Introduction

Small molecules of biothiols are closely related to the occurrence and development of various diseases [1,2,3]. For example, abnormal levels of cysteine (Cys) can result in slow growth, skin lesions, lethargy, liver damage, severe neurotoxicity, and cardiovascular diseases [4,5,6]. Therefore, the determination of Cys in a physiological system is of great significance to early clinical diagnosis of the above diseases. In general, several methods [7,8,9,10,11,12,13,14,15,16,17,18] have been applied for biothiols quantitation such as high-performance liquid chromatography (HPLC) [7], capillary electrophoresis [8], and fluorescence method [10].

As a point-of-care testing system, microfluidic paper-based analytical devices (μPADs) provide a promising platform for biochemical assays with the advantages of low cost, portability, and disposability [19,20,21,22,23,24,25,26,27,28]. μPADs are usually integrated with colorimetric systems because of its simplicity. As the nanoparticles can change their color depending on their shape, size, and distance between the particles, they have been widely employed in colorimetric determination [25, 29,30,31,32,33,34,35,36]. Some reported nanomaterial-based colorimetric determinations for Cys were listed in Table 1. However, the surrounding conditions in the solution can affect the aggregation or disaggregation of the particles. To overcome the limitation, Prasetia et al. formed nanoparticles on a hydroxyapatite solid support [36]. To the best of our knowledge, few studies on nanoparticles for quantification of Cys on μPADs using colorimetric system have been reported to date.

Table 1 An overview on recently reported nanomaterial-based optical determination of Cys
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Aspartic acid (Asp) modified gold nanoparticles (Asp-AuNPs) were synthesized and used as a colorimetric indicator. Using the Asp-AuNPs, Cys was determined on paper-based devices via a simple ratiometric color intensity method. Different from general colorimetric determination, the results were recorded using a cell phone. The color intensity was obtained by the Photoshop software. A ratiometric color intensity method was designed for the analysis. The effects of assay conditions were investigated systemically. Cys concentration of human blood plasma was obtained under the optimum conditions.

Materials and methods

Apparatus and reagents

DL-aspartic acid (98%), DL-cysteine (97%), DL-homocysteine (95%), ascorbic acid (≥ 99%), and chloroauric acid (HAuCl4·3H2O, 99.9%) were all from Shanghai Aladdin Bio-Chem Technology Co., LTD (https://www.aladdin-e.com/, Shanghai, China). Glutathione (≥ 98%) was obtained from TCI (Shanghai) Development Co., LTD (https://www.tcichemicals.com/zh/cn/, Shanghai, China). Trisodium citrate and sodium sulfite were purchased from Tianjin Sheng’ao Chemical Reagent Co., Ltd. (http://5583800.1024sj.com/, Tianjin, China). Chitosan was from Shanghai Hefeng pharmaceutical Co., Ltd. (http://www.shharvest.com/, Shanghai, China). All the reagents were analytical grade unless otherwise stated. Blood plasmas of healthy volunteers were obtained from the Department of Medical Technology, Xi’an Medical University. Deionized water was used in all the experiments. Whatman No. 1 qualitative papers were purchased from Whatman (Maidstone, Kent, http://whatman.utopbio.com/, UK). Nanoparticle size and zeta potential were analyzed with Malvern Zetasizer Nano zen3600 (Malvern Instruments, Worcestershire, http://www.malvern.com/, UK). Huawei 5s cell phone was from Huawei Technologies Co. Ltd. (https://www.huawei.com.cn/, Guangdong, China). Adobe Photoshop CS2 (PS 9.02) was from Adobe Systems Incorporated (California, https://www.adobe.com/, USA).

Experimental procedures

Synthesis of the Asp-AuNPs

The AuNPs were synthesized based on the sodium citrate synthesis method similar as reported previously [29]. The syntheses in adequate detail were given in the Electronic Supporting Material (ESM). Then, 20 μL of 2 mM aspartic acid (Asp) was added into 5 mL above AuNP solution. The solution was stirred for 2 h at room temperature to obtain the Asp-AuNP solution. The obtained Asp-AuNP solution was stored in refrigerator at 4 °C, and filtered with 0.45 μm pore membrane filters prior to use.

