Competitive Coordination of Cu2+ between Cysteine and

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Competitive Coordination of Cu2+ between Cysteine and Pyrophosphate Ion: Toward Sensitive and Selective Sensing of Pyrophosphate Ion in Synovial Fluid of Arthritis Patients Jingjing Deng, Ping Yu, Lifen Yang, and Lanqun Mao* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, The Chinese Academy of Sciences, Beijing 100190, China ABSTRACT: Direct selective and sensitive sensing of pyrophosphate ion (PPi) in synovial fluid of arthritis patients is of great importance because of its crucial roles in the diagnosis and therapy of arthritic diseases. In this study, we demonstrate a sensitive and selective method for PPi sensing in synovial fluid of arthritis patients with gold nanoparticles (Au-NPs) as the signal readout based on the competitive coordination chemistry of Cu2+ between cysteine and PPi. Initially, Au-NPs stabilized with cysteine are red in color and exhibit absorption at 519 nm in the UV−vis spectrum. The addition of an aqueous solution of Cu2+ to the Au-NPs dispersion containing cysteine causes the aggregation of Au-NPs, resulting in the wine red-to-blue color change and the appearance of a new absorption at 650 nm in the UV− vis spectrum of the Au-NPs dispersion. The subsequent addition of PPi to the Au-NPs aggregation well solubilizes the aggregated Au-NPs with the changes in both the color and the UV−vis spectrum of the Au-NPs dispersion. These changes are ascribed to the higher coordination reactivity between Cu2+ and PPi than that between Cu2+ and cysteine. On the basis of this, the concentration of PPi can be visualized with the naked eyes through the blue-to-wine red color change of the Au-NPs dispersion and quantitatively determined by UV−vis spectroscopy. Under the optimized conditions, the ratio of the absorbance at 650 nm (A650) to that at 519 nm (A519) shows a linear relationship with PPi concentration within a concentration range from 130 nM to 1.3 mM. The method demonstrated here is highly sensitive, free from the interference from other species in the synovial fluid, and is thus particularly useful for fast and simple clinic diagnosis of arthritic diseases.

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chondrocalcinosis and hypophosphatasis.3c,d Although PPi could be determined by some elegant methods including chromatographic and fluorescent methods and so forth,4 the high requirements from the fast clinic diagnosis and therapy of arthritis substantially necessitate a new method for PPi sensing both in method simplicity and sensitivity. In this study, we demonstrate a technically simple yet selective and sensitive method for direct colorimetric sensing of PPi in the synovial fluid of arthritis patients. Inspired by the unique optical properties of gold nanoparticles (Au-NPs) and the excellent surface/interface recognition ability that benefited from the rational design of the surface chemistry, Au-NPs based assays have been demonstrated to be very attractive and effective for the detection of various analytes ranging from small molecules to enzyme activity.5 This kind of method is anticipated to be particularly attractive for direct sensing of PPi in the synovial fluid of arthritis patients because of the high extinction coefficient of Au-NPs principally enabling a highly sensitive detection of low concentration of PPi in the synovial fluid, which is of great importance for clinic applications.

n recent years, considerable attention has been drawn in arthritis because, as one kind of the most frequent disorders, arthritis is directly correlated with age, has already been a significant health care problem, and will become even more of an economic burden in the coming years, especially with a quick increase in the proportion of the elderly in the population.1 On the basis of the data from National Health Interview Survey (NHIS), more than 21% of U.S. adults (46.4 million persons) have been found to have self-reported doctordiagnosed arthritis. Moreover, in the next 25 years, the number of arthritis patients is projected to increase by 40% in the U.S.2 Therefore, development of a direct, sensitive, and selective method for monitoring the clinically important species that can be used as a biomarker for arthritis is of great importance in the clinic diagnosis and therapy of arthritic diseases. As one kind of the most important biological anions, pyrophosphate ion (P2O74‑, PPi) plays significant roles in the pathological processes of arthritis.3 Early efforts have suggested that PPi could be used as a potential biomarker for the clinic diagnosis and therapy of arthritic diseases.3c,d For instance, the high level of PPi in the synovial fluid has been proposed as an index for arthritis, closely relating to the pathogenesis of calcium pyrophosphate dehydrates deposition diseases (CPDD) and the diseases associated with CPDD such as © 2013 American Chemical Society

Received: December 20, 2012 Accepted: January 22, 2013 Published: January 22, 2013 2516

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of arthritis patients with Au-NPs as readout signal, which is envisaged to offer a new route to the arthritis diagnosis in a simple fashion.

