Introduction

Uric acid (UA) is a pure, odorless crystalline component of protein digestion, found in trace amounts across various organs in the body, as well as in blood and urine1. In plasma and serum, UA concentrations typically range from 100 to 400 µM, while adult urine contains about 250–750 mg/L2,3,4. In the environment, biological, and clinical systems, UA is among the most significant biological analytes considering its clinical and toxicological importance. As an essential waste product of nitrogen-containing purine synthesis in humans, UA also acts as a therapeutic antioxidant, with its concentrations increasing in response to oxidative stress5. Normally, UA is present in minimal amounts within the body, however, elevated UA levels in the blood, a condition known as hyperuricemia, can lead to the development of gout and kidney stones6. Abnormal UA levels are also associated with various disease states, including hypertension, pneumonia, and cardiovascular conditions, and may contribute to the formation of crystals or stones in the body7. Given its dual role and clinical relevance, accurate monitoring of UA concentrations in body fluids is vital for the early diagnosis of diseases linked to its abnormal levels. Back in years, UA detection in clinical analysis relied on the enzymatic conversion of UA to allantoin or to CO2 and H2O2, using the uricase enzyme in combination with colorimetric measurement or ultraviolet spectrophotometry3. However, these conventional methods are often associated with several limitations, including the use of expensive reagents, time-consuming, and complex sample preparation steps8. With advancements in analytical technologies, novel sensors incorporating nanomaterials have emerged as promising alternatives, offering stable, cost-effective, and highly sensitive methods for UA detection.

A novel group of carbon nanomaterials known as carbon-based quantum dots, which include graphene quantum dots and carbon quantum dots (CQDs), have emerged with dimensions typically below 10 nm9,10. Initially obtained through preparative electrophoresis during the purification of single-walled carbon nanotubes in 200411, CQDs were later synthesized in 2006 by laser ablation of graphite powder and cement12. These CQDs have the properties of both quantum dots and carbon and is easier to be handle as their chemical and optical properties can be altered. Unlike traditional carbon structures, which have low water solubility and weak fluorescence, CQDs exhibit high solubility and strong luminescence13,14,15. This shows that CQDs have unique optoelectronic properties compared to other quantum dots. Additionally, CQDs are highly soluble in various solvents, low in toxicity, and easily functionalized with diverse functional groups at their edges, which enhances their applicability16. These advantageous properties have driven extensive research, enabling CQDs to be explored for applications across fields such as photovoltaics17, electronics18, bio-imaging19,20, and optical sensors21.

CQDs have also found extensive application as sensing probes for UA detection, integrated with various types of sensors. Some examples of the sensors are electrochemical22,23,24, chemiluminescence25, colorimetric26, and fluorescence sensors27,28. However, some drawbacks have been encountered by these methods, such as the need for complex optical instruments, complicated operational procedures, and long measurement times. To address these drawbacks, the surface plasmon resonance (SPR) sensor emerges as an ideal alternative for this work, as it is renowned as one of the leading sensor technologies due to its high sensitivity, label-free, and real-time detection capabilities29,30. Moreover, SPR offers additional advantages as it is cost-effective, requires simple sample preparations, allows rapid measurements, and does not need a reference solution31,32,33. To generate SPR, several approaches has been introduced, such as metal-insulator-metal waveguides34, metal thin films35, and noble metal nanoparticles36. Among the plasmonic materials, metals and metal nanoparticles were extensively used in the fabrication of SPR sensors, particularly gold (Au)37, silver38, copper39, and Au nanoparticles40,41. Compared to other metals, Au and Au nanoparticles remain the most widely used plasmonic materials due to their excellent chemical stability, biocompatibility, and resistance to oxidation42. In this study, an Au layer was selected for the integration with the SPR method to ensure reliable plasmon excitation.

While Au-based SPR sensors have been widely explored for various analytes, their application for UA detection remains largely underexplored. Thus, this study introduces an innovative approach by integrating CQDs with SPR for UA detection. CQDs were synthesized from lemon juice, which notably has one of the highest citric acid content among all citrus fruits, constituting up to 8% of the dry weight of lemons43. Due to its high citric acid content, lemon juice is considered a promising precursor for producing CQDs with high photostability and low toxicity44. To date, reports on the structural and optical properties of citric acid-based CQDs derived specifically from lemon juice are greatly limited.