Sample preparation

The sample preparation was similar as reported previously [37]. In briefly, acetone (300 μL) was added to 100 μL fresh plasma and centrifuged at 8000 rpm (214.6 g) for 10 min. Then, the supernatant was dried with N2 gas and 1000 μL deionized water was added to obtain the plasma sample.

Preparation of paper-based platform

Round-shaped Whatman No. 1 qualitative papers were cut with a hole puncher, each with a diameter of 6 mm. To avoid the rapid leaching of nanoparticles from the paper surface, 6.0 μL of 0.25 mg mL−1 chitosan solution was spotted in the paper and dried at room temperature initially. Figure 1a displays the preparation procedure of the paper-based analysis platform.

Fig. 1
figure 1

a Preparation of the paper-based analysis platform. b Experimental procedure and illustration of the ratiometric color intensity method

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The performance of the assay

Asp-AuNP solution with volume of 7 μL was dropped on above paper-based platform. They were entrapped onto the surface through the hydrogen bonding between chitosan and Asp. Then, 7 μL analyte was spotted. After action for 2 min, the results were recorded using a Huawei 5s camera under a lamp (LED lamp, 3 W power). The distance between paper-based platform and the light source was 20 cm. These conditions make the same brightness when taking a picture. For multiple sample tests, the analyses were conducted one by one. The experimental procedure and ratiometric color intensity method are shown in Fig. 1b.

The ratiometric color intensity method

The images were analyzed using Adobe Photoshop CS2 as follows. First, the images were scanned using the CS scanning application software (IntSig Information Co., Ltd) that was installed on the Huawei 5s. The image is similar to the result of the scanner after CS software processing. Second, the color intensity was obtained by measuring “mean” value of the corresponding channel with Photoshop CS2. The procedure of Photoshop analysis is as follows. Open “Histogram” in the “Window” menu, and then select “Red” (or “Blue”) channel. “Pixels” was chosen as 380. The “mean” intensity is obtained when the keys “control” and “I” were pressed at the same time. At last, the Red/Blue value was calculated by dividing the “mean” value of Red channel to that of Blue channel.

Results and discussion

Principle of the determination

The approach is based on the aggregations of Asp-AuNPs. Resulting from the aggregation of Asp-AuNPs, the color gradually changes from red to blue as the concentration of Cys is increasing. It is attributed to the ion pair interactions between Cys and Asp. The interaction strength is directly related to the molecular structure and the charged groups [29]. Amino group and carboxyl group of Cys can change the intermolecular ion pair interaction of Asp-AuNPs. The higher the concentration of Cys, the faster aggregation and the more obvious color change. According to the results of Adobe Photoshop CS2, the color intensity of “Red” and “Blue” channels was all gradually changing. It is found that the Red/Blue ratio is proportional to the Cys concentration. Therefore, the average Red/Blue ratio was used to quantify the concentration of Cys in the sample.

Characterization of Asp-AuNPs

The size and zeta potential of AuNPs and Asp-AuNPs were analyzed on Malvern Zetasizer Nano zen3600 instrument. The particle size of AuNPs and Asp-AuNPs was (37.3 ± 0.1 nm) and (54.4 ± 0.2 nm), respectively. The zeta potential of AuNPs and Asp-AuNPs was (− 20.1 ± 1.0 mV) and (− 33.3 ± 0.7 mV), respectively. The successful bonding of Asp to AuNPs can be proved by the increased particle size. The dispersion of Asp-AuNPs is stable which may attribute to its higher absolute value of zeta potential.

To confirm the existence of Asp on AuNP surface, UV-visible absorption spectrum was also investigated (Fig. 2). The maximum absorption of Asp-AuNPs is 521 nm. Compared with AuNPs (518 nm), it has a red shift. The color and the maximum absorption of Asp-AuNPs have no obvious changes after 30 days. It shows the good stability of the Asp-AuNPs.

Fig. 2
figure 2

UV-Vis spectra of AuNPs and Asp-AuNPs. Inset is the photographs of AuNPs and Asp-AuNPs

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Optimization of method

The following parameters were optimized: (a) the Asp-AuNP volume; (b) the sample (Cys) volume; (c) action time. Respective text and figures on optimizations were given in the ESM. In short, the following experimental conditions were found to give best results: (a) Best volume of Asp-AuNPs was 7 μL; (b) best volume of Cys sample was 9 μL; and (c) best action time was 2 min.

