1 Introduction

Phenylketonuria (PKU) stands out as one of the common inherited metabolic disorders affecting children and adults worldwide. Globally, it is estimated that 0.45 million people have PKU, with a global prevalence estimated at 1:23,930 live births [1]. If it is left untreated, excessive blood Phe accumulation can cause physiological, neurological and intellectual disabilities [2]. The primary cause of this disorder is the deficiency of the phenylalanine hydroxylase (PAH) enzyme, which plays a significant role in converting phenylalanine (Phe) to tyrosine to stabilize the body condition [2, 3]. The inability to metabolize Phe, an essential amino acid, leads to its accumulation in the body and can result in potential permanent or temporary neurological and physical damage. Only a small percentage of the population carrying PKU genes must be homozygous dominant to exhibit symptoms [4]. PKU can cause various developmental disorders such as hyperactivity, seizures, microcephaly, and changes in skin pigmentation [5, 6]. Traditionally, monitoring and treatment of metabolic errors have been a central focus of major scientific centers. These centers delay monitoring and unnecessarily make patients and their families dependent on medical authorities. The gap between centers and patients should be narrowed with a more focused approach to alleviate the daily challenges of these chronic diseases [7]. However, the gap between centers and patients should be narrowed with a more focused approach to alleviate the daily challenges of these chronic diseases. Moreover, recent studies have raised serious concerns about the safety of high blood Phe levels, which are often relied upon in PKU screenings through costly, time-consuming and frequent blood tests [8].

Methods for diagnosing PKU in patients include newborn screening procedures, the Guthrie bacterial inhibition assay conducted within the first days of life [9], high-performance liquid chromatography [10, 11], and tandem mass spectrometry analysis [12]. However, these detection methods are expensive for regular monitoring and involve cumbersome sample preparation processes. Additionally, analyzing the test results can take several days, delaying treatment for many PKU patients. Therefore, it is ideal to develop a fast, easy, low-cost, and instrument-free test to monitor Phe levels [13]. The current treatment for PKU revolves around a strict dietary regimen aimed at maintaining blood Phe levels between 120 and 360 µM, significantly lower than levels observed in individuals without PKU [14]. This dietary management begins in infancy and continues throughout life, involving the intake of low- Phe foods and nutritional supplements [15]. The foods with high protein, such as meat, fish, chicken, milk, eggs, beans, and nuts cause high level of Phe for the people with PKU disease. Recent research has shown the benefits of lifelong implementation of this, predominantly childhood dietary management, as it not only controls blood Phe levels but also reduces the risk of various physical and mental health problems [4, 15]. However, concerns about potential neurological complications persist after discontinuation of Phe -restricted diets [16,17,18]. Regular monitoring of Phe levels, particularly during infancy, is achieved through blood tests that become less frequent as the child grows older [14]. Given the detrimental effects of high Phe levels, especially on the central nervous system, new approaches aimed at improving existing treatment methods are being explored. On the other hand, recent studies on enzyme-based diagnosis and treatment of PKU have been noteworthy. Particularly, phenylalanine ammonia lyase (PAL) is considered a potential substitution therapy [13, 19, 20]. However, the complexity of the extraction process, low availability, and poor stability have limited the use of PAL in the detection and treatment of PKU [13]. Additionally, although enzyme-inorganic hybrid nanoflower (HNF) technology, presented as a new enzyme immobilization method, has gained prominence, it has been reported that it can obstruct mass transfer between enzymes and substrates, leading to conformational changes in enzymes, which in turn results in low immobilization efficiency and activity [21, 22].