Previous studies have reported the synthesis of CQDs from lemon juice using the solvothermal method for applications such as luminescent probes in both in vitro and in vivo bioimaging45, and as a teaching tool for introducing nanoparticle synthesis to students using carbonization method46. In terms of sensing applications, lemon juice-derived CQDs have predominantly been synthesized using the hydrothermal method and successfully incorporated with photoluminescence-based sensors for detecting V5+ ions47, Fe3+ ions48, and Mo6+ ions49. These hydrothermally synthesized CQDs have also been utilized in fluorescent sensors for the detection of Cu2+ ions50, Hg2+ ions51, Fe2+ ions52, Fe3+ ions53, and tannic acid54. In addition, CQDs derived from lemon juice have been prepared using a thermal decomposition approach for Hg2+ ions detection via photoluminescence sensing55. Moreover, a fluorescence detection of Fe3+ ions has been conducted using lemon juice-derived CQDs synthesized through microwave-assisted method56. However, to date, no studies have reported the synthesis of CQDs from lemon juice using the hydrothermal method and their subsequent integration with SPR optical sensor for UA detection.

Therefore, in this study, lemon juice-derived CQDs were synthesized using the hydrothermal method. Their structural and optical properties were explored, and the synthesized CQDs were employed as a sensing layer in an Au-integrated SPR sensor for the detection of UA. The resulting CQDs exhibited excellent optical properties and when incorporated with SPR sensor, they demonstrated a low detection limit of 0.002 µM and high sensitivity towards UA. These findings highlight the strong potential of this eco-friendly sensing platform for future sensing applications.

Materials and methods

Reagent and materials

Lemon fruit was purchased from local market. Disodium ethylenediaminetetraacetic acid (Na2EDTA, M = 292.24 g/mol) was purchased from Bio Basic. SnakeSkin™ Dialysis Tubing with a diameter of 22 mm and molecular weight cut-off of 3.5 kDa was purchased from Thermo Scientific. UA powder (C5H4N4O3, M = 168.11 g/mol) was purchased from Sigma-Aldrich. Deionized water (H2O) was used throughout the experimentation and characterization. A prism with refractive index, n = 1.77861 and the substrate, glass cover slips 24 × 24 mm with thickness 0.13–0.16 mm, were purchased from Menzel-Glaser. All chemicals were used without further purification.

Preparation of CQDs solution

Firstly, the CQDs were synthesized from lemon juice by a simple hydrothermal treatment at relatively low temperatures and through a less time-consuming process. 3 ml of pulp-free lemon juice was mixed with 1.875 ml of Na2EDTA. The mixture was then dissolved in 30 ml of deionized water. The solution was then transferred into a polytetrafluoroethylene-equipped stainless-steel autoclave and heated in the oven at three different temperatures which are 120 °C, 150 °C, and 180 °C for 3 h. After the reaction, the dark brown product of CQDs was obtained after natural cooling to room temperature. The CQDs solution was collected by dialysis against deionized water through a dialysis membrane.

Preparation of UA solution

The UA powder was firstly diluted using deionized water to standard solution with a concentration of 1 M. Then, the standard solution was further diluted with deionized water using the dilution formula of M1V1 = M2V2 with deionized water to produce UA solutions with concentration of 0.05 µM, 0.1 µM, 0.5 µM, and 1.0 µM.

Preparation of thin film

To fabricate highly luminescent CQDs thin film, the spin coating approach was used. First of all, the glass slips were cleaned using acetone in order to make sure the surface of the glass slips was adequately clean and also to remove dirt and suspensions. Next, the glass slips were firstly coated with Au using the SC7640 sputter coater machine. The duration set determined the thickness of the Au layer on the glass slip. In order to get the thickness of 50 nm, the time was set to 67 s57.

After finish coating the glass slip with Au, it was then brought to the spin coating process. Approximately 1000 µL of diluted CQDs solution was placed on the Au coated glass slip to cover almost all part on the surface. Then the glass slip was spun one by one at 3000 rpm for 30 s to produce the Au/CQDs thin film.

