Color-Coded Single-Particle Pyrophosphate Assay with Dark-Field

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Color-Coded Single-Particle Pyrophosphate Assay with Dark-Field Optical Microscopy Fang Qi, Yameng Han, Zhongju Ye, Hua Liu, Lin Wei, and Lehui Xiao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03211 • Publication Date (Web): 17 Aug 2018 Downloaded from http://pubs.acs.org on August 27, 2018

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Analytical Chemistry

Color-Coded Single-Particle Pyrophosphate Assay with Dark-Field Optical Microscopy Fang Qi,1 Yameng Han,1 Zhongju Ye,1 Hua Liu,1 Lin Wei,2 and Lehui Xiao1,* 1. State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin, 300071, China, www.xiaolhlab.cn; 2. Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research, Key Laboratory of Phytochemical R&D of Hunan Province, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, 410082, China. ABSTRACT: In this work, we demonstrate a convenient yet sensitive color-coded single-particle detection (SPD) method for the quantification of pyrophosphate (PPi) by using single gold nanoparticle (GNP) as the probe. The design is based on GNPdependent catalytic deposition of Cu onto the surface of GNPs with reduced nicotinamide adenine dinucleotide (NADH). Without PPi, Cu2+ can be directly reduced to Cu0 through the gold-catalyzed oxidization of NADH. In the presence of PPi, the coating process is impeded due to the strong coordination capability of PPi with Cu2+. The selective coating of Cu shell onto the GNPs surface results in the extraordinary red-shift of localized surface plasmon resonance (LSPR) from individual GNPs. By quantitatively counting the fraction of yellow particles with color-coded dark-field optical microscopy (DFM), the trace amounts of PPi in solution can be accurately quantified. The limit-of-detection (LOD) is as low as 1.49 nM with a linear dynamic range of 0-4.29 µM, which is much lower than the spectroscopic measurements in bulk solution. In artificial urine sample, good recovery efficiency was achieved. As a consequence, the method demonstrated herein will find promising applications for the ultra-sensitive detection of target biomolecules under biological milieu in the future.

M

any biological processes are associated with anionic biomolecules. Among those anionic compounds, the selective detection of anion pyrophosphate (P2O74−, PPi) has aroused global concern due to their pivotal roles in biochemistry such as cellular metabolism. For example, PPi was formed by the hydrolysis of nucleoside triphosphates, including adenosine 5’-triphosphatedi sodium salt (ATP) hydrolysis, DNA/RNA polymerase reactions, and synthesis of cyclic adenosine monophosphate (c-AMP) catalyzed by DNA polymerase and adenylate cyclase.1 The elevated level of PPi has been found in synovial fluid or urine of patients with all kinds of diseases, including osteoarthritis, urolithiasis and kidney related diseases. Moreover, some studies have demonstrated that abnormal expression of PPi in the cells could be adopted as a sign for cancer diagnosis. Taking into account the significance, considerable efforts have been devoted to design different probes for the quantification of PPi and its derivatives in the past decades.2,3 Until now, many interesting techniques for PPi detection have been reported, including chemiluminescence, surfaceenhanced Raman scattering (SERS) assay, colorimetric sensing, as well as fluorescence spectroscopic measurement.4-10 Although the above mentioned methods are well developed for PPi quantification, some limitations still exist such as unsatisfied detection limit and sophisticated operation procedures. For example, the detection accuracy of fluorescent assays is impeded by the limited photo-physical properties of the fluorescent dyes such as photobleaching, background autofluorescence, self-quenching as well as poor water-solubility. For the electrochemical sensing, it is usually time-consuming and labor-intensive due to the requirement of frequent calibration. As a consequence, it is significant to develop an envi-