The complexity, special requirements and impracticality of existing laboratory-based monitoring methods for resource-constrained environments have led to the development of test formats [23, 24]. Therefore, the development and adoption of adaptive test formats, such as sensors, offer promising alternatives with advantageous features. Their advantages include low cost, biodegradability, biocompatibility, flexibility, ease of manufacturing, immobilization, speed, accuracy, high sensitivity and specificity [25, 26]. Biosensors have garnered attention as promising analytical devices for the detection, quantification, and monitoring of specific chemical species in clinical, environmental, and industrial analyses [27]. Although paper-based biosensors are known to have garnered significant interest due to their easy-to-use systems [23], Polycaprolactone (PCL) sensors, one of these biosensors, have the advantage of being easy to prepare, having excellent chemical resistance, biological compatibility, and good physical properties. Due to these properties, PCL is suitable for polymer regeneration and medical applications. PCL has excellent properties for nanofiber tissue, bone regeneration, gum regeneration, and drug delivery systems due to its chemical characteristics. PCL has a melting point of 60 °C and glass transition temperature of -60 °C, with semi-crystalline aliphatic thermoplastics. PCL is non-toxic, biodegradable in soil, has a wide range of miscibility, mechanical compatibility with other polymers, and can be greatly combined with a wide range of polymers [28]. In a study where PLC-based biosensors were developed to monitor the stress levels of fibroblast cells, it was emphasized that the low-cost biocompatible polymeric sensor contributed to the in-situ examination of various cellular events through enzyme immobilization and that its successful use paved the way for the development of new biosensors [29]. Additionally, highly sensitive PLC-based biosensors have been constructed for the specific detection of carcinoembryonic antigen glycoprotein, an important biomarker for the detection of Lvarious malignancies, and satisfactory results have been achieved [30]. In another study, an enzyme-modified biosensor based on phenylalanine dehydrogenase (PDH) is also used for the detection of phenylalanine [31]. This facilitates the use of doped modifications that can be applied to sensors for high efficiency and sensitivity in PCL-based biosensors. Although the role of the PCL-based biosensor systems in the diagnosis of PKU is still unknown, it is believed that iron(III) chloride homogenized with highly porous PCL structure is the primary reason of detecting the Phe levels.

Given the global prevalence of PKU and the challenges associated with current diagnostic and therapeutic approaches, there is an urgent need for innovative solutions for the levels of Phe in blood, urine, sweat and saliva. This study aims to meet this need by developing a low-cost, user-friendly urine test biosensor for PKU patients. Iron (III) chloride and porous PCL-based biosensors were used to detect PKU and enhance sensitivity to the presence of L-phenylalanine in urine. Biosensors were developed and tested with PKU diagnostic solutions based on color changes. Tests were performed with various concentrations of PKU diagnostic solutions. PKU detection was achieved by quantitative measurement of colors using red, green, and blue (RGB), hue, saturation, and lightness (HSL), and cyan, magenta, yellow and black (CMYK). The additional accuracy of this test method was achieved by applying a specialized device controlling light. Additionally, the effect of iron (III) chloride combination with PKU diagnostic solution concentrations at different ratios on confirming the presence of L-phenylalanine was investigated. Test results indicate that the proposed method can be easily, safely, and affordably implemented by an ordinary individual at home. By utilizing advanced technologies and new methodologies, such biosensors aim to facilitate timely intervention and improve patient outcomes by enabling real-time monitoring of Phe levels.

The novelty of this work is that for the first time, iron (III) chloride – based liquid and porous PCL biosensors were produced using a salt-leaching method and their sensitivity values were analyzed using RGB, HSL and CMYK color models. Test results indicated that any small changes in the Phe level of the solutions could be predicted using the color charts. It is believed that this approach can accelerate the Phe detection levels in the urine of PKU patients with high accuracies in fast and affordable manners. In the future, when the process is fully developed, color models can be integrated into a smartphone to detect the Phe level using the urine samples instantly.

2 Experiment

2.1 Materials

The materials used in the experimental procedure include dimethylformamide (DMF), with a density of 0.948 g/mL and a molecular weight of 73.10 g/mol; 98% pure iron (III) chloride (FeCl3) and poly(caprolactone) (PCL) with an average molecular weight of 70,000 GPC purchased from Scientific Polymer Products, Inc., polyvinylpyrrolidone (PVP) with an average molecular weight of 40,000 GPC purchased from BioPLUS Chemicals, at least 98% pure L-phenylalanine purchased from Sigma Aldrich, sodium chloride (NaCl) from Morton, and deionized water. These materials were used as-is without any further purification or modification.

2.2 Production of biosensors

The liquid-based biosensor is based on an iron (III) chloride solution sensitive to the Phe solution. The iron (III) chloride solution is prepared by adding iron (III) chloride to DI water. The concentrations of the iron (III) chloride solution depend on the amount of FeCl3 added to DI water. During the preparation of the biosensor, FeCl3/DI concentrations containing 0.1%, 1%, and 10% FeCl3 by weight were prepared. Detailed weight measurements of the concentrations are provided in Table 1. Additionally, the schematic steps of the experimental procedure are illustrated in Fig. 1.