Moreover, the unique sensing features of the Au-NP-based colorimetric assays such as direct visualization with the naked eyes and concise quantification simply with UV−vis spectrometry largely facilitates a quick and technically simple clinic diagnosis of arthritic diseases. The rationale for the PPi sensing demonstrated here is essentially based on the competitive coordination of Cu2+ between cysteine and PPi (Scheme 1A). Cysteine bears a

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EXPERIMENTAL SECTION Chemicals and Reagents. Chloroauric acid (HAuCl4·3H2O), trisodium citrate, cupric sulfate anhydrous, and sodium pyrophosphate were purchased from Beijing Chemical Reagent Co. (Beijing, China). L-Cysteine was obtained from Kanto Chemical Corporation. Baker’s yeast inorganic pyrophosphatase (PPase, EC 3.6.1.1) was from Sigma. All chemicals were of at least analytical grade reagents and used without further purification. All aqueous solutions were prepared with Milli-Q water (18.2 MΩ cm−1). Unless otherwise noted, all experiments were carried out at room temperature. Synthesis of Au-NPs. Au-NPs with a diameter of about 13.5 nm were synthesized as reported before.7 Briefly, trisodium citrate (10 mL, 38.8 mM) was added to a boiling solution of HAuCl4 (100 mL, 1 mM), and the resulting solution was kept continuously boiling for another 30 min to give a wine red mixture. The mixture was cooled to room temperature and then filtrated through a Millipore syringe (0.45 μm) to remove the precipitate, and the filtrate was stored in a refrigerator at 4 °C for use. The size of the synthetic nanoparticles was about 13.0 nm as confirmed by TEM image and UV−vis spectroscopy (TU-1900 spectrophotometer, Beijing Purkinje General Instrument Co. Ltd., China) with an absorption peak at around 519 nm. Colorimetric PPi Sensing. For mechanistic investigation on colorimetric PPi sensing, initially, an aqueous solution of cysteine (70 μL, 25 μM) was added into a dispersion of AuNPs (270 μL, 5 nM), and the resulting mixture was then incubated in a 25 °C water bath for 30 min. After that, 60 μL of Cu2+ solution (4 mM) was then added into the as-prepared AuNPs dispersion (340 μL). After 3 min, 60 μL of different concentrations of PPi was added to the reaction mixtures (400 μL). The resulting mixtures were allowed to stand by for 3 min, and then used for photographing with a digital camera (Canon IXUS 951S, Japan) and UV−vis spectroscopic measurements. The final concentrations of PPi in the resulting mixtures (460 μL) were 5 μM, 10 μM, 50 μM, 100 μM, 500 μM, 1 mM, and 5 mM. For the colorimetric detection of PPi, the order of chemical addition was different from that employed for the mechanistic studies. In this case, 60 μL of different concentrations of PPi was first added into the as-prepared Au-NPs dispersion containing cysteine (340 μL). The mixtures containing Au-NPs, cysteine, and PPi were first allowed to stand by for 3 min, and then an aqueous solution of Cu2+ (60 μL, 4 mM) was added. After 3 min, the resulting mixtures were photographed and used for UV−vis spectroscopic measurements. The final concentrations of PPi in the resulting mixtures (460 μL) were 130 nM, 650 nM, 1.3 μM, 6.5 μM, 13 μM, 65 μM, 130 μM, 650 μM, and 1.30 mM. Colorimetric Sensing of PPi in Synovial Fluids of Arthritis Patients. Prior to the colorimetric sensing, the synovial fluids of arthritis patients were purified by perfusing the fluids through a microdialysis probe (4 μm length) at 2.0 μL/min. The recovery of the microdialysis probe for PPi was determined to be about 21.0%, as evaluated with our colorimetric method. To avoid the possible aggregation of Au-NPs induced by the salts in the synovial fluid dialysates, the dialysates were 2-fold diluted with water prior to the