The calibration plot of Cys

To determine the concentration of Cys in samples, a calibration plot was fabricated. First, 7 μL Asp-AuNP was added onto the round paper, and then 9 μL Cys with different concentrations was added. The results were recorded at 2 min by Huawei 5s camera. The calibration plot is shown in Fig. 3, and the inset is the corresponding photographs. The ΔRed/Blue ratio (excluding blank value) was proportional to the concentration of Cys. The calibration function was y = 0.1652x + 0.0761, and the linear correlation coefficient (R) was 0.9984. The results indicate that ΔRed/Blue is linear with the concentration of Cys over the range of 99.9–998.7 μM with a LOD of 1.0 μM. In experiments, it is found that when the average Red/Blue ratio (0.7425) is greater than the blank value (0.7410) by 0.0015, the color difference between them is visible. Therefore, the Cys concentration in this case (1.0 μM) was defined as the LOD. In order to evaluate the validity of the calibration plot, a simple comparison of the experimental and theoretical Cys concentrations based on the calibration plot was conducted (Table 2). The recovery was determined as reported previously [37]. The results show that the recovery was between 98.8% and 102.0%. It reveals the accuracy of the method.

Fig. 3
figure 3

Calibration curve and LOD of Cys detection. a Calibration curve and photographs of Cys detection obtained by Huawei 5s at 2 min. b Photographs of Cys detection at 2 min. Asp-AuNPs and Cys were fixed at 7 μL and 9 μL, respectively

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Table 2 Comparison of the experimental and theoretical results of Cys based on the calibration plot
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Selectivity and the repeatability of the method

The selectivity of the Cys assay method was investigated. As shown in Fig. 4, compared with the remarkable color intensity ratio of Cys, the ratios show no obvious responses of several other amino acids. The selectivity of the method was further evaluated by comparing the results in the absence and presence of coexistent interfering ions and molecules. Equal amount of ions or molecules were added to the Cys sample (0.50 mM), and the Red/Blue ratio was recorded and analyzed (Table 3). The results show that equal amounts of Zn2+, glucose, SO32−, and vitamin C, et al. have no interference in the determination of Cys. All substances had a deviation in the range of − 3–+ 3%. It reveals that the coexistent substances will not interfere with the Cys determination.

Fig. 4
figure 4

Selectivity test of the Cys compared with other amino acids. Asp-AuNPs and amino acids were fixed at 7 μL and 9 μL, respectively. The concentration of amino acids and action time were 0.78 mM and 2 min, respectively

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Table 3 Effects of coexistence interfering substances on the reliability of test results
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The repeatability of the method was also discussed. The relative standard deviation (RSD) of intra-day (n = 5) and inter-day (n = 5) was all smaller than 2% for Cys samples with different concentrations (Table 4). It reveals that the method has good repeatability. The better RSD which is attributed to the average color intensity of one image is used [37].

Table 4 Repeatability of Cys detection in intra-day and inter-day
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Determination of Cys in real samples

In order to show the potentiality of the method, the human plasma was chosen as sample. And to confirm the reliability of the data, sample recovery tests were conducted. As shown in Table 5, Cys concentration was (0.27 ± 0.02 mM) and the recoveries was from 99.2 to 101.1%. The satisfactory recoveries reveal the good validity of the method. The results prove that the method can be used to determine Cys in real sample. There were no severe interferences in these samples for Cys determination. According to the linear range of the calibration plot, pre-concentration is needed for urine samples because of its lower concentration [36].

Table 5 Determination of Cys in human plasma sample
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Conclusion

Through amino groups, Asp modified AuNPs were synthesized. The colorimetric quantitation of Cys was obtained on the surface of a small paper patch (i.e., 6 mm) using the Asp-AuNPs. The method allows the Cys determination in the absence of UV-visible or fluorescence spectrophotometer. Its determined principle was the aggregation of Asp-AuNPs in the presence of Cys. With the aggregation, the color of Asp-AuNPs will change from red to blue. The results were recorded by a cell phone. The color intensity was analyzed by the Adobe Photoshop software. The ratio of color intensity value at red and blue channel (Red/Blue) increases with Cys concentration linearly. The assay method has the advantages of paper-based analytical devices and AuNPs. It shows good selectivity, portability, low cost, and etc.