Table 1 Weights of materials used to make different concentrations of liquid-based biosensors
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Fig. 1
figure 1

Experimental sematic flowchart of the fabrication process: a production of biosensor, b preparation of PKU diagnostic solution, and c RGB, HSL, and CMYK color-based methodology

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For the PKU biosensor, a polymeric solution was prepared in a glass beaker using 7 g of PCL in 20 g of DMF with magnetic stirring at 60 °C. After the PCL was completely dissolved, 3 g of PVP was added to the solution. The final polymeric solution is obtained when all polymers are completely dissolved in DMF, and then 5 g of NaCl is added to the polymeric solution to make the porous biosensors. After adding NaCl, the polymeric solution is continuously stirred with a magnetic stirrer for 20 min. DMF cannot dissolve NaCl particles in the polymeric solution, thus increasing the porosity of the biosensor. The prepared polymeric solution, along with NaCl particles, was transferred to a lip-cylinder glass container in a thin layer. The polymeric solution was left in a laboratory oven at 60 °C for 15 h for the evaporation of DMF. The resulting product was then cut into rectangular pieces measuring 2.25 × 1 cm2 in length and width. The cut rectangular pieces were then soaked in DI water for 12 h to dissolve the NaCl particles to prepare highly porous PCL structures (approximately 25–35% porosity), and then dried at room temperature for 1 h to remove the DI water in the biosensor. The prepared polymer layer was immersed in iron (III) chloride solutions for 12 h to add iron (III) chloride at different concentrations (complete soaking). The polymer layer with added iron (III) chloride was dried in an industrial oven for 12 h. The dried polymer layer completes the production of the PKU biosensor in its final form (Fig. 1a).

2.3 Preparation of PKU diagnostic solutions

The PKU or Phe solutions were prepared by heating it in the laboratory to 60 °C and then placing it into DI water using a magnetic stirrer. To increase the solubility of the solution, L-phenylalanine was added to the heated DI water and thoroughly mixed well. The PKU diagnostic solution, once completely dissolved, appeared quite clear and resembled water. The obtained solution was cooled to room temperature and made ready for testing (Fig. 1b). Table 2 provides information for different PKU diagnostic solutions with varying percentages.

Table 2 PKU diagnostic solutions with various percents by weight
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2.4 Color-based biosensors

Color changes were tested by dripping the prepared Phe solutions on the PKU diagnostic biosensors. After dripping the Phe at intervals, the PKU biosensor was removed from the solution, dried at room temperature, and the surfaces were recorded using a digital DSLR camera. The color changes on the biosensors were examined using images taken at different time intervals during the experiments. Tests of each biosensor with different iron (III) chloride solution concentrations were conducted. Subsequently, to increase test sensitivity and facilitate obtaining test results within hours compared to the typically longer laboratory tests lasting several hours and even days, the RGB, HSL, and CMYK values were determined using the online image analyzer and evaluated. These color space analysis approaches accelerate color extraction capability and are known to expedite the progress of colorimetric technology, thereby eliminating unnecessary time wastage [32]. In this part of the study, RGB, HSL, and CMYK color analyses were subjected to images via smartphone software to determine the level of PKU disease at various concentrations. The polymer percentage was varied in the preparation of the biosensor, and the biosensors were tested again following a similar process on the PKU diagnostic solutions with different percentage values.

3 Results and discussion

3.1 Effects of FeCl 3 concentration on liquid biosensors

The response of FeCl3 concentration on the biosensor solution was examined by adding only 4% PKU to the solution [8]. The observed color changes in the liquid-based biosensor, contributed by 0.1%, 1%, and 10% FeCl3 by weight, before and after the addition of Phe solutions, are shown in Fig. 2. It can be observed from the "before" images that there is a noticeable color change in the biosensor concentration with the increase of FeCl3 concentration from 0.1% to 10%. When Phe solution is added to the 0.1% iron (III) chloride solution, a noticeable color change in the solution over time can be observed, indicating that PKU disease can be rapidly diagnosed with a mixture of 0.1% FeCl3/Phe solution (Fig. 2a). Upon increasing the solution concentration to 1% FeCl3, it was observed that the color change in the solution occurred immediately after the addition of the PKU diagnostic solutions, albeit somewhat difficult to detect from some images depending on the waiting time and optimum color changes, slight differences in color transitions were detected during the application. When the Phe solution was added to the liquid biosensor with 10% FeCl3 by weight, it became increasingly difficult to track the physical color transitions (Fig. 2c). However, the changes that were actually present but not visible to the naked eye were re-examined through RGB, HSL, and CMYK color analyses. As a result, the liquid-based biosensor with FeCl3 can significantly change the colors of the solution and can be easily influenced by the concentration of iron (III) chloride/L-phenylalanine solution with different time intervals. These color changes depending on Fe+3 concentration are known to affect the sensitivity and response time of the biosensor [33]. Besides, it was also reported that the use of FeCl3 enhances the stability and functionality of implantable biosensors for various patients [34].