Characterization

For optical characterization, LS 55 Photoluminescence Spectrometer (PerkinElmer, Waltham, MA, USA) was used to analyze the photoluminescence (PL) spectra over the wavelength range of 200–800 nm. The Fourier transform infrared (FTIR) spectrometer model spectrum 100 (PerkinElmer, Waltham, MA, USA) was used to determine the functional groups of the Au/CQDs thin film, at the wavelength range from 3800 to 1000 cm−1. The morphological characteristics, including the shape and size distribution of the CQDs, were analyzed using JEM-2100 F transmission electron microscopy (TEM). The acquired TEM images were further analyzed using ImageJ software to determine the particle size distribution.

Potential sensing application using SPR

An optical spectroscopy was designed to identify the potential of the citric acid-based CQDs, derived from lemon juice, for sensing different concentrations of UA which is based on the principle of SPR. Figure 1 shows the schematic diagram of the SPR instrument setup. SPR is a technique used for noticing changes in the refractive index at the surface of a sensor. In this study, the sensor consists of thin films made of Au and Au/CQDs. Basically, when light beam passes through a surface with different refractive index, the light will partially reflect and partially refracted. Total internal reflection happens when no light is refracted since the angle of incident light is greater than the critical angle. The excitation of surface plasmons occurs through total internal reflection, which takes place when a monochromatic, p-polarized light beam strikes a metal-coated thin film58.

In the SPR sensor setup, a p-polarized He-Ne laser with a specific wavelength (632.8 nm) is directed to a prism. On one side of the prism, a glass cover slip coated with either Au or Au/CQDs is attached, and the UA solution being tested is placed in contact with this surface. In order to hold the UA solution, a cell was constructed and linked to the thin film. In the middle of the cell, an O-ring was used to seal the solution, ensuring proper contact between the solution and the sensor surface while allowing the laser light to interact with the thin film. The incident light is controlled by placing the prism and cell on a rotating plane to adjust the angle of incidence. Thus, a large area photodiode will detect the reflected beam and then it will be processed by the lock-in-amplifier. Next, the results retrieved from SPR sensor were analyzed using a simulation study. For this, the Winspall software was employed, which simulates SPR performance theoretically based on Fresnel’s equations59. By inputting the thickness and refractive index of the materials involved, Winspall generates SPR curves, which can then be compared with the experimental data.

Fig. 1
Fig. 1
Full size image

Experimental setup of SPR sensor60.

Results and discussion

PL analysis

The PL intensity of CQDs synthesized at three different temperatures with an excitation wavelength of 350 nm is shown in Fig. 2. From the spectrum, CQDs synthesized at 120 °C exhibited an emission wavelength of 454 nm. The PL spectra of CQDs prepared at 150 °C reached an intense emission peak at 457 nm, while the CQDs synthesized at 180 °C displayed the emission peak at 455 nm, with the highest intensity among the three samples. This suggests that CQDs synthesized at the higher temperature of 180 °C produced the most intense emission compared to the other two temperatures. The stronger PL intensity suggests that the CQDs possess better surface properties, such as better surface passivation and fewer non-radiative recombination sites, which may contribute to better sensitivity when applied in the SPR sensor.

The strong fluorescence emission observed in the CQDs synthesized at 180 °C is likely attributed to surface defect states introduced by oxygen-functional groups, which are formed through the partial oxidation of lemon juice during the synthesis process61. These functional groups create multiple energy levels on the surface of the CQDs, resulting in a series of emissive traps62. These traps facilitate the radiative recombination of the photoexcited electrons, which in turns dominate the emission process47. This mechanism significantly contributes to the PL behavior of the CQDs.

It was also observed that the PL spectrum for the 180 °C sample exhibited a slight red shift toward longer wavelengths, which may be due to the distribution of various emissive sites and distinct particle sizes of the CQDs63. Due to the excellent properties of the CQDs treated at 180 °C, this sample was continued to be used in the following characterizations.

Fig. 2
Fig. 2
Full size image

PL spectra of CQDs treated at various temperatures.

