Article pubs.acs.org/IECR
Gold Nanoparticle-Based Colorimetric Recognition of Creatinine with Good Selectivity and Sensitivity Hong Du,† Ruiyi Chen,† Jianjun Du,* Jiangli Fan, and Xiaojun Peng State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, Dalian, 116024, P. R. China S Supporting Information *
ABSTRACT: A gold nanoparticle-based colorimetric sensor for the determination of creatinine was developed as an important index for early diagnosis of kidney function and corresponding renal diseases. Because of the unique synergistic coordination capability of adenosine and creatinine with Ag+ on a particle surface, our system exhibits an excellent selectivity to creatinine among various ions and biomolecules. There are good linear relationships of absorption changes (A630 nm/520 nm) over creatinine concentrations, so both colorimetric qualitative detection by the naked eye and quantitative determination by UV−vis spectrometer could be realized with an excellent limit of detection compared with that of other methods. Finally, by testing creatinine in practical samples, such as urine mimic and bovine serum, good recoveries were obtained with proper relative standard deviations.
1. INTRODUCTION Creatinine, the ultimate metabolites of nitrogen element in the human body, is chiefly removed from the blood by kidneys, specifically by glomerular filtration. If there was a marked damage on functioning nephrons, the dysfunction of filtration could result in an obvious rise of creatinine concentration in blood and a decrease in urine. Therefore, a relationship of creatinine concentration in body fluids is built with kidney function1 and corresponding renal diseases (e.g., chronic kidney disease, CKD)2 which millions of patients suffer every year all over the world. Usually the content of creatinine in serum is a more important factor, being 44−106 μM for a healthy person in a clinical trial, while reaching above 1000 μM for those suffering kidney malfunction. Besides, compared with serum creatinine concentration, the glomerular filtration rate (GFR) is a more accurate index for assessment of renal functions, which is calculated depending on not only the serum creatinine concentration but also urine creatinine concentration and urine volume.3 The GFR in healthy young men and women is approximately 130 mL/min per 1.73 m2 and 120 mL/min per 1.73 m2, respectively.4 CKD is defined as either kidney damage or a GFR < 60 mL/min per 1.73 m2 for 3 months or more, irrespective of cause, and classified stages of CKD severity are based on GFR. When GFR is less than 15 mL/min per 1.73 m2, it is a sign and symptom of uremia, which requires dialysis or kidney transplantation.5 A variety of methods,6 therefore, have been developed for quantitative detection of creatinine, such as capillary electrophoresis, gas chromatography, liquid chromatography, mass spectrometry, and so on besides the mostly used strategy of Jaffe’s method and enzymatic digestion in clinical trials.7−17 Actually, the colorimetric method, such as © XXXX American Chemical Society
Jaffe’s method, is one of simplest, most convenient, and promising methods with not only fast naked eye-distinguishable results but quantitative determination by UV−vis spectrometer. However, picric acid forms an orange complex not only with creatinine but also with many other biomolecules (e.g., metabolins and drugs) in alkaline media. Therefore, there is an unmet need in developing an easy colorimetric method for creatinine with enough sensitivity and especially good selectivity. In last two decades, plasmonic nanoparticles (NPs), especially gold NPs (AuNPs), have attracted much attention as an ideal transducer of colorimetry for sensing, recognizing, and determination of ions and small biomolecules, as well as proteins and enzymes because of their distinct surface plasmonic resonance (SPR) which brings them 1000-fold greater absorption extinction coefficient than normal dyes.18−24 In recent, inspired from synergistic effect,25−28 which combines respective advantages with various functional collaborators,29,30 we developed an AuNP system for selective recognition of creatinine in body fluid samples with a naked-eye distinguishable red-to-blue color change based on synergistic coordination effect of creatinine and uric acid to Hg2+ on AuNP.31 Though it exhibited very good selectivity and sensitivity creatinine determination, Hg2+ is needed in the assay. Concerning the potential toxicity of Hg2+ used in our former system, not only creatinine-selective synergistic complexes but mercury-free Received: September 5, 2016 Revised: November 4, 2016 Accepted: November 10, 2016
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DOI: 10.1021/acs.iecr.6b03433 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
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Industrial & Engineering Chemistry Research
Scheme 1. Illustration of Colorimetric Assay of Creatinine Based on AuNPs via Synergistic Coordination Chemistry of Creatinine with Adenosine and Ag+
Figure 1. (a) UV−vis spectra of adenosine/Ag+−AuNP system in the absence (red line) and presence (blue dash line) of creatinine (5 μM); (inset) photo of adenosine/Ag+−AuNP solution in the absence (left) and presence (right) of creatinine (5 μM); (b) UV−vis spectra of adenosine/Ag+− AuNP system in the presence of different creatinine (0.2, 0.6, 0.8, 1.0, and 1.4 μM); (c) A630 nm/520 nm values versus creatinine concentrations over the range of 0.2−1.4 μM, error bar means 5 parallel tests for each point; (d) A630 nm/520 nm values of adenosine/Ag+−AuNP system in the absence and presence of different molecules with similar structures as creatinine ([creatinine] was 10 μM, others were 50 μM).