Fig. 2
figure 2

Before and after the additions of 4% Phe solutions to different weight percentages of FeCl3 concentrations: a 0.1%, b 1%, and c 10%

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3.2 Effects of Phe solution concentration on biosensors

The effects of Phe solution concentrations on the liquid biosensor was tested with a 10% FeCl3 addition, and the physical color changes are provided in Fig. 3 [8]. It is observed that dripping 0.25% PKU diagnostic solution onto the biosensor did not produce any color change over time (Fig. 3a). Furthermore, it is seen that a 1% concentration of Phe solution also did not induce many changes in the liquid sensor (Fig. 3b). However, a significant color change occurred when using a 4% concentration of PKU diagnostic solution (Fig. 3c). Allowing the FeCl3/4% PKU diagnostic solution to sit for up to 120 min did not lead to any further physical color change. For rapid diagnosis of PKU disease, it is evident that additional color analyses would be required for 0.25% and 1% PKU diagnostic solutions for the real test conditions. It can be suggested that a noticeable speed of PKU diagnosis can occur when the PKU diagnostic solution concentration is relatively high. In conclusion, it is observed that PKU diagnostic solution at low concentrations does not contribute significantly to the color change of the liquid-based biosensor with the current conditions. New approaches, materials, methods, and more sensitive color change analysis systems should be incorporated into the new model with the higher detection limits.

Fig. 3
figure 3

Before and after the addition of different percentages of the PKU diagnostic solutions to 10% FeCl3 biosensor: a 0.25%, b 1%, and c 4%

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3.3 RGB, HSL, and CMYK color analyses on biosensors

The color change induced by the PKU diagnostic solutions in the liquid biosensors formed the basis of color mode analyses. The RGB, HSL, and CMYK analyses of the samples were calculated at regular intervals from images taken from the surfaces, and this method represents a new, accurate, and fast technique for early diagnosis of PKU disease. Additionally, in previous studies, the color variations of bloodstains have also been examined using similar approaches, and it has been observed that the reduction in the brightness of the blood sample over time, up to 11 days, has been carried out using similar color analyses [35]. In another study, the quantitative analysis of urinary glucose was conducted simply and quickly by using RGB analysis of the solution's color values without the need for special materials or scientific instruments, specifically for diabetic patients. The results emphasized that this method could be used by diabetic patients for daily self-monitoring of their urinary glucose levels [36]. Therefore, directly reflecting the color values has been pursued as a quick way instead of making detections with a spectrophotometer or other characterization methods. The color analyses of the 0.1% FeCl3/4% PKU diagnostic solution are presented in Fig. 4. In the RGB color model (Fig. 4a), concentrations after PKU addition changed as the waiting time increased for a better saturation. The R parameter showed minimal change over time, while the G and B parameters exhibited a decreasing trend. In another study where color mode analyses were introduced as a new technique in analytical chemistry, it was emphasized that the HSL model is more intuitive than the RGB model [37]. In the HSL model in Fig. 4b, it can be observed that the S parameter reached saturation after a 120-min waiting period, while the values of H and L modes decreased. In the CMYK model, the C parameters could not be detected on the images and were recorded as zero (Fig. 4c). However, while the Y parameter reached saturation between 20–120 min, the M parameter reached saturation between 40–120 min. No significant change was observed in the K parameter after the addition of the PKU diagnostic solution. Furthermore, a "dominant color" template showing approximately how the color of the solution changed over time is provided in Fig. 4d. From the dominant color template, it can be observed that the color of the solution darkened over time. The changes in color modes and the physical color of the solution after the addition of 4% PKU diagnostic solution to the biosensor containing 0.1% FeCl3 are evidence of the rapid detection of PKU disease in a patient.