FTIR analysis

The IR spectrum of CQDs in the range of 3800 cm−1 to 1000 cm−1 is shown in Fig. 3. The spectrum reveals broad absorption bands at 3282 cm−1, 3350 cm−1, and 2858 cm−1, corresponding to the stretching vibrations of O–H, N–H, and C–H bond, respectively63. A broad peak at 1631 cm−1 is attributed to the stretching bond of the C = O64, while a medium-weak C = C stretching bond is observed at 1416 cm−165. Additionally, the absorption peak at 1227 cm−1 represents the C–O group, indicating the presence of carboxyl (–COOH) groups in the CQDs66. These results confirm that the CQDs exhibit characteristic absorption peaks for O–H, C–H, C = O, and C–O bonds. Overall, the presence of carboxylic acids, carboxyl, and hydroxyl functional groups in the CQDs thin film was successfully verified. The FTIR spectra also demonstrate the successful incorporation of the plentiful amino and hydroxyl groups on the surface of CQDs, contributing to their high polarity and excellent water solubility.

Fig. 3
Fig. 3
Full size image

FTIR spectrum of CQDs.

TEM analysis

The morphology and particle size of the lemon juice-derived CQDs were examined using TEM. As illustrated in Fig. 4 (a) and (b), captured at magnifications of approximately 150,000x and 300,000x, respectively, the CQDs appeared predominantly spherical and were randomly dispersed. Moreover, a particle size analysis based on a statistical evaluation of at least 30 CQDs from Fig. 4 (a) revealed a size distribution ranging from 2 to 8 nm, as presented in Fig. 4 (c). The average particle diameter of the CQDs was calculated to be 4.73 nm, which is in a good agreement with a previous study by Ding et al. (2017), who synthesized CQDs from pulp-free lemon juice through a solvothermal method and obtained an average size of about 4.6 nm45.

Fig. 4
Fig. 4Fig. 4
Full size image

TEM image of lemon juice-derived CQDs at (a) ~ 150,000x magnification and (b) ~ 300,000x magnification. (c) Particle size distribution of lemon juice-derived CQDs.

Additionally, energy dispersive X-ray (EDX) analysis was performed to identify the elemental composition of the citric acid-based CQDs synthesized from lemon juice, as shown in Fig. 5. The analysis revealed that the CQDs primarily consisted of carbon, with a high content of 98.9%, indicating the dominance of carbonaceous structures in the sample. A small amount of oxygen (1.1%) was also detected, which is likely attributed to the presence of oxygen-containing functional groups (O–H, C–O, C = O)67. This finding is consistent with the FTIR analysis, which previously confirmed the presence of these functional groups in the CQDs.

Fig. 5
Fig. 5
Full size image

EDX spectrum of lemon juice-derived CQDs.

Optical sensing analysis

SPR signal for UA on Au single layer

Firstly, a preliminary SPR test was conducted with the Au thin film in contact with deionized water. Approximately 2 ml of deionized water was injected into the cell to ensure that it was in contact with the Au layer thin film. The resonance angle obtained from the graph is 53.47°. Further analysis was done by fitting this experimental data of SPR reflectivity curve for Au film in contact with deionized water to the theoretical data. Both theoretical and experimental SPR curves are shown in Fig. 6. The refractive index parameters obtained for the Au layer were 0.16014 for the real part, n68 and 3.70 for the imaginary part, k69. Moreover, the thickness of the Au layer was found to be approximately 50 nm. Meanwhile, the refractive index value obtained for deionized water was found to be 1.333. This SPR curve was used to compare the changes in resonance angle for different concentrations of UA solution.

Fig. 6
Fig. 6
Full size image

Fitted and experimental SPR curves of Au thin film exposed to deionized water.

The SPR experiment was then continued using different concentrations of UA in aqueous solution. Starting from the lowest concentration (0.05 µM) until the highest (1.0 µM), the UA solutions were injected one after another into the cell and this experiment was repeated three times in order to achieve data accuracy. Then, the resonance angles for all distinct concentrations of UA were compared using the SPR curves. The SPR curves for UA solutions ranged from 0.05 µM to 1.0 µM in contact with Au thin film is shown in Fig. 7. From the figure, it can be observed that the resonance angles for all concentrations are quite similar. This might due to the little quantity of active ions exist in the concentration solutions that were attached to the Au surface.

Fig. 7
Fig. 7
Full size image

SPR curves of Au thin film exposed to different concentrations of UA ranging from 0 µM to 1.0 µM.

