Introduction

Hyperuricemia, a metabolic disorder, is a state of elevated uric acid levels in the blood [1, 2]. This can lead to a variety of serious diseases, such as gout [3, 4], uric acid nephropathy [5, 6], and kidney disease [7, 8]. These diseases can cause chronic pain and health problems, resulting in a reduced quality of life for the patient. Some of the factors mentioned as causes of hyperuricemia include the consumption of high purine-containing foods and an unhealthy lifestyle [9, 10]. However, the influence of these factors on elevated uric acid levels varies widely from individual to individual [11]. Therefore, monitoring the uric acid levels of individual patients is very important to take appropriate actions corresponding to their individual constitutions and lifestyles. In addition, for patients already being medically treated for hyperuricemia, monitoring of uric acid levels is also beneficial in implementing an appropriate treatment design.

In designing a measurement technique that would allow routine monitoring of uric acid levels in private homes, regardless of the size of the medical facility, micro-sized particles are an attractive material. Because of their high visibility, changes in optical properties (color, absorption, scattering, etc.) of microparticles can be easily determined by the naked eye. Enrichment of micro-sized particles with marker dye molecules in solution, i.e., dyes produced in an analyte concentration-dependent manner, has the advantage of improving high sensitivity of measurement techniques. This is a unique feature of micro-sized particles rather than nano-sized particles. In the case of nanoparticles, the narrow surface area limits the number of concentrated marker molecules. Even if marker dyes are concentrated on nanoparticles in a dispersed state, visibility is unlikely to be improved compared to the case in which marker molecules are present in solution. In addition, microparticles have the advantage of easy interface design. When various biomolecules are immobilized on microparticles by chemical bonding or physisorption as molecular recognition or signal conversion elements, unreacted biomolecules can be easily removed only by centrifugation process. Previously, we have demonstrated the potential utility of these features of micro-sized particles in measurement techniques by developing a colorimetric sensor for the antiviral drug favipiravir [12]. This method is based on the formation of a blue complex between favipiravir and ferrihydrite on the surface of the microparticles. A limited number of microparticles was successfully used to increase the enrichment effect of favipiravir in serum samples onto the microparticles, achieving higher sensitivity. Further, the color change is highly visible due to the high density of the colored microparticles.

In this study, micro-sized particles with a red color in response to uric acid were prepared and applied to the development of a suspension-based assay for the measurement of uric acid. Resorufin is generated from Amplex red through the mediator reaction of uricase or horseradish peroxidase (HRP) immobilized on two different micro-sized polymer particles with uric acid in sample solution [13,14,15]. Resorufin is immediately concentrated on the surface of the enzyme-immobilized microparticles by hydrophobic interactions, causing the particles to change a red color. AmplexRed is also adsorbed on the polymer particles, but the adsorbed AmplexRed is not easily converted to resorufin. Therefore, in this assay, the contribution of adsorbed AmplexRed to the red color change of the microparticles is considered to be slight. This suspension-based assay provides a highly visible colorimetric analysis to estimate uric acid concentrations, allowing us to monitor uric acid levels at home, albeit semi-quantitative analysis. Quantitative analysis is also possible by eluting the concentrated resorufin with ethanol and measuring the fluorescence intensity of the eluate. When testing multiple samples, uric acid levels can be measured with a fluorescent plate reader. On the other hand, a portable fluorometer can be used to inexpensively measure uric acid levels on-site.