Fig. 4
figure 4

Color index analysis for 0.1% FeCl3 / Phe solution-based biosensors: a RGB, b HSL, and c CMYK color values

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The RGB, HSL, and CMYK color values along with the dominant colors of the 1% FeCl3 / PKU diagnostic solution are provided in Fig. 5. Following the addition of the Phe, a decrease in the R, G, and B parameters of the RGB values was observed. These dips correspond to color changes from bright to dark colors [35]. There was almost no significant change in the R, G, and B parameters over time after the addition of the Phe (Fig. 5a). This indicates that the concentration of the biosensor containing 1% iron (III) chloride almost remained unchanged over time after the addition of the PKU diagnostic solution (Phe). When the HSL color chart data was evaluated, it was observed that although the H, S, and L parameters showed decreased values compared to before the PKU diagnostic solution, they did not exhibit a clear behavior depending on the waiting time after the addition of the PKU diagnostic solution (Fig. 5b). Therefore, a brief interpretation can be made from the HSL color analysis of the biosensor containing 1% iron (III) chloride immediately after the addition of the PKU diagnostic solution for the interpretation of the disease. In the CMYK analysis, it was observed that the M parameter increased after the addition of the Phe solution (Fig. 5c). Although the Y parameter appeared to decrease compared to the beginning, no significant change was observed when considering the waiting times. Conversely, there was an increase in the K parameter after the addition of PKU, but again, no significant change over time was observed. Upon examining the dominant colors occurring in the biosensor, it was found that a noticeable color change occurred immediately after the addition of the Phe solution. Furthermore, there was no gradual change observed in color concentration depending on the waiting time.

Fig. 5
figure 5

Color index analysis for 1% FeCl3 /Phe—based biosensors: a RGB, b HSL, and c CMYK color values

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The results of color analyses after adding the Phe to the 10% FeCl3 concentrated liquid biosensor are presented in Fig. 6. In the RGB analysis, a decrease in the R and G parameter values was observed after the Phe solution, while an increase was noted in the B parameter (Fig. 6a). However, no significant changes in parameter values were observed over time. A similar trend of decrease and increase was identified in the HSL and CMYK color analyses of the 10% FeCl3 / Phe, and similarly, no significant time-dependent differences were found in color parameters (Fig. 6b-c). The dominant color change table in Fig. 6d indicates that the absence of a noticeable color transition after the addition of the Phe solution confirms the parameter stability obtained in the color analysis depending on the waiting time. The initial state of color change after adding the Phe solution to the 10% FeCl3 biosensor could signify the presence of the disease process. Evaluating the results through time-dependent color analyses and capturing similar parameter changes will provide definitive information regarding the patient's PKU disease for the fast and affordable detection.

Fig. 6
figure 6

Color index analysis for 10% FeCl3 / Phe solution-based biosensors: a RGB, b HSL, and c CMYK color values

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Color analyses depending on the waiting time of the biosensor before and after the penetration of 1% Phe solution onto the liquid biosensor containing 10% FeCl3 are presented in Fig. 7. When examining the time-dependent RGB values of the biosensor, apart from a slight increase in the B parameter compared to before PKU penetration, there was no significant change in other parameters (Fig. 7a). Although a slight decrease was observed in the H and S parameters, the S parameter remained almost constant in pre- and post-penetration states (Fig. 7b). When examining the CMYK data, fluctuations in the Y index depending on the waiting time and unstable data in the M and K indices have limited the use of the biosensor with 10% FeCl3/1% PKU additive ratios in diagnosing PKU disease (Fig. 7c). When analyzing the dominant color groups, it is observed that the color spectrum is almost similar tones with each other, which has made it difficult to use this sensor for disease diagnosis based on color change (Fig. 7d). It is also known that some sensitivities were not captured in studies based on color histogram analysis. In a study presented for the automatic detection of bleeding regions based on color analysis, it was observed that it could not eliminate some areas considered non-bleeding but very small [38]. It is also known that researchers focus on pixel-based different feature extraction techniques to solve these problems for a precise detection [39].