Scheme 1. (A) Competitive Coordination of Cu2+ with Surface-Confined Cysteine and PPi and (B) Schematic Illustration of Colorimetric Sensing of PPi with Au-NPs as the Signal Readout

thiol group that interacts with Au-NPs through the formation of Au−S bond. Meanwhile, the surface-confined cysteine bears α-amino and α-carboxyl groups and could form a five membered ring with Cu2+.6 These properties enable the aggregation of Au-NPs triggered by the addition of Cu2+ into the Au-NPs dispersion containing cysteine (Scheme 1B). Upon the addition of PPi to the as-formed aggregated Au-NPs dispersion, the aggregated Au-NPs tend to solubilize into the aqueous solution because of the stronger binding ability between PPi and Cu2+ than that between cysteine and Cu2+. As reported previously, the stability constant (K) of the complex formed by Cu2+ and S-methyl-cysteine is log KCu‑Cysteine = 7.88,6c and that for the complex formed by Cu2+ and PPi is log KCu‑PPi = 12.45.6d These properties essentially form a straightforward basis for PPi sensing with Au-NPs as readout signal based on competitive coordination chemistry of Cu2+ between cysteine and PPi. To enable such properties to constitute an analytical protocol for sensitive sensing of PPi, various concentrations of PPi are first added into the Au-NPs dispersion containing cysteine, and Cu2+ is then added into the mixtures to evoke the different changes in the dispersion/ aggregation states of Au-NPs that can be used for PPi sensing (Scheme 1B). To the best of our knowledge, this is the first report on the fast and direct sensing of PPi in the synovial fluids 2517

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not lead to an observable aggregation of Au-NPs, maintaining the initial color and UV−vis spectrum of well dispersed AuNPs. These results suggest that the sole addition of cysteine or Cu2+ did not induce the aggregation of Au-NPs. However, transmission electron microscopy (TEM) image (Figure 2A,B) and UV−vis spectroscopic (Figure 2C) results reveal that the addition of cysteine to the initially prepared citrate-stabilized Au-NPs slightly increases the diameter of Au-NPs from 13.5 ± 3.3 nm to 15.1 ± 2.3 nm (Figure 2A,B, inset) and shifts the surface plasmon band from 519 to 522 nm in the absorption spectrum (Figure 2 C, inset), presumably due to the damping effect of the thiol anchoring group on the gold surface.8 Both features essentially confirmed the formation of cysteinestabilized Au-NPs. Unlike those triggered by the sole addition of cysteine or Cu2+, the addition of Cu2+ (60 μL, 4 mM) into the aqueous dispersion of Au-NPs (270 μL, 5 nM) containing cysteine (70 μL, 25 μM) clearly turns the color of the resulting dispersion from initially wine red to blue (Figure 1, vial 4, blue curve) and produces a new absorption peak at 650 nm (Figure 1, blue curve), indicative of the aggregation of Au-NPs. The aggregation of Au-NPs was not considered to be induced by the salt effects since, in a control experiment, we added NaCl (60 μL, 4 mM) into the aqueous dispersion of the cysteinestabilized Au-NPs (340 μL). We found that such a procedure did not lead to the change in either the color or the UV−vis spectrum of the Au-NPs dispersion, as displayed in Figure 3A. This result substantially rules out the possibility of the saltinduced aggregation of Au-NPs, further verifying that the changes observed in Figure 1 (vial 4, blue curve) were essentially induced by Cu2+. This phenomenon was elucidated by the coordination chemistry between Cu2+ and cysteine on the surface of the Au-NPs; the N atom in the α-amino group and the O atom in the α-carboxyl groups of cysteine molecule coordinate with Cu2+, forming a stable five-membered ring of Cu-cysteine complex (Scheme 1A).6a,b,9 Such a surface coordination interaction eventually leads to the aggregation of Au-NPs, being responsible for the changes in both the color and the UV−vis spectrum of the aqueous dispersion of Au-NPs containing cysteine (Figure 2, vial 4, blue curve). We have also investigated the effect of Cu2+ concentrations on the detection system (Figure 3B) and found that 600 μM of Cu2+ was essential to trigger quick aggregation of Au-NPs. As depicted in Figure 4, the further addition of PPi (60 μL, 38.3 mM) into the dispersion of Cu2+-triggered aggregated AuNPs turns the dispersion color from blue (vial 3) to dark red

measurements. For the colorimetric sensing of PPi in the synovial fluids, 60 μL of the 2-fold diluted synovial fluid dialysates was first added into 340 μL of the aqueous dispersion of Au-NPs containing cysteine (70 μL, 25 μM), and then 60 μL of Cu2+ (4 mM) was added into the resulting mixtures. After being allowed to stand by for 3 min, the mixtures were photographed, and the concentrations of PPi in the synovial fluids were determined with UV−vis spectroscopy.