2. MATERIAL AND METHODS 2.1. Materials. Gold(III) chloride trihydrate (>99.9%) and sodium citrate dehydrate (>99%) were purchased from Aladdin Reagent Company and Energy Chemical Reagent Company, respectively. Solutions of metal ions and anions were prepared from Zn(NO3)2·6H2O, CaCl2, BaCl2, Na2SO4, KNO3, MgSO4, CoCl2, AgNO3, Cu(CH3COO)2·H2O, Ni(NO3)2·6H2O, Mn(CH3COO)2·4H2O, Pb(NO3)2, HgCl2·0.5H2O, Cr(NO3)3, FeCl3·6H2O, and CH3COONa, NaNO3, NaClO4, Na3PO4, NaSCN, Na2S2O3, Na2HPO4, NaHCO3, Na2SO4, NaCl, NaF, NaBr and Na2SO3 by separately dissolving each salt in DI water. Solutions of amine acids and small molecules were prepared from lysine, aspartic acid, proline, arginine, histidine, phenylalanine, glycine, asparagine, glutamine, serine, methionine, tyrosine, tryptophan, cysteine, and urea, 1-methylcaprolactam, niacin, glucose, pyrrolidone hydrotribromide, phenylboronic acid, sucrose, melamine, imidazole, glutathione, uric acid, sodium ascorbate, folic acid, uracil, creatinine, Nhydroxysuccinimide (NHS), hydantoin, 1-methyl hydantoin,
should be developed for a fast, sensitive, and convenient determination of creatinine in both serum and urine for further calculating GFR. Herein, to obtain a mercury-free but creatinine-respond synergistic system, dozens of biomolecules and metal ions are selected for screening synergistic complexes on the platform of AuNP. By an orthogonal experiment, besides creatinine/uric acid/Hg2+ system in our former work, a group of creatinine/ adenosine/Ag+ is found possessing a synergistic effect on gold surface (Scheme 1 and Table S1). Compared with Hg2+, Ag+ shows not only little toxicity at low concentration but some kind of bactericidal capability.32 After data on creatinine concentration in both urine and serum has been obtained, then GFR could be easily calculated for early diagnosis of kidney disease. As a proof-of-concept, sensor of adenosine/ Ag+−AuNP was developed for selectively recognizing creatinine as shown in Scheme 1, wherein the creatinine-involved complexes act as receptor and Au NPs is transducer. B
DOI: 10.1021/acs.iecr.6b03433 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Article
Industrial & Engineering Chemistry Research
3.2. Selectivity of the Assay. Successful creatinine assay in practical urine and serum samples counts on good selectivity, because both urine and serum samples are a complicated system involving various metal ions, anions, amino acids, and other biomolecules, some of which could affect the creatinine recognition potentially. So we examined our system with multitudinous metal ions (Ag+, Na+, K+, Hg2+, Mn2+, Cu2+, Mg2+, Co2+, Ca2+, Ba2+, Pb2+, Ni2+, Zn2+, Fe3+, and Cr3+), anions (NO3−, CH3COO−, F−, Cl−, Br−, ClO4−, HCO3−, SO32−, SCN−, SO42−, S2O32−, HPO42−, and PO43−), amino acids (lysine, aspartic acid, proline, arginine, histidine, phenylalanine, glycine, asparagine, glutamine, serine, methionine, tyrosine, tryptophan, and