Fig. 7
figure 7

Color index analysis for 10% FeCl3 / 1% Phe solution-based biosensor: a RGB, b HSL, and c CMYK color values

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Time-dependent color analysis results when 0.25% Phe is dropped onto the liquid biosensor are presented in Fig. 8. Similar to the biosensors with 1% Phe, the color analysis results of the 0.25% Phe could not achieve stability with a better detection. In the RGB analysis, there were slight increases in the parameters compared to the pre-PKU dropping state, while decreases were observed depending on the waiting time (Fig. 8a). The parameter values in other color analyses did not provide the expected response despite the presence of the PKU diagnostic solutions and waiting time for a better saturation (Fig. 8b-c). Furthermore, the close proximity of the color tones in the dominant color group can be allowed for the use of the 10% FeCl3-doped biosensor for disease diagnosis (Fig. 8d).

Fig. 8
figure 8

Color index analysis for 10% FeCl3 / 0.25% Phe solution-based biosensors: a RGB, b HSL, and c CMYK color values

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3.4 Evaluation of PCL-based highly porous solid biosensors

Following the assessment of liquid-based biosensors, the surfaces of PCL-based polymer (solid) biosensors were examined to detect the presence of L-phenylalanine in the PKU diagnostic solutions. The presence of L-phenylalanine in the solutions was detected through color changes in PCL-based biosensors. These color changes also indicate that polymer sheets doped with FeCl3 serve as a biosensor for the diagnosis of PKU disease. Images taken from the surfaces after the color changes resulting from doping the biosensors with different concentrations of FeCl3 and PKU diagnostic solutions were identified as is shown in Fig. 9. Considering the color changes in the liquid-based biosensor, significant color changes did not occur on the surfaces of the PCL-based biosensors containing 0.1% and 1% FeCl3 and droplets of 4% PKU diagnostic solution depending on the waiting time (Fig. 9a-b). The immediate and time-dependent changes after dripping the same concentration of PKU diagnostic solution onto the 10% FeCl3-based biosensor are provided in Fig. 9c. Similar color changes were observed when 1% PKU diagnostic solution was added to the same biosensor (Fig. 9d). Furthermore, noticeable changes in color tones are evident when the PKU ratio is further reduced (Fig. 9e). The summary of these color changes implies that even in solutions containing low levels of L-phenylalanine, detection is possible, suggesting that the diagnosis of PKU disease can be made at earlier stages; however, more studies are needed for further explorations.

Fig. 9
figure 9

Images showing the PLC based biosensors' surfaces after dropping the Phe solutions

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To reveal the differences in color changes in PCL-based biosensor surface examinations, RGB, HSL, and CMYK index analyses of the samples were conducted. Among the samples with similar color tones in the index analyses of the biosensors, PCL-based biosensors with concentrations of 0.1% FeCl3 / 4% PKU, 10% FeCl3 / 4% PKU, and 10% FeCl3 / 0.25% PKU were selected after surface examinations. The index analyses of the 0.1% FeCl3 PLC biosensor, depending on the waiting time, are provided in Fig. 10. Noticeable differences in index parameters are observed in the first 30 min of RGB analysis (Fig. 10a). Furthermore, after 30 min, the values of the index parameters begin to decrease over time. In HSL analyses, there is a significant increase in the H parameter during the 30–120 min waiting periods (Fig. 10b). However, there is almost no change in the S and L parameters. This indicates that the low FeCl3 addition in the HSL analysis did not induce a change in all index parameters. A single HSL test conducted alone may not be sufficient for diagnosing the disease in this case. In CMYK tests, fluctuations in index parameters over time are observed (Fig. 10c). These changes can be associated with the progression of the disease when all index analyses are evaluated together. When the dominant colors of the samples are visually examined, it seems difficult to physically detect color changes (Fig. 10d). However, the changes in color index analyses actually serve as evidence of existing color changes. To increase the sensitivity of these biosensors, urine samples can be evaporated to increase the concentrations of the phenylalanine level for a better detection. Evaluating colored spots where markers localized in specific subcellular regions, such as the nucleus, cell membrane, cytoplasm, provides important information for cancer assessment. Hence, analyzing color models for microscopic digital image processing in the literature is used for the diagnosis of cardiovascular risk factors, lung, breast, and colon cancers, and diseases such as brain tumors [4, 40,41,42,43].

Fig. 10
figure 10

Color index analysis for 0.1% FeCl3 / 4% PKU PLC-based biosensors: a RGB, b HSL, and c CMYK color values

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Color analyses of the PCL biosensors with the highest FeCl3 ratio are presented in Fig. 11. In the RGB tests of the sample, it is observed that the green and blue indices show a continuous increase while the Red index tends to remain constant (Fig. 11a). In the H parameter, an increase related to the waiting time is observed, while decreases are observed in the S and L parameters (Fig. 11b). In CMYK analyses, while the M and Y indices decrease over time, the K parameter follows an almost constant value (Fig. 11c). When the dominant colors of the biosensor over time are examined, the color change within the first 30 min is clearly evident (Fig. 11d).

