adsorption capacities, highlighting the MIP-PAD’s enhanced selectivity and sensitivity. The imprinting factor (IF) obtained was 1.7 ± 0.2, explaining the superior performance of the MIP-PAD compared to the NIP-PAD. These findings underscore the MIP-PAD’s effectiveness as a tool for precise and reliable detection of SMX, demonstrating its practical advantages in environmental monitoring and analytical applications, where accurate and sensitive detection of specific compounds is essential.

3.4. Smartphone Detection

A range of SMX concentrations were evaluated using MIP-PAD, with each concentration tested in triplicate for precision. Color intensities were captured using a smartphone camera and analyzed with ImageJ software (Java 1.8.0_345), focusing on the RGB (red, green, blue) channels. Figure 4a illustrates the correlation analysis of red, green, and blue values with SMX concentrations ranging from 1 to 25 ppm, revealing that the green channel had the strongest correlation with the pink product, making it the most effective for quantifying SMX concentrations. Consequently, future experiments will utilize the green channel for measurement. The calibration curve for SMX detection using the PAD method is shown in panel Figure 4b, and the calibration curve for the smartphone-based MIP-PAD platform is shown in panel Figure 4c. The calibration curve was performed by plotting green intensities against SMX concentrations from 1 μg/mL to 25 μg/mL. The limits of detection (LOD) and quantification (LOQ) were calculated as 0.8 μg/mL and 2.4 μg/mL, respectively, highlighting the method’s sensitivity and accuracy for detecting SMX. The LOD and LOQ were calculated according to the formula LOD = 3σ/b and LOQ = 10 σ/b, where σ is the standard deviation of the intercept and b the slope of the calibration curve.

3.5. Selectivity Study

The selectivity evaluation of the synthesized MIP-PAD demonstrated its successful formation of recognition sites specifically tailored for SMX. This was evidenced by the significantly higher uptake capacity of the MIP-PAD for SMX compared to other structurally similar sulfonamides, which exhibited much lower adsorption capacities, as presented in Figure 5. The marked difference in adsorption can be attributed to the presence in template-shaped cavities within the MIP matrix, designed to match the size, shape, and functional groups of SMX. These cavities result from the imprinting process, where the SMX molecules served as templates during polymerization, creating highly specific binding sites. The high affinity of the MIP for SMX, as opposed to other sulfonamides, confirms the effectiveness of the imprinting process and underscores the MIP’s potential as a selective material for the recognition and extraction of SMX from complex mixtures. This selective binding is a critical feature, as it enhances the MIP’s application in environmental monitoring and analytical detection, providing a reliable tool for detecting SMX with minimal interference from other similar compounds.

The imprinting factor (IF) highlights the MIP-PAD’s enhanced binding affinity for SMX over the NIP-PAD, validating the effectiveness of the imprinting process. Meanwhile, the selectivity coefficient (α) served to quantify the MIP-PAD’s ability to differentiate SMX from other closely related compounds. As shown in

Table 1. The imprinting factor and selectivity coefficient for SMX relative to SMZ, SDZ, and SPD.

	Equation	Value
SMX IF	$IF={\displaystyle \frac{Q\left(MIP\right)}{Q\left(NIP\right)}}$	1.7
SMZ (α)	$\mathsf{\alpha }={\displaystyle \frac{Q\left(SMX\right)}{Q\left(SMZ\right)}}$	3.47
SDZ (α)	$\mathsf{\alpha }={\displaystyle \frac{Q\left(SMX\right)}{Q\left(SDZ\right)}}$	2.44
SPD (α)	$\mathsf{\alpha }={\displaystyle \frac{Q\left(SMX\right)}{Q\left(SPD\right)}}$	3.04

. an α value greater than 1 indicates that the MIP exhibits significantly higher affinity and selectivity for SMX compared to other structurally similar molecules. This selectivity is essential for the MIP-PAD’s effective performance in complex matrices containing potential interfering substances.

3.6. Repeatability

In this study, we evaluated the repeatability of the MIP-PAD by conducting a series of adsorption tests to determine its consistency and reliability, as illustrated in Figure 6. The tests involved exposing the MIP-PAD to a 25 ppm SMX solution, and the adsorption process was repeated seven times under identical conditions. To quantify the repeatability, we calculated the relative standard deviation (RSD) of the adsorption results from these seven tests, which yielded an RSD of 3.26%. This low RSD value indicates that the MIP-PAD exhibits high precision and consistency in its performance. The reliable and reproducible results confirm the MIP-PAD’s effectiveness as a sensing tool. It is well suited for applications requiring accurate and dependable measurements, such as environmental monitoring and analytical detection. The study highlights the robustness of the MIP-PAD and its capability to deliver consistent performance in practical settings.