cysteine), and a variety of biomolecules (urea, 1-methyl caprolactam, niacin, glucose, pyrrolidone, hydrotribromide, phenylboronic acid, sucrose, melamine, imidazole, glutathione, uric acid, sodium ascorbate, folic Acid, thymine, and uracil). Results show that none of these ions and molecules, except for creatinine, could lead to obvious changes in A630 nm/520 nm values as well as solution color (Figure S3). Furthermore, we particularly selected molecules with similar chemical structure as creatinine, such as N-hydroxysuccinimide (NHS), 2-pyrrolidone, hydantoin, 1-methylhydantioin, and creatine, to verify the distinct capability of adenosine/Ag+− AuNP in distinguishingly recognizing creatinine. None of those interferents result in an obvious increase of A630 nm/520 nm value (Figure 1d). Besides, more complicated system involving mixed interfering ions/molecules and creatinine were tested as shown in Figures S4 and S5, which further exhibited that our system had a strong resistance to interfering ions/molecules.34−36 The above strict selectivity experiments prove our system can identify creatinine among multitudinous interferents in the complicated biological fluid sample. TEM images demonstrate the changes in microscopic field in which AuNPs decorated with adenosine/Ag+ are dispersed(Figure 2a) and then
2-pyrrolidone, and creatine by separately dissolving each molecule in DI water. All other chemicals were supplied by Aladdin Reagent Company and Energy Chemical Reagent Company and were used as received. 2.2. Characterization. UV−vis spectra were recorded by using a UV−vis spectrophotometer (UV−vis 2501 PC) with the baseline correction. Nanoparticles dispersion and aggregation were characterized by transmission electron microscopy (TEM, JEM2010). The zeta potentials of AuNP before and after modification were measured with a Zetasizer Nano-ZS90 instrument. 2.3. Synthesis of 13 nm AuNP. AuNP (13 nm) was prepared by sodium citrate reduction of a HAuCl4 solution as described in the literature.33 2.4. Preparation of Urine Mimic. Urine mimic solution was prepared as follows: CaCl2 (0.089 g), MgSO4 (0.100 g), NaHCO3 (0.034 g), Na2C2O4 (0.003 g), Na2SO4 (0.258 g), NaH2PO4 (0,100 g), Na2HPO4 (0.011 g), NaCl (0.634 g), KCl (0.450 g), NH4Cl (0.161 g), sodium citrate (0.297 g), urea (2.427 g), uric acid (0.034 g), and creatinine (15 μM, 20 μM, 25 μM, 30 μM, and 35 μM) in 200 mL of DI water. 2.5. Pretreatment of Bovine Serum Samples. The bovine serum samples spiked with different concentrations of creatinine were pretreated as follows: a couple of drops of trichloroacetic acid (300 g/L) were added into 2 mL of bovine serum, and the mixture was shaken for 2 min. The white deposits that appeared were separated by filter (0.22 μm). NaOH (2 M) was added to adjust pH to about 7 as determined by pH testing strip.