Experimental

Reagents

Water was purified by a Direct-Q system (Nippon Millipore, Tokyo, Japan) and all chemical reagents were of analytical grade. Uricase, horse radish peroxidase (HRP), Amplex red, ureic acid, and human serum control were purchased from Fujifilm Wako Pure Chemical Corp. (Osaka, Japan). For the preparation of a methacrylate base resin, glycidyl methacrylate (GMA, FUJIFILM Wako Pure Chemical) and ethylene glycol dimethacrylate (EGDM, Tokyo Chemical Industry, Japan) were used as monomer and cross-linking agent, respectively. N-Butyl acetate (BuOAc), 2,2’-azobisisobutyronitrile (AIBN), and methyl cellulose 50 (MC, 40 ~ 60 mPa s at 2% solution, average molecular weight: 50 000) were purchased from FUJIFILM Wako Pure Chemical. Sodium dodecyl benzene sulfonate (DBS, Tokyo Chemical Industry) was also used.

Preparation of methacrylate base resin

Methacrylate base resin was prepared according to the procedure of previous papers [16,17,18,19,20]. A methacrylate base resin was prepared by suspension copolymerization of GMA and EGDM. AIBN (0.4 g) was dissolved in a mixture of 12 g of GMA, 28 g of EGDM and 48 g of BuOAc. The resulting solution (The organic mixture) was added to a mixture of 200 mL of deionized water, 50 mL of 1% (w/v) MC solution, and 0.4 mL of 1% (w/v) of DBS solution. After stirring at 400 rpm for 20 min, the copolymerization was conducted at 80 °C for 7 h with stirring at 300 rpm. The suspension was cooled to room temperature and the obtained methacrylate base resin was washed with deionized water, acetone, and methanol, in that order, under ultrasonic irradiation. After drying, the methacrylate base resin was classified at 53 ~ 90 µm of particle size by standard sieves.

Modification of enzymes on the surface of polymer particles

Uricase-modified microparticles and HRP-modified microparticles were prepared by the same procedure. Uricase or HRP was dissolved in 0.1 M phosphate buffer (pH 7.4) (abbreviated as PB) to prepare an enzyme solution of 0.1 mg/mL. Fifty mg of methacrylate-based microparticles were suspended in 2.5 mL of enzyme solution and stirred for 10 min at room temperature. The suspension was centrifuged at 2300g for 30 s to precipitate the microparticles, and the supernatant was aspirated with a micropipette to remove any unreacted enzyme. After removing the supernatant, 2.5 mL of PB solution was added to the precipitate and allowed to percolate gently. This centrifugal washing process was repeated three times.

Procedures for a suspension-based assay of uric acid

Since the optimum pH of HRP is 5.0–8.0 [21] and that of uricase is 8.5 [22], measurement of uric acid was performed at pH 8.0. Uric acid was dissolved in 0.1 M Tris buffer at pH 8.0 (abbreviated as a Tris buffer solution) to prepare a 1 mg/mL uric acid solution (abbreviated as uric acid solution). This solution was diluted with a Tris buffer solution as necessary.

Amplex red was adjusted to 100 µM by dissolving in Tris buffer solution. In a microtube, 5 mg each of both uricase and HRP-modified microparticles were added and suspended in 300 µL Tris buffer solution. To prepare suspensions with final concentrations of 0–1.2 µg/mL for the uric acid solution and 10 µM for Amplex red, respectively, 100 µL of uric acid solution and 100 µl of Amplex red solution were added to the microtube containing the suspension. The suspension was agitated for 5 min and centrifuged at 2300g for 30 s. The centrifugation procedure precipitated microparticles which turned red due to the adsorption of resorufin produced by the mediator reaction. For colorimetric analysis, the precipitation of the colored microparticles was photographed with a smart phone from the upper direction of the microtubes.

For quantitative analysis of uric acid levels, the following procedure was used to desorb the resorufin adsorbed on the microparticles. The red-colored microparticles were precipitated by centrifugation of the suspension after the enzyme-mediator reaction at a rotation rate of 2300 g for 30 s. After the supernatant was removed with a micropipette, 500 µL of ethanol was added to the precipitate of microparticles and stirred at room temperature for 5 min. The suspension after desorption of resorufin was centrifuged at a rotation rate of 2300g for 30 s. The supernatant was collected and 300 µl of it was added to a 96-well plate, and the fluorescence intensity of the solution in the wells was measured with a microplate reader (SH-9000, Corona Electric, Ibaraki, Japan). When using a portable fluorometer (name of the instrument FC-1, Tokai Kogaku, Japan), 300 µL of the supernatant after resorufin desorption was collected with a micropipette. The supernatant was diluted with 2.7 mL of Tris buffer and the fluorescence intensity of the diluted solution was measured.