Further analysis was done by fitting these experimental data to the theoretical models using the WinSpall software. Figure 8 shows the SPR fitted and experimental curves for different concentrations of UA. The refractive indices of the UA solutions and the Au layer, obtained from the fitting, are listed in Table 1. The results indicate that the n of refractive index of the UA solutions was almost identical to that of deionized water, around 1.333, with a slight increase observed as the UA concentration rose70. However, this small increment did not significantly affect the refractive index of the Au layer, and thus did not result in any noticeable shift in the resonance angle. These observations are consistent with those of Fen and Yunus (2013), who reported no changes in the resonance angle for metal ions across various concentrations when in contact with a bare Au film71.

Fig. 8
Fig. 8
Full size image

Fitted and experimental SPR curves of Au thin film exposed to different concentrations of UA from (a) 0.05 µM, (b) 0.1 µM, (c) 0.5 µM, and (d) 1.0 µM.

Table 1 Refractive index of different concentrations of UA ranging from 0 µM to 1.0 µM and Au thin film exposed to the UA solutions.
Full size table

Next, a graph was plotted for all different concentrations of UA against their respective resonance angle as shown in Fig. 9. The results confirmed that no changes in resonance angle occurred when only Au layer was used in SPR analysis, that is due to the similarity of refractive index for the low concentration of analytes72.

Fig. 9
Fig. 9
Full size image

Resonance angle of Au thin film exposed to different concentrations of UA ranging from 0.05 µM to 1.0 µM.

SPR signal for UA using lemon-based CQDs on Au surface

The SPR experiment was then pursued for the detection of UA by replacing Au layer thin film with CQDs coated on top of the Au layer thin film. It was observed that the resonance angle slightly shifted to the right when the Au/CQDs thin film was in contact with deionized water, showing that the resonance angle increased compare to only Au layer thin film. The resonance angle for Au/CQDs film in contact with deionized water is 53.65° compared to 53.47° when it is in contact with Au layer thin film only. Further analysis was done by fitting these experimental data to the theoretical data by inserting the values of thickness and refractive index of Au film and UA solutions obtained earlier. The experimental and fitted SPR curves of the Au/CQDs thin film exposed to deionized water are shown in Fig. 10. From the simulation, the value of refractive index and thickness for the CQDs layer were obtained where the values of n and k of the refractive index were 1.343 and 0.1137, respectively. Meanwhile, the thickness of the CQDs layer was found to be 12 nm.

Fig. 10
Fig. 10
Full size image

Fitted and experimental SPR curves of Au/CQDs thin film exposed to deionized water.

The SPR experiment was further continued for different concentrations of UA solutions ranging from 0.05 µM to 1.0 µM. The UA solution was injected one after another into the cell. The SPR curves for different concentration of UA solution in contact with the Au/CQDs thin film are shown in Fig. 11. The resonance angles were determined from the SPR curves of 0, 0.05, 0.1, 0.5, and 1.0 µM, respectively. Slight shifts to the right corresponding to the increasing resonance angles were observed as the concentration of UA solution increases.

The lower concentrations of UA solution were used to compare and find the limit of quantitation (LOQ) of the Au/CQDs thin film by comparing the SPR reflectivity curves of the various UA concentrations with deionized water. LOQ refers to the lowest UA concentration that the SPR sensor can reliably detect and differentiate from the baseline signal. From Fig. 11, the resonance angle of the 0.05 µM UA showed a slight shift from the resonance angle of deionized water when in contact with the Au/CQDs thin film. From this observation, it can be concluded that the LOQ of Au/CQDs thin film is 0.05 µM of UA.

It is believed that the CQDs layer deposited on the Au surface played a significant role in enhancing the detection of UA73. The interaction between UA and the CQDs thin film likely involved the formation of a pair of shared electrons, where the positively charged UA molecules interacted with the negatively charged amine functional groups present in the CQDs. Furthermore, the CQDs contributed to increased optical absorption in both the visible and infrared regions, further enhancing the sensitivity of the sensor. This improvement in sensitivity with the CQDs layer was markedly higher compared to the use of the Au surface alone.

Fig. 11
Fig. 11
Full size image

SPR curves of Au/CQDs thin film exposed to different concentrations of UA ranging from 0 µM to 1.0 µM.