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RESULTS AND DISCUSSION Mechanistic Investigation on Colorimetric PPi Sensing. Initially, Au-NPs synthesized with citrate as the stabilizer were red in color and exhibit absorption at 519 nm (Figure 1,

Figure 1. (A) Schematic illustration of Cu2+-triggered aggregation of Au-NPs through the coordination chemistry between Cu2+ and surface-confined cysteine. (B) UV−vis spectra and photographs (left corner) of the mixtures prepared by separate addition of pure water (60 μL) (vial 1, black curve), cysteine (70 μL, 25 μM) (vial 2, red curve), Cu2+ (60 μL, 4 mM) (vial 3, cyan curve), or cysteine (70 μL, 25 μM) + Cu2+ (60 μL, 4 mM) (vial 4, blue curve), into citratestabilized Au-NPs (270 μL, 5 nM). The final volume of each resulting mixture was adjusted to 460 μL with Milli-Q water.

vial 1, black curve), both features of well-dispersed 13.5 nm AuNPs.7 The sole addition of cysteine (70 μL, 25 μM) (vial 2, red curve) or Cu2+ (60 μL, 4 mM) (vial 3, cyan curve) into the dispersion of the citrate-stabilized Au-NPs (270 μL, 5 nM) did

Figure 2. TEM images of the citrate-stabilized (A) and cysteine-stabilized (B) Au-NPs. The size distribution histograms of the citrate-stabilized (A, inset) and cysteine-stabilized (B, inset) Au-NPs were made from the TEM images by counting more than 80 particles. (C) UV−vis spectra of citratestabilized (black curve) and cysteine-stabilized (red curve) Au-NPs in water. Inset, enlarged UV−vis spectra of Au-NPs. 2518

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Figure 3. (A) UV−vis spectra and photographs (left corner) of the mixtures prepared by separate addition of pure water (60 μL) (vial 1, black curve), NaCl (60 μL, 4 mM) (vial 2, red curve), cysteine (70 μL, 25 μM) + NaCl (60 μL, 4 mM) (vial 3, cyan curve), or cysteine (70 μL, 25 μM) + Cu2+ (60 μL, 4 mM) (vial 4, blue curve), into citrate-stabilized Au-NPs (270 μL, 5 nM). The final volume of each resulting mixture was adjusted to 460 μL with Milli-Q water. (B) UV−vis spectra and photographs (left corner) of the mixtures prepared by separate addition of pure water (60 μL) (vial 1, black curve), Cu2+ (60 μL, 1 mM) (vial 2, red curve), Cu2+ (60 μL, 2 mM) (vial 3, blue curve), Cu2+ (60 μL, 3 mM) (vial 4, cyan curve), or Cu2+ (60 μL, 4 mM) (vial 5, brown curve), into citrate-stabilized Au-NPs (270 μL, 5 nM) containing cysteine (70 μL, 25 μM). The final volume of each resulting mixture was adjusted to 460 μL with Milli-Q water.

This property, along with the change in the dispersion color triggered by the PPi addition, strongly suggests that the addition of PPi eventually alleviates the Cu2+-induced aggregation of Au-NPs. Note that the addition of PPi into the aqueous dispersion of the cysteine-stabilized Au-NPs did not cause an obvious change either in the dispersion color (Figure 4, vial 2) or in the UV−vis spectrum (red curve), further confirming the PPi-induced alleviated aggregation of Au-NPs, as observed in Figure 4 (vial 4, cyan curve). The protective effect of PPi toward the Cu2+-triggered aggregation of Au-NPs was understood by the strong coordination between PPi and Cu2+ (Scheme 1A), in which the stability constant (K) of the complex formed by Cu2+ and PPi was higher than that of the complex formed by Cu2+ and cysteine analogue (i.e., Smethyl-cysteine), as mentioned above.6d,e These features substantially form a straightforward basis for the direct colorimetric sensing of PPi through the changes in the color and UV−vis spectra of the Au-NPs dispersion. Sensitivity and Selectivity. To evaluate the sensitivity of our method, in a first try, we added different concentrations of PPi into the aggregation of Au-NPs triggered by Cu2+. As depicted in Figure 5A, the addition of various concentrations of PPi to the Au-NPs aggregation gradually turns the color of the dispersion from blue to dark red. Meanwhile, the addition of PPi also leads to a dramatic change in the UV−vis spectra of the dispersion; with increasing the concentration of PPi added into the aqueous dispersions of the aggregated Au-NPs, A519 increases, while A650 decreases (Figure 5A). The ratio of A650/ A 519 decreases with the logarithm (log) of the PPi concentration and shows a linear response toward PPi within a concentration range from 5 μM to 5 mM (A650/A519 = 1.124 − 0.197 log C/μM, R = 0.995). Interestingly, we found that the linear range for the PPi sensing was dependent on the order employed for addition of the chemicals into the Au-NPs dispersion; the first addition of both cysteine and different concentrations of PPi into the Au-NPs dispersion followed by the addition Cu2+ significantly broadens the linear range for the PPi sensing. With such an order, the linear range for PPi sensing was broadened from 130 nM to 1.30 mM still with a good linear efficiency (A650/A519 = 1.013 − 0.2798 log CPPi/ μM, R = 0.9979, Figure 5B). Moreover, the change of the order