When measuring uric acid in control serum, 100 µl each of the 20 fold diluted control serum solution and Amplex red solution was added to 300 µl of the micro-sized particle suspension to achieve a 100 fold dilution of the control serum in the suspension. Other steps of the experiment were performed using the same protocol as for the measurement of uric acid in a Tris solution.

Results and discussion

Color change of micro-sized polymer particles caused by the mediator reaction

Since methacrylate base resin has been focused on as a substrate for solid-phase extraction [16,17,18,19,20], resorufin produced by the mediator reaction with uricase, HRP, and uric acid is expected to be adsorbed on this resin. Therefore, the uricase-modified microparticles and HRP-modified microparticles mixed at a weight ratio of 1:1 were suspended in uric acid solution, and the suspension was light-shielded and agitated for 5 min. Figure 1a shows a photograph of the precipitation of the microparticles by centrifugation. Although no color change could be visually observed in the supernatant, the precipitated particles turned red in color. On the other hand, no such color change was observed in the microparticles themselves without uric acid addition or without the uricase/HRP modification. These indicate that the red resorufin produced by the mediator reaction was adsorbed on the particle surface by passing through the gaps between the enzymes, despite the modification of the enzymes on the microparticles. To determine the type of interaction causing adsorption of resorufin onto the enzyme-modified microparticles, ethanol was added to the microparticles collected by removing the supernatant of the suspension after the enzyme– mediator reaction. The fluorescence intensity of the supernatant was measured in a fluorescent microplate reader, revealing that resorufin was desorbed from the microparticles (Fig. 1b). On the other hand, no resorufin could be desorbed from the microparticles in 500 mM NaCl. These results suggest that the enzyme product, resorufin, hydrophobically interacts with the polymer backbone, causing the color change to red in the presence of uric acid, regardless of the type of enzyme immobilized, such as uricase or HRP. Absorbance spectrophotometry showed that AmplexRed, the substrate of HRP, is similarly adsorbed onto the enzyme-immobilized micro-sized particles (Fig, S1). When AmplexRed in the bulk solution accesses the particles, it is immediately converted to resorufin by HRP on the surface of the particles. Therefore, resorufin will preferentially be adsorbed on the particle surface. Further, when the enzyme-immobilized micro-sized particles with pre-adsorbed AmplexRed were mixed with the uric acid solution, the particles were only slightly colored (Fig. S2). AmplexRed on the beads may be less accessible to the HRP immobilized on the particles, resulting in a lower apparent enzymatic activity. The color change of the microparticles to red due to the enzyme-mediated reaction indicates its potential application in colorimetric analysis of uric acid. In addition, the desorption of resorufin from the microparticles with ethanol suggests that the same experimental process can be used not only as a colorimetric method but also for quantitative analysis by fluorometer.

Fig. 1
figure 1

Coloration of microparticles by the mediator reaction. a Image of the coloration of microparticles. The image was obtained by stirring the suspension containing 5 mg each of uricase and HRP-modified microparticles for 5 min in the presence (1) or absence (2) of 0.7 mg/mL of uric acid. b Desorption of resorufin, which is generated by the mediator reaction, from the surface of microparticles. After sedimentation of the colored microparticles, they were treated with 500 µL of ethanol (1) or 500 mM NaCl (2). After the treatment, the fluorescence intensities of 300 μL of the supernatant were measured (ex. 570 nm, em. 585 nm). Averages of three measurements are plotted. Error bars indicate mean ± standard deviation (n = 3)