Fig. 11
figure 11

Color index analysis for 10% FeCl3 / 4% PKU PLC-based biosensors: a RGB, b HSL, and c CMYK color values

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When the concentration of the Phe solution is 0.25%, the response of the 10% FeCl3-doped PCL-based biosensor over time is shown through color analysis in Fig. 12. It is observed that the behavior exhibited by the sensor in color index analyses is almost identical to that exhibited by 4% Phe solution (Fig. 12a-c). In other words, it is understood that the PCL-based biosensor containing 10% FeCl3 provides positive signals for the diagnosis of the disease in both high and low PKU concentrations. In the representative dominant colors of the test sample, differences in color tones are observed, indicating a promising result for monitoring PKU disease (Fig. 12d). After the full development of these liquid and solid biosensors and optimization processes, an App can be created and downloaded on the smartphone for the fast and affordable detections of Phe levels in the body using a simple urine sample. In this way, PKU patients can balance their diet continuously and comfort their health for the rest of their lives.

Fig. 12
figure 12

Color index analysis for 10% FeCl3 / 0.25% PKU PLC-based biosensors: a RGB, b HSL, and c CMYK color values

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3.5 Cost analysis for biosensors of PKU disease

Concerns regarding the cost-effectiveness of frequently collected fee-for-service samples from patients for PKU screenings and their reflection of disease outcomes have highlighted the necessity for a low-cost and rapid PKU disease diagnostic sensor. Therefore, following the successful results obtained from liquid and solid (PCL-based) biosensors designed for PKU disease, it has become imperative to determine the estimated costs of biosensors, as well. Assuming the establishment of biosensor production facilities, an approximate cost table for a biosensor sample is provided in Table 3. The total cost of materials to be used in the PKU metabolic disorder detection system is $7.99 for producing 10 biosensor samples. The operational cost of running the equipment for producing ten samples is approximately $35. The storage cost for ten samples is $6.82. It is assumed that there may be various costs totaling approximately $11.17 for the biosensor. The total cost of producing 10 test samples is $60.98. The ultimate cost of producing a PKU detection sample/biosensor under current conditions is approximately $6–8. Since material and labor costs can vary monthly with the regions, disease and level, the cost analysis is an estimated cost for production and not an exact amount. Considering that in the US, the annual cost per individual is estimated to be between $50,000 and $120,000, the results of the obtained low-cost analysis offer a highly effective and economical solution for the diagnosis of PKU disease for various patients [44, 45].

Table 3 Estimated cost of producing 10 biosensor test samples
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4 Conclusions

This study aimed to develop low-cost and effective diagnostic methods for phenylketonuria (PKU) disease using liquid and PCL-based solid biosensors. In the experiments conducted, the ability of iron (III) chloride and porous PCL-based solid biosensors to detect the presence of L-phenylalanine in PKU diagnostic solutions through color changes was demonstrated. The effect of FeCl3 concentration on the color change of the biosensor was observed to increase with the increase in FeCl3 concentration, indicating an increase in sensitivity in PKU detection. Furthermore, it was determined that the color change also increased with the increase in the concentration of the Phe solution, and this change could be monitored over time to diagnose the disease. Color analyses confirmed the usability of liquid and PCL-based biosensors in diagnosing PKU disease. Analyses conducted using RGB, HSL, and CMYK color models were shown to be important tools in identifying the presence of L-phenylalanine in Phe solutions. Specifically, a reliable method for diagnosing the disease was provided by examining time-dependent color changes. Cost analysis demonstrated that the developed biosensors are a more cost-effective alternative compared to existing methods for diagnosing PKU disease. This represents an important step towards accessibility to a wider population and early diagnosis of the disease. In conclusion, this study has demonstrated the potential of liquid and PCL-based solid biosensors as low-cost and effective diagnostic methods for PKU disease. These developed sensors could be a significant step in meeting the needs of patients and healthcare systems in many countries. More research efforts should aim to evaluate the effectiveness, sensitivity, selectivity, and reliability of these sensors under different conditions.