Further analysis was performed by fitting the experimental data with theoretical data. The initial values of the refractive index and thickness of the CQDs layer, obtained from fitting the SPR curve of deionized water, were used as the reference for other UA concentrations up to 1 µM. Table 2 shows the parameters calculated by the Winspall software after conducting the simulation.

Table 2 Refractive index and thickness of au/cqds thin film exposed to different concentrations of UA ranging from 0 µM to 1.0 µM.
Full size table

The comparison between the simulated SPR curves and the experimental SPR curves for different concentrations of UA ranging from 0.05 µM to 1 µM are displayed in Fig. 12. In the figure, the experimental SPR curve is represented by the dotted line, while the simulated SPR curve is depicted as a solid line. The simulation results suggest that the thickness of the sensing layer, specifically the CQDs thin film, increases slightly as higher concentrations of UA solution are introduced into the SPR sensor cell. Additionally, the n of refractive index of the UA solution shows a gradual increase with increasing concentrations. As tabulated in Table 2, when 0.05 µM of UA solution was injected, the value of n of refractive index of the CQDs thin film increased slightly to 1.344, with a constant thickness of 12 nm. However, the k value decreases to 0.1087. For the higher concentrations of UA (0.1, 0.5, and 1.0 µM), the n of refractive index of the CQDs thin film continued to rise, while the k values decreased. The thickness of the CQDs layer remained constant until the 1.0 µM concentration was reached, where it increased slightly to 12.2 nm.

Fig. 12
Fig. 12
Full size image

Fitted and experimental SPR curves of Au/CQDs thin film exposed to different concentrations of UA from (a) 0.05 µM, (b) 0.1 µM, (c) 0.5 µM, and (d) 1.0 µM.

As expressed, it can be concluded that the interaction between the CQDs sensing layer and varying concentrations of UA played a key role in the significant increase in the n of refractive index of the CQDs layer. As the concentration of UA increases, the refractive index of the CQDs layer also rises, indicating a proportional relationship between the two as the binding process occurs. This can be observed in the enhanced sensitivity of the SPR sensor, as the resonance angle shifts in response to the increasing refractive index, reflecting a more sensitive detection of UA74. In addition, the CQDs layer becomes slightly thicker as the concentration of UA approaches 1 µM. This shows that as the active layer interacts with higher concentrations of UA, the adsorption process occurs at a higher rate, leading to an increase in the thickness of the layer. This results also contributes to the rightward shift of the SPR curves, with an increasing resonance angle observed as the concentrations of UA increases75.

Sensitivity and limit of detection of Au/CQDs thin film

The resonance angle shift (Δθ) was introduced as a parameter to calculate the sensor sensitivity76. Since CQDs is believed to have a significant contribution in the detection of UA, the Δθ was used in this study to further investigate the sensitivity of the sensor thin film. Δθ is determined by taking the difference between the resonance angle of sample and deionized water as reference. The resonance angle and shift of resonance angle for different concentrations of UA in contact with Au/CQDs thin film is shown in Table 3.

Table 3 Resonance angle and shift of resonance angle for au/cqds thin film exposed to different concentrations of UA ranging from 0 µM to 1.0 µM.
Full size table

The sensitivity of this sensor can be determined by plotting the resonance angle shift against the concentration of UA77. For further analysis, the plotted data were fitted linearly. In order to achieve the best linear regression coefficient R2, two different regions were fitted separately, as shown in Fig. 13. The R2 for the first region (0 to 0.1 µM) was 1.000 and the relationship between the SPR angle shift (ΔθSPR) and the UA concentration was governed by the equation ΔθSPR = 3.698[UA] + 1.359−15. For the second region (0.1 to 1 µM), the R2 obtained was 0.995 with the equation ΔθSPR = 0.4098[UA] + 0.02617. The gradient of each regression line, 3.968° µM−1 for the first region and 0.4098° µM−1 for the second region, represents the sensitivity of the sensor across these ranges. Overall, the active layer thin film showed an excellent sensitivity towards UA up to 1 µM. Above this concentration, the sensitivity declined, likely due to surface saturation from UA accumulation. These findings confirm that the Au/CQDs thin film demonstrates high sensitivity towards UA, especially at lower concentrations.