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Optimization of the mediator reaction between uricase/HRP micro-sized particles and uric acid

Higher activity in the mediator reaction leads to a colorimetric method in which the analyte can be measured in a shorter time and with higher sensitivity. Therefore, we decided to optimize the activity in the mediator reaction. If both enzymes are immobilized on the same micro-sized polymer particle, the actual ratio of the immobilized enzymes cannot be identified. Therefore, in this study, we decided to use micro-sized polymer particles in which each of uricase and HRP is immobilized separately. To quantitatively evaluate the activity, resorufin was desorbed with ethanol, and the fluorescence intensity of the supernatant was used as an indicator of activity. The effect of the weight ratio of uricase to HRP-modified microparticles on the activity of the enzyme-mediator reaction was examined (Fig. S3).

When the weight ratio (w/w) of uricase to HRP was larger than 1.0, the activity of the mediator reaction was higher. Therefore, using the weight ratio of 1.0 w/w, we investigated the activity of the mediator reaction modulated by a total weight of the uricase-modified microparticles and HRP-modified microparticles (Fig. 2). The fluorescence intensity at a total weight of 20 mg was slightly lower than that at 10 mg. In the reaction between the immobilized enzyme and the substrate in the bulk solution, i.e., heterogeneous reaction, agitation of the solution is important for the enzyme activity to proceed effectively. In the case of 20 mg total particle weight, too many particles may have inhibited effective agitation of the uric acid solution and enzyme-modified microparticles in the microtubes, resulting in slightly lower fluorescence intensity values. From these results, the optimal condition was found to be a weight ratio of 1 between uricase-modified microparticles and HRP-modified microparticles, with a total weight of 10 mg.

Fig. 2
figure 2

Optimization of a total weight of uricase and HRP-modified microparticles (weight ratio 1:1, w/w). After precipitation of the colored microparticles, they were treated with 500 µL ethanol. Fluorescence intensities of 300 μL of the supernatant after desorption of resorufin from microparticles were measured (ex. 570 nm, em. 585 nm). Averages of three measurements are plotted. Error bars indicate mean ± standard deviation (n = 3)

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Time course of the mediator reaction

To determine the stirring time for the suspension-based assay, the time course of the enzyme-mediator reaction was examined. After the mediator reaction under the previously determined conditions, the resorufin was desorbed from the enzyme-modified microparticles and the fluorescence intensity of the supernatant was measured (Fig. 3). The time, at which a mixture of uric acid and Amplex Red was added to the suspension of enzyme-modified microparticles, was used as the reaction start time. Because the mediator reaction proceeded in suspension without packing the enzyme-modified microparticles, the rate of resorufin formation was so rapid and the fluorescence intensity immediately after the reaction (t = 0 min) was difficult to measure. Therefore, the fluorescence intensity was measured from 2.5 min after the start of the reaction. The fluorescence intensity increased significantly at t = 2.5 min of reaction time, and then gradually increased with time. In determining the assay time, although a shorter time for fluorescent detection of uric acid would be desirable, the mediator reaction time was set to t = 5 min for ease of use.

Fig. 3
figure 3

Amount of resorufin adsorbed on microparticles produced by the mediator reaction depending on the stirring time. The suspension contains two micro-sized particles modified with uricase and HRP (weight ratio 1:1, w/w; total weight 10 mg) and 0.7 µg/ml uric acid. After stirring for a given time and precipitation of the colored micro-sized particles, the suspension was treated with 500 µL ethanol. Fluorescence intensities of 300 μL of the supernatant were measured (ex. 570 nm, em. 585 nm). Averages of three measurements are plotted. Error bars indicate mean ± standard deviation (n = 3)

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Colorimetric measurement of uric acid in control serum