Figure 4. (A) Schematic illustration of the change in the dispersion/ aggregation states of Au-NPs evoked by the competitive coordination chemistry of Cu2+ between surface-confined cysteine and PPi. (B) UV−vis spectra and photographs (left corner) of the mixtures prepared by separate addition of pure water (60 μL) (vial 1, black curve), PPi (60 μL, 38.3 mM) (vial 2, red curve), cysteine (70 μL, 25 μM) + Cu2+ (60 μL, 4 mM) (vial 3, blue curve), or cysteine (70 μL, 25 μM) + Cu2+ (60 μL, 4 mM) + PPi (60 μL, 38.3 mM) (vial 4, cyan curve), into citrate-stabilized Au-NPs (270 μL, 5 nM). The final volume of each resulting mixture was adjusted to 460 μL with Milli-Q water.

(vial 4). Meanwhile, such a procedure also leads to a change in the UV−vis spectrum of the initially Cu2+-induced aggregated Au-NPs; upon the addition of PPi, the absorbance at 519 nm (A519) increases, while that at 650 nm (A650) decreases (blue and cyan curves). As demonstrated previously,5b−i the ratio of A650 to A519 (A650/A519) could be considered as an indicator for the degree of dispersion/aggregation state of Au-NPs. As could be easily seen from Figure 4 (blue and cyan curves), the addition of PPi into the dispersion of Cu2+-triggered aggregated Au-NPs remarkably decreases the A650/A519 value. 2519

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Figure 5. (A) (Upper) Schematic illustration of PPi sensing with first addition of Cu2+ into Au-NPs dispersion containing cysteine followed by the addition of PPi. (Lower) UV−vis spectra and photographs (upper corner) of the mixtures prepared by the addition of various concentrations of PPi (60 μL) to the Cu2+-triggered aggregation of Au-NPs. The final concentrations of PPi in the resulting mixtures (460 μL) were 0 μM (vial 1), 5 μM (vial 2), 50 μM (vial 3), 500 μM (vial 4), 5 mM (vial 5). Inset: Plot of A650/A519 against logarithm (log) of the PPi concentration. (B) (Upper) Schematic illustration of PPi sensing with first addition of PPi into Au-NPs dispersion containing cysteine followed by the addition of Cu2+. (Lower) UV−vis spectra and photographs (upper corner) of the mixtures prepared by the addition of Cu2+ (60 μL, 4 mM) to dispersions of Au-NPs (270 μL, 5 nM) containing cysteine (70 μL, 25 μM) and various concentrations of PPi (60 μL). The final concentrations of PPi in the resulting mixtures (460 μL) were 0 nM (vial 1), 130 nM (vial 2), 1.3 μM (vial 3), 13 μM (vial 4), 130 μM (vial 5), 1.30 mM (vial 6). Inset: Plot of A650/A519 against logarithm (log) of the PPi concentration. Each point was the average of three independent experiments. Error bars indicate standard deviations.