Fig. 13
Fig. 13
Full size image

Resonance angle shift of Au/CQDs thin film exposed to different concentrations of UA ranging from 0 µM to 1.0 µM.

Additionally, the limit of detection (LOD) for UA detection was determined using the following equation:

$$LOD=\frac{{3\sigma }}{S}$$
(1)

where σ represents the standard deviation of the blank measurements (n = 3) and S denotes the sensitivity of the sensor. The sensitivity obtained from the lower UA concentration range was used in this calculation, as it yielded a higher sensitivity value compared to the higher concentrations. Based on this, the LOD of the Au/CQDs thin film-integrated SPR sensor for UA detection was determined to be 0.002 µM. This finding demonstrates a significantly lower LOD compared to a previous study, which reported a value of 0.27 µM78. This indicates that the Au/CQDs thin film incorporated with SPR technique displays great promise as a potential sensor for monitoring biochemical compounds in the future.

Binding affinity of Au/CQDs thin film

Another parameter that can be obtained from the SPR analysis is the binding affinity constant, K. In this study, Langmuir isotherm model was used to calculate the K using the following equation:

$$\Delta \theta =\frac{{\Delta {\theta _{\hbox{max} }}C}}{{\frac{1}{K}+C}}$$
(2)

where Δθmax is the SPR angle shift at the saturation, C is the concentration of the UA, and K is the affinity constant. Figure 14 shows the plot that fitted the Langmuir isotherm model for Au thin film and Au/CQDs thin film in contact with different concentrations of UA. The fitting of calibration curve for the Au/CQDs thin film using Langmuir isotherm model yielded a R2 value of 0.97588 and Δθmax of approximately 0.79744°, which is about 0.057° higher than experimental maximum shift of the SPR angle, i.e., 0.7402°. From the graph, the value of \(\frac{1}{K}\) for the Au/CQDs thin film obtained was 0.143935 µM thus, the calculated K was 6.94754 MM−1. The same analysis was also conducted on the calibration curve for the Au thin film where the values of \(\frac{1}{K}\) and K were 1.01905 µM and 0.938 MM−1, respectively.

In conclusion, a higher affinity constant was calculated for UA when using the Au/CQDs thin film compared to the Au thin film alone, suggesting that the CQDs thin film exhibits stronger binding and higher sensitivity toward UA79. This shows that the CQDs layer has a greater attraction to UA than the Au layer, enhancing the performance of the SPR sensor in UA detection.

Fig. 14
Fig. 14
Full size image

Langmuir isotherm model of Au thin film and Au/CQDs thin film exposed to different concentrations of UA ranging from 0 µM to 1.0 µM.

Conclusions

In this study, a citric acid-based CQDs thin film has been successfully synthesized using lemon juice. The PL spectrum of the CQDs under the excitation of 350 nm showed a maximum emission peak at 450 nm. The presence of functional groups in the thin film, including amino, carboxylic acid, carboxyl, and hydroxyl groups was confirmed by FTIR analysis. TEM analysis indicated that the CQDs had an average diameter of 4.73 nm. Additionally, the Au/CQDs thin film was integrated with a SPR technique for the detection of UA in the concentration range of 0.05 to 1 µM. The incorporation of the lemon juice-derived CQDs enhanced the sensitivity of the SPR system compared to the bare Au film. Using Winspall simulation, the refractive index and thickness of the CQDs layer on the Au thin film were also determined. The sensing layer exhibited an increasing refractive index in response to higher UA concentrations, which is indicative of improved sensitivity in the SPR measurements. The shift of resonance angle displayed a linear relationship for the Au/CQDs thin film in contact with UA, yielding sensitivities of 3.968° µM−1 and 0.4098° µM−1. The sensor also demonstrated a LOQ and LOD of 0.05 µM and 0.002 µM, respectively. Furthermore, a higher binding affinity constant was calculated for UA using the Au/CQDs thin film compared to the Au thin film, with values of 6.94754 MM−1 and 0.938 MM−1, respectively. Thus, these results confirmed that the Au/CQDs thin film has high potential sensing for UA using SPR technique. Future studies may focus on investigating the selectivity and anti-interference ability of the Au/CQDs thin film towards UA in the presence of potentially interfering organic compounds or biomolecules, as well as validating the performance of the sensor in real sample matrices.