The concentration dependence of the color change to red was examined using samples of uric acid dissolved in buffer solution under the conditions previously determined (Fig. 4a). In the concentration range of 0–1.2 µg/mL, the red color became deeper in dependence on the concentration of uric acid. The reference value for uric acid in human serum is 70 µg/mL, and in Japan, levels above 80 µg/mL are subject to drug treatment [23]. Considering the practicality of the uric acid level monitoring method, the ideal colorimetric method should possess the ability to visually determine uric acid levels in the range of 70–100 µg/mL. The concentration range of this method is about two orders of magnitude smaller than this range. Therefore, as shown by the concentration-dependent color change, this method can visually discriminate uric acid at concentrations of 0.7 µg/mL or higher, i.e., corresponding to the reference value of 70 µg/mL for serum uric acid, by diluting the serum 100-fold.

Fig. 4
figure 4

Image of the change in color tone of the microparticles in response to uric acid. a uric acid in buffer solution, b uric acid in control serum

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To demonstrate applicability to human serum, colorimetric analysis of uric acid in control serum was performed. The assay results using control serum as a sample are useful for evaluating the accuracy of the analytical method, since the amounts of the components in the serum are described. Control serum containing 105 µg/mL of uric acid was colorimetrically examined by this method (Fig. 4b). The concentration of uric acid in the control serum was about 1 µg/ml, which corresponds to 100 µg/mL in the control serum, judged visually against the image of the concentration-dependent color change. This was in almost the same level as the concentration of uric acid in the control serum. The control serum contains many contaminants, which may interfere with our colorimetric method. However, the 100 fold dilution of the control serum may have reduced these Interferences.

A colorimetric method using 1,10-phenanthroline and 2,4,6-tripyridyl-s-triazine (TPTZ) was proposed by Morin et al. The method is based on the reduction of Fe(III) to Fe(II) by uric acid in the sample and its subsequent complexation with TPTZ [24, 25]. Instead of TPTZ, ferrozine may be used as a complexing agent [26]. The molar absorbance, solubility, and stability of ferrozine-iron(II) complexes are greater than those of TPTZ [27]. Furthermore, a highly sensitive colorimetric method based on uric acid concentration-dependent inhibition of the oxidative etching reaction of silver nanoparticles (AgNPs) by HAuCl4 has been proposed [28]. In addition to these colorimetric methods based on the reducing properties of uric acid, a colorimetric method using uricase and peroxidase has been proposed. This method has high selectivity for uric acid determination due to the substrate specificity of the enzyme. Colorimetric methods have also been proposed that utilizing organic chemicals such as organometallic frameworks [29,30,31,32] and nanoparticles such as silver [33], gold [34,35,36,37], and copper [38] as substances with peroxidase-like activity. These approaches are characterized by high storage stability of the materials. Although these principles of changing the color tone of the solution in response to uric acid have been widely proposed, our proposed method is an approach for improving the visibility of colorimetric analysis, which distinguishes it from other methods.

Quantitative analysis of microparticle-based uric acid assays with a 96-well microplate reader

Colorimetric assays allow for easy semi-quantitative determination of analytes by visual examination. Therefore, they are attractive techniques for realizing on-site analysis. On the other hand, many researchers are addressing the issue that colorimetric analysis lacks quantitative performance. In particular, with the advancement of image processing technology, approaches to quantify color intensity from images captured by digital cameras, smartphones, and other devices have been reported extensively. This approach is now becoming the golden standard. On the other hand, in our method, since resorufin can be easily desorbed with ethanol, we investigated the possibility of quantifying the color intensity obtained by colorimetric analysis by fluorescence measurement. After colorimetric assays performed with different uric acid solutions as samples, red micro-sized particles were treated with ethanol. The fluorescence intensity of the supernatant was measured with a 96-well microplate reader and the obtained concentration dependence is shown in Fig. 5. The fluorescence intensity increased with increasing uric acid concentration, and the calibration curve showed a good linear relationship with r2 = 0.9876. The detection limit (3σ) and RSD (n = 3) were estimated to be 2.2 × 10–2 μg/mL and ≤ 12.1%, respectively. These results indicate that the performance in this method is sufficient for measuring serum uric acid levels. In addition, Resorufin fluorescence intensity is constant up to 2 h at room temperature (Fig. S4). This suggests that the images should be taken or the fluorescence intensity should be measured within approximately 2 h after incubation of the sample with the enzyme-immobilized micro-sized particle suspension.