for chemical addition enables the PPi sensing down to the nanomolar level. While the elucidation of the mechanisms underlying these excellent analytical properties simply benefited from the change of the order for the chemical addition still needs more experimental studies, we considered that the enhanced electrostatic repulsion among Au-NPs caused by the adsorption of PPi onto the surface of Au-NPs presumably constitutes one of the main reasons for the above properties. We reasoned this from our control experiments by adding Cu2+ into the aqueous dispersion of initially prepared (i.e., citratestabilized) Au-NPs containing PPi. In this case, we did not observe the aggregation of Au-NPs. Moreover, PPi molecule is highly charged and may coadsorb onto the surface of Au-NPs with the stabilizers employed. These properties suggest that the presence of trace amount of PPi in the Au-NPs dispersion could remarkably enhance the electrostatic repulsion among Au-NPs, enabling the low detection of PPi with our method. The excellent analytical properties of this method substantially validate its application for direct sensing of PPi in the synovial fluids of arthritis patients, as will be demonstrated below. To investigate the selectivity of our method, different kinds of species that were considered to possibly interfere with the PPi sensing were first added into the aqueous dispersion of AuNPs containing cysteine, and Cu2+ was then added into the resulting mixtures. As illustrated in Figure 6, the separate addition of each kind of anion including Cl−, NO3−, H2PO4−, HCO3−, SO42‑, HPO42‑, PO43‑, and CO32‑ into the aqueous dispersion of Au-NPs containing cysteine did not result in an obvious change either in the color (B, inset) or in the UV−vis spectra (A) of the dispersions, as compared with those of the aggregation triggered by Cu2+. These results substantially suggest that these anions did not interfere with the PPi sensing with our method. The addition of Cu2+ into the mixtures containing Au-NPs and cysteine with the presence of ATP

Figure 6. (A) UV−vis spectra recorded for the mixtures prepared by the addition Cu2+ (60 μL, 4 mM) to dispersions of Au-NPs (270 μL, 5 nM) containing cysteine (70 μL, 25 μM) with the presence of various anions (60 μL, 1 mM). (B) Values of A650/A519 and photographs (inset) obtained by the addition of Cu2+ (60 μL, 4 mM) to dispersions of Au-NPs (270 μL, 5 nM) containing cysteine (70 μL, 25 μM) with the presence of various anions (60 μL, 1 mM). The final concentration of these anions in the resulting Au-NPs dispersions (460 μL) was 130 μM.

(Figure 6A, red curve), ADP (Figure 6A, blue curve), or AMP (Figure 6A, cyan curve) leads to the changes both in the color and the spectra of the resulting mixtures, suggesting that our method was also responsive to ATP, ADP, and AMP, causing potential interference toward the PPi sensing. Nevertheless, our control experiments with yeast inorganic pyrophosphatase (PPase) eventually rule out such interference in the synovial fluids. It has been well documented that PPase specifically catalyzes the hydrolysis of PPi into orthophosphate with the presence of Mg2+.10 This enzyme has been previously employed to demonstrate the specificity of the methods for PPi determination both in plasma and in synovial fluids.10b−d In 2520

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our control experiments, 5 μL of PPase (0.02 U/μL, containing 1.2 mM MgCl2) was added into the purified 2-fold diluted synovial fluid of the arthritis patients (60 μL), the resulting mixture was incubated at 25 °C for 6 min, and then 60 μL of the resulting mixture was added into the aqueous dispersion of Au-NPs (270 μL, 5 nM) containing cysteine (70 μL, 25 μM). As displayed in Figure 7, the addition of Cu2+ into the resulting

simple yet effective approach to direct sensing of PPi biomarker in synovial fluids of arthritis patients. Colorimetric Sensing of PPi in Synovial Fluid. To demonstrate the validity of our colorimetric method for PPi sensing in the synovial fluids, the purified 2-fold diluted synovial fluid (60 μL) was first added into the aqueous dispersion of Au-NPs (340 μL) containing cysteine (70 μL, 25 μM), to which Cu2+ (60 μL, 4 mM) was then added. As shown in Figure 8 A, the addition of synovial fluid dialysate leads to a slight change in the color of the Au-NPs dispersion from blue (vial 3) to somewhat purple blue (vial 2), and to a clear decrease in the A650/A519 value in the UV−vis spectra (red curve), followed by subsequent Cu2+ addition, suggesting the existence of PPi in the synovial fluids of the arthritis patients. To verify that the aggregation of Au-NPs was triggered by competitive coordination interaction of Cu2+ between cysteine and PPi in the synovial fluids, the diluted synovial fluid dialysate was added into the aqueous dispersion of pure Au-NPs (i.e., citrate-stabilized Au-NPs) and Au-NPs containing cysteine (i.e., cysteine-stabilized Au-NPs) without subsequent addition of Cu2+. In this case, no change either in the color or in the UV− vis spectra was observed (Figure 8B), demonstrating that the competitive coordination interaction remains very essential for the direct selective sensing of PPi. We note that cysteine and Cu2+ endogenously existing in the synovial fluid do not interfere with our detection because of their lower concentrations than those added into the dispersion of Au-NPs.11 According to the calibration curve discussed above, the initial value of PPi level in synovial fluids of arthritis patients was detected to be 23.5 ± 5.3 μM (n = 9), which was consistent with the values reported in literature (Table 1).10c,d These