3. RESULTS AND DISCUSSION 3.1. System Fabrication and Creatinine Determination. AuNP (13 nm) was prepared by sodium citrate reduction of a HAuCl4 solution as described in the literature.33 After adenosine (0.5 μM) was introduced to the AuNP solution, the solution was centrifuged (10000 rpm, 10 min) and then dispersed in DI water, giving a red color with a typical localized SPR (LSPR) band at 520 nm. Different Ag+ concentrations were added to fabricate an adenosine/Ag+−AuNP system based on electrostatic effect and coordination chemistry in different pH values. Finally, Ag+ (5 μM) in PBS (pH 7.4, 10 mM) was proven to be the optimal condition for creatinine recognition in fluid samples (Supporting Information, Figure S1). As expected, an introduction of creatinine (5 μM) results in a decrease of LSPR intensity at 520 nm while a new peak appears at around 630 nm (Figure 1a), and meanwhile a red-to-blue color change could be observed by the naked eye in a minute (Figure 1a inset). Our colorimetric system is so fast for recognizing creatinine that A630 nm/520 nm values become stable within 5 min (Figure S2). Further, a quantitative titration experiment was performed for studying ratiometric changes in UV−vis spectra by increasing creatinine gradually in the adenosine/Ag+−AuNP system, and the obtained ratiometric changes in the UV−vis spectra had an isoabsorptive point at 550 nm (Figure 1b). Furthermore, a good linear relationship (R2 = 0.994) is obtained between the SPR intensity ratio of A630 nm/520 nm values and creatinine concentrations over the range of 0.2−1.4 μM (Figure 1c), exhibiting very good repeatability and acceptable relative errors (five parallel tests for each point, respectively). The limit of detection (LOD) of this system reaches as high as 12.7 nM (calculated by 3σ/ slope), which is sensitive enough for both qualitative and quantitative creatinine recognition in practical samples.
Figure 2. TEM images of the adenosine/Ag+−AuNP system in the (a) absence and (b) presence of creatinine (5 μM); scale bar, 50 nm.
aggregated heavily after introduction of creatinine (Figure 2b), which is consistent with colorimetric and spectroscopic changes. Therefore, good performance of this system gives it potential in further qualitative and quantitative assays of creatinine in more complicated media, such as urine and serum samples. 3.3. Creatinine Detection in Urine Mimic Samples. An accurate and colorimetric situ assay needs excellent selectivity, fast response time, and naked eye distinguishable color changes. Compared to methods demanding complicated instruments, the AuNP-based colorimetric method exhibits potential in a new generation of fast, convenient, and portable assay, and integration with a smart phone makes them portable testing C
DOI: 10.1021/acs.iecr.6b03433 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
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Figure 3. A630 nm/520 nm values of adenosine/Ag+−AuNP system versus creatinine concentrations in (a) urine mimic samples and (b) bovine serum samples.
devices.37,38 We then apply our system in creatinine assay in urine mimic samples by artificially adding quantitative concentrations of creatinine (4−40 mM) as prepared in the literature,23 which were further diluted 200 times by DI water for tests. Diluted urine samples (5 μL) were added to adenosine/Ag+−AuNP solution (500 μL) in optimized conditions (10 mM PBS, pH 7.4, [Ag+] = 5 μM, [AuNP] = 5.4 nM), followed by UV−vis spectroscopy recording after 6 min incubation. Therefore, the creatinine concentrations should be calculated by [creatinine]test × 200 × 100. As shown in Figure 3a, a good linear relationship is obtained (R2 = 0.982) between A630 nm/520 nm values and creatinine concentration over the range of 0.2−2.0 μM (urine samples were diluted 200 × 100 fold in testing systems). Furthermore, to verify this linear relationship in Figure 3a, we examined two urine mimic samples with the artificial addition of different creatinine contents (16 mM and 36 mM). The detected means with standard deviations reached 20.2 ± 0.8 mM and 33.2 ± 2.6 mM with acceptable recoveries and RSD% as shown in Table 1.
were diluted 100-fold in the testing system), which covers the critical point of normal creatinine concentration (130 μM) in healthy people. Moreover, bovine serum with 125.0 μM and 200.0 μM creatinine were prepared as normal and diseased serum samples artificially. Recoveries of 88.3% and 122.6% were achieved by using the linear equation in Figure 3b with an RSD% of 15.14% and 6.5%, respectively (Table 2), meaning our system could quantitatively distinguish a healthy serum sample from a diseased one. Table 2. Determination of Creatinine in Bovine Serum Samples
samplea
detected mean ± SDb of [creatinine] by AuNPs (mM)
recovery (%)
RSDc (%)
1 2
16.0 36.0
20.2 ± 0.8 33.2 ± 2.6
127.6 92.0
3.9 7.8
detected mean ± SDb of [creatinine] by AuNPs (μM)
recovery (%)
RSDc (%)
1 2
125.0 200.0
110.4 ± 17.0 245.0 ± 15.9
88.3 122.6
15.4 6.5
a
The samples were prepared as described in the experiment section, wherein different concentrations of creatinine are added artificially. b SD = standard deviation, the mean of six parallel tests. cRelative standard deviation of mean recovery RSD (%) = (SD/mean) × 100.