Fig. 5
figure 5

Dependence of uric acid concentration measured in the suspension-based assay using 96-well fluorescence plate reader. After precipitation of the colored microparticles, they were treated with 500 µL ethanol. Fluorescence intensities of 300 μL of the supernatant were measured (ex. 570 nm, em. 585 nm). Averages of three measurements are plotted. Error bars indicate mean ± standard deviation (n = 3)

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When the fluorescence assay was performed using a 100 fold diluted control serum in the same manner as the colorimetric assay was performed, the uric acid level in the control serum was 0.95 ± 0.12 µg/mL The recovery rate was calculated from the uric acid level in the control serum to be 91 ± 11%. This result demonstrates that the assay is not significantly interfered with by various biomolecules in the control serum. These indicate the possibility of quantitative fluorescent measurement of uric acid in human serum. When using a 96-well microplate reader, blood samples collected from many patients can be used to perform blood tests simultaneously. Therefore, the method is expected to be highly useful not only for colorimetric analysis but also for quantitative fluorescent measurement of serum uric acid levels, although this approach is limited to large medical facilities and laboratories capable of purchasing microplates.

Simple quantitative analysis of uric acid using a portable fluorescent analyzer

In small- and medium-scale medical facilities, this method may enable easy fluorescent measurement of serum uric acid levels by utilizing an inexpensive portable fluorometer. Therefore, we investigated the measurement of uric acid in 100 fold diluted control serum using a portable fluorometer. Prior to this experiment, the detectable concentration range of resorufin in the portable fluorometer was investigated. The portable fluorometer uses a cuvette-type square cell with 1 cm on each side as a sample cell, and thus has the potential to measure resorufin with higher sensitivity than a 96-well microplate reader. Comparing the dependence of resorufin concentration obtained with each instrument (Fig. S5), the portable fluorometer was found to be able to measure fluorescence intensity at concentrations, about an order of magnitude lower. Therefore, the fluorescence intensity of the supernatant of a tenfold dilution of the colored particulate suspension was measured although the assay was performed using the same procedure as when measured in a 96-well microplate reader. The uric acid level in the control serum was calculated from the fluorescence intensity obtained by measuring 0.7 µg/mL uric acid solution and control serum. The results showed that the uric acid level in the control serum was 1.2 ± 0.06 µg/mL, and the recovery rate was 114 ± 5.7%. This uric acid value was nearly identical to that obtained using a 96-well microplate reader. This suggests that a portable fluorescent device can also be used to measure uric acid levels in serum by this assay method.

Conclusion

We have developed a suspension-based assay for uric acid using uricase and HRP-modified microparticles. The experimental protocol is simple, requiring only the addition of a sample to a suspension containing these microparticles and stirring for 5 min, enabling anyone to easily measure uric acid levels visually. In this study, a centrifuge was used to completely sediment these microparticles, but the microparticles settled spontaneously after the suspension was allowed to stand for about 1 min. Therefore, we believe that uric acid levels can be measured anywhere, even in a non-laboratory setting. Further, a suspension-based assay can be combined with a fluorescence spectrophotometer to quantitatively measure uric acid levels. Since both a fluorescent microplate reader and a portable fluorometer can be used with this assay, we expect that it can be used at various facilities regardless of their scale. The proposed method is designed to allow measurement of uric acid in serum with the same procedure, and the user only selects either photography or fluorescence measurements according to the measurement location, such as at home or in a medical facility.