Figure 7. UV−vis spectra and photographs (left corner) of the mixtures prepared by the addition Cu2+ (60 μL, 4 mM) to dispersions of Au-NPs (270 μL, 5 nM) containing cysteine (70 μL, 25 μM) with the presence of PPi (60 μL, 10 mM) (vial 1, black curve), pure water (60 μL) (vial 2, blue curve), or PPase-incubated synovial fluid (60 μL) (vial 3, red curve).

mixture containing Au-NPs, cysteine, and PPase-incubated synovial fluid did not cause an obvious change either in the color (vial 3) or in the A650/A519 value calculated from the UV− vis spectrum (red curve) of the mixture, as compared with those achieved with the addition of Cu2+ into the aqueous dispersion of Au-NPs containing cysteine (vial 2, blue curve). This comparison demonstrates that the method developed in this study possesses a high selectivity against not only ATP, AMP, and ADP but also other kinds of compounds endogenously existing in the synovial fluids. This property, along with the good linearity, substantially enables the utilization of competitive coordination chemistry of Cu2+ between cysteine and PPi, essentially offering a technically

Table 1. Comparison of Pyrophosphate (PPi) in Synovial Fluid

a

analytical methods

PPi (μM)

our colorimetric method enzymatic analytical method P32 chromatographic method

23.5 ± 5.3 (n = 9)a 23.9 ± 10.5 (n = 12) 22.7 ± 13.8 (n = 11)

n = number of patients having samples tested.

Figure 8. (A) UV−vis spectra and photographs (left corner) of the mixtures prepared by the addition Cu2+ (60 μL, 4 mM) to dispersions of Au-NPs (270 μL, 5 nM) containing cysteine (70 μL, 25 μM) with the presence of PPi (60 μL, 10 mM) (vial 1, black curve), 2-fold diluted synovial fluid (60 μL) (vial 2, red curve), or pure water (60 μL) (vial 3, blue curve). (B) UV−vis spectra and photographs (left corner) of the mixtures prepared by separate addition of pure water (60 μL) (vial 1, black curve), 2-fold diluted synovial fluid (60 μL) (vial 2, red curve), or cysteine (70 μL, 25 μM) + 2fold diluted synovial fluid (60 μL) (vial 3, blue curve) into citrate-stabilized Au-NPs (270 μL, 5 nM). The final volume of each resulting mixture was adjusted to 460 μL with Milli-Q water. 2521

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properties substantially demonstrate that the colorimetric assay developed in this study by exploring the competitive coordination chemistry of Cu2+ between cysteine and PPi would offer an effective way to directly monitoring PPi in the synovial fluids of the patients with arthritis.

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CONCLUSIONS In summary, by rationally tailoring the surface chemistry of AuNPs through the competitive coordination interaction of Cu2+ between cysteine and PPi and fully exploring the excellent optical property of Au-NPs, we have successfully demonstrated a highly sensitive and selective method for direct sensing of PPi in synovial fluid of arthritis patients. The method bears advantages in the low technical and instrumental demands, high sensitivity, and selectivity and could thus be very promising for the reliable clinic sensing of PPi in the synovial fluids. This study essentially offers a new approach to simple monitoring of the biomarker of arthritic disease, which is of great importance in early diagnosis and therapy of arthritic diseases.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86-10-62559373. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the NSF of China (Grants 20975104, 20935005, 21127901, 21210007, and 91213305 for L.M., and 91132708 for P.Y.), the National Basic Research Program of China (973 programs, 2010CB33502, 2013CB933704), and The Chinese Academy of Sciences (KJCX2-YW-W25 and Y2010015).

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dx.doi.org/10.1021/ac303698p | Anal. Chem. 2013, 85, 2516−2522