3.7. Stability

To evaluate the stability of the MIP-PAD over time, we conducted a one-month experiment comparing its performance at two different storage temperatures, room temperature and +4 °C, as highlighted in Figure 7. The MIP-PADs were stored under these conditions, and their performance was assessed regularly throughout the month. The results demonstrated that the MIP-PAD exhibited stable and consistent performance when stored at 4 °C, indicating that this cooler temperature is conducive to maintaining the device’s effectiveness and reliability over time. In contrast, at room temperature, the performance of the MIP-PAD began to decline after one week, suggesting that higher temperatures affect its stability. This decrease in performance highlights the importance of optimal storage conditions for ensuring the MIP-PAD’s long-term functionality. The findings underscore that 4 °C is preferable for preserving the MIP-PAD’s performance, ensuring accurate and dependable results, while room temperature storage may compromise its efficacy, necessitating careful temperature control for effective environmental monitoring and analytical applications.

3.8. Reusability

To evaluate the reusability of the MIP-PAD, we conducted extensive testing to determine its effectiveness over multiple cycles of use. The MIP-PAD underwent a series of adsorption and desorption processes involving exposure to sulfamethoxazole (SMX) solutions and subsequent regeneration with the solvent. This cycle was repeated more than seven times, and the results demonstrated that the MIP-PAD maintained consistent performance throughout these cycles. The ability of the MIP-PAD to function reliably for over seven cycles is of significant importance, as it highlights the device’s durability and efficiency in repeated applications, as presented in Figure 8. Reusability enhances the cost-effectiveness of the MIP-PAD by reducing the need for frequent replacements and extending its practical utility in ongoing environmental monitoring and analytical tasks. This characteristic ensures that the MIP-PAD remains a valuable tool for accurate and reliable detection and quantification of target molecules, such as SMX, across multiple uses, thereby contributing to more sustainable and economical analytical practices.

3.9. Real Samples

To assess the real-world applicability of the developed method for detecting SMX using the MIP-PAD as the sensing platform in combination with a smartphone readout tool, we conducted tests on river water samples. This testing was essential for determining the method’s effectiveness in complex, real-world environments that might include various interfering substances. The river water samples were spiked with known concentrations of SMX, and detection was performed using the MIP-PAD. The results were promising, with recovery rates ranging from 81.24% to 99.09%, demonstrating that the MIP-PAD can accurately detect and quantify SMX in environmental samples. Furthermore, the method showed good precision, as evidenced by the low RSD, which was below 6%. These low RSD values indicate the method’s consistency and reliability across multiple trials, emphasizing its robustness in real-world scenarios. The high recovery rates and low RSDs confirm that the MIP-PAD method paired with smartphone-based colorimetric detection provides a precise and accurate tool for monitoring SMX in environmental waters. This approach offers a cost-effective and portable solution for on-site environmental analysis and ensures high reproducibility, making it a practical option for widespread environmental monitoring. As shown in

Table 2. Data collected from analyzing river water samples for SMX extraction using MIP-PAD. Results are presented as means ± standard deviation based on three independent measurements (n = 3).

Sample	Added (µg·mL−1)	Found (µg·mL−1)	Recovery (%)	RSD (%) (n = 3)
	5	4.95	99.09	5.74
River Water	10	8.12	81.24	2.25
	25	22.82	91.29	3.08

, the data further support the method’s effectiveness, highlighting its potential for practical application in detecting pharmaceutical contaminants in water.

4. Conclusions

This study introduces an innovative approach for the rapid and scalable production of lab-on-paper devices integrated with MIPs, using SMX as a proof of concept. We achieved precise molecular recognition and enhanced detection accuracy in just 10 min by utilizing an in situ photopolymerization technique. This rapid synthesis accelerates the mass production process and improves the efficiency of template removal. The extraction process is notably quick and uses minimal solvents, enhancing the method’s practicality.

Due to its specific recognition sites, the MIP-PAD exhibited significantly higher adsorption capacities for SMX than NIP-PAD. Stability testing showed that storing the device at 4 °C preserved its performance, whereas storage at room temperature resulted in a performance decline after one week. Real-world testing on spiked river water samples demonstrated recovery rates ranging from 81.24% to 99.09% and low RSDs below 6%, underscoring the method’s precision and reliability.

Overall, the MIP-PAD offers a cost-effective, scalable solution with high sensitivity and precision, making it a valuable tool for environmental monitoring and analytical applications. This technology holds promise for further expansion beyond SMX detection, with potential applications for other molecules and analytes in various fields, including food safety, healthcare, and industrial monitoring. Future research will focus on optimizing the platform for a broader range of target compounds and enhancing its integration into diverse analytical systems.