Table 1. Determination of Creatinine in Urine Mimic Samples [creatinine] added artificially (mM)
samplea
[creatinine] added artificially (μM)
When the creatinine concentrations in both urine and serum were obtained, the GFR was calculated and gave more accurate information on kidney function according to the classification of CKD in clinical tests: at least 60 mL/min per 1.73 m2, 45 to 59 mL/min per 1.73 m2 (stage 3a), 30 to 44 mL/min per 1.73 m2 (stage 3b), 15 to 29 mL/min per 1.73 m2 (stage 4), and less than 15 mL/min per 1.73 m2 (stage 5).39 Besides, identification of chronic kidney disease rely on measurement of not only GFR but also albuminuria40 which was studied in our former works by fluorescent probes.41,42 And cystatin C, as an alternative filtration marker, is also being explored to estimate GFR.43,44 Compared with the methods based on complicated and expensive instruments, our system exhibits obvious advantages: (1) it has very good selectivity toward creatinine than nearly all tested biomolecules, even with similar structures; (2) the sensitivity could compete with and be even better than that of some of instrument-based methods, which is compared as shown in Table 3; (3) it can be conveniently operated without professional experts. 3.5. Recognition Mechanism. In our earlier work, uric acid/creatinine/Hg2+ complexes on the AuNP surface were presented for recognizing creatinine because uric acid has a similar binding site as thymine that is always used for Hg2+ detection.45 Adenosine/Ag+ complexes on the AuNP surface, similarly, can functionally recognize creatinine because
a
The samples were prepared as described in the experiment section, wherein different concentrations of creatinine are added artificially. b SD = standard deviation, the mean of six parallel tests. cRelative standard deviation of mean recovery RSD (%) = (SD/mean) × 100.
3.4. Creatinine Detection in Serum Samples. The serum component is much more complicated and greater than that in urine, so the creatinine recognition in serum is more difficult. However, the creatinine level in serum is even more important to show how well the kidney works. For example, a high level of creatinine means the kidney is not working as it should. For creatinine determination in practical serum, bovine serum is used instead, wherein the creatinine concentration is around 42.4 μM detected by classical Jaffe’s method (Figure S6). Then bovine serum samples spiked with different concentrations of creatinine (50−400 μM) were pretreated using trichloroacetic acid (300 g/L, 150 μL) to remove bovine serum inside. The A630 nm/520 nm values are found increasing linearly (R2 = 0.990) with more creatinine addition over the range of 50−400 μM (Figure 3b; creatinine concentrations D
DOI: 10.1021/acs.iecr.6b03433 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
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Industrial & Engineering Chemistry Research Table 3. Different Sensor and Parameters for Creatinine Detection
a
sensor
LODa
response time
real sample
ref
conducting-polymer electrochemical sensor by Wei and Veale et al. enzyme-amperometric sensor by Chemnitius et al. photonic crystal sensor by Asher et al. electrochemical sensor by Banks et al. tandem mass spectrometry by Reynolds and Creaser et al. potentiometric sensor by Andrade and Ballester et al. amperometric sensor by Lin et al. capillary and gravitational chip by Wang et al. ion mobility spectrometric sensor by Ebrahimzadeh et al. multichanllel kinetic spectrometric sensor by Dasgupta et al. portable microfluidic sensor by Laiwattanapaisal AuNPs based colorimetric sensor
0.46 mg/dL (40.4 μM) 0.06 mg/dL (5.3 μM) 6 μM 0.27 mM 0.4 μg/mL (3.5 μM) 0.6 μM 6.8 μg/dL (0.6 μM) 10 mg/dL (900 μM) 0.6 mg/L (5.3 μM) 0.76 mg/L (6.7 μM) 3.3 mg/L (29.2 μM) 12.7 nM