ronmental-friendly sensor for PPi detection, which is easy operation as well as low cost. In order to meet these demands, the recently developed single-particle detection (SPD) strategy has attracted much more attention because of the capability to analyze the target object at single-particle (or -molecule) level by a regular dark-field optical microscope (DFM).11-15 Distinct from the traditional approaches, which were founded on the calculated signals taken from multiple nanoparticles, SPD produces the signal throughout single-particle and each particle is considered as an independent signal output.16,17 The signal is not calculated by numerous molecules, avoiding the ensemble averaging effect from the sample which greatly improves the detection limit as well as specificity. Therefore, the signal-to-noise ratio and the sensitivity could be greatly improved through SPD. For DFM-based SPD assay, many robust scattering-based nanoprobes have been developed, such as gold nanoparticles, gold nanorod, silver nanoparticles, silver cube and so on.18-25 Among these particles, GNPs have been intensively investigated owing to their simple synthesis, good water-solubility, non-bleaching, outstanding catalysis, and so on.26,27 Especially, individual GNP is endowed with distinctive color and strong scattering intensity close to the peak of LSPR spectrum due to the large optical absorption and scattering cross-section, which can be effectively gathered through a DFM. In the previous works, with plasmonic nanoparticles, we have developed several DFM-based SPD for biomolecules assay under biological milieus with greatly improved detection limit, including short strand DNA and cancer biomarkers.28-31

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Scheme 1. Schematic diagram of the light path for the optical microscopic imaging of single nanoparticle and the principle of the color-coded SPD for PPi assay.

Encouraged by these attractive merits of SPD, in this work, we demonstrate a convenient strategy for PPi detection with quantitative color-coded SPD, which is based on the color change of individual GNPs after the deposition of Cu shell through the gold-catalyzed reduction of NADH. Owing to the strong coordination effect of PPi with Cu2+ (i.e., Cu(P2O7)26−), the Cu2+ cannot be reduced to the GNP surface and the number of green particles in the solution will change as a function of the PPi concentration. Through quantitatively counting the fraction of yellow particles on the cover glass surface with DMF, the concentration of PPi can be accurately determined. The LOD is as low as 1.49 nM with a linear dynamic range of 0-4.29 µM, which is much lower than the spectroscopic measurements in bulk solution. Furthermore, according to the artificial urine sample assay, satisfactory recoveries between 93% and 103% were obtained, which is in good consistent with the results determined from the aqueous solution. Because of the merits described above, this color-coded SPD method will open up a variety of prospects for the ultra-sensitive detection of biomolecules in diverse areas.

EXPERIMENTAL SECTION Chemicals and Materials. Chloroauric acid (HAuCl4·3H2O) was purchased from Aladdin (Shanghai, China). 3aminopropyltriethoxysilane (APTES) was purchased from J&K Scientific Ltd. Chemical Company (Beijing, China). Reduced nicotinamide adenine dinucleotide (NADH) was purchased from Shanghai Yisheng biotechnology Co., Ltd. (Shanghai, China). Adenosine 5’- triphosphatedi sodium salt (ATP), adenosine 5’-diphosphate sodium salt (ADP), adenosine 5’-monophosphate sodium salt (AMP), trisodium citrate, cupric sulfate (CuSO4), sodium pyrophosphate (Na4P2O7), sodium chloride (NaCl), potassium chloride (KCl), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), sodium phosphate (Na3PO4), sodium sulfate

(Na2SO4), sodium nitrate (NaNO3), calcium sulfate (Ca2SO4), magnesium sulfate (Mg2SO4), carbamide (CO(NH2)2) and creatinine (C4H7N3O) were obtained from Sinopharm Group (Shanghai, China). All other reagents were of analytical purity. Unless otherwise noted, all experiments were carried out at room temperature. All aqueous solutions were prepared with ultrapure water. Synthesis of GNPs and Detection of Pyrophosphate at Single-Particle Level. The GNPs were prepared by the seedmediated growth method as described before.32 Briefly, the method involves in two steps, including seed preparation and particle growth processes. For seed particle preparation (with diameter ~18 nm), 10 mL of sodium citrate (14.55 mM) and 1.03 mL of HAuCl4·3H2O (24.28 mM) were added into 98.97 mL H2O under vigorous stirring at 110 °C for 20 min. The color of the mixture gradually changed after 5 min from colorless to pale red, pale purple, and finally wine red. When the color change process was complete, the solution was kept stirring at room temperature for another 15 min. To generate larger size particles, 30 mL of sodium citrate solution (13 mM) was added into the above seed solution step by step. The mixture was heated to 120 °C for 10 min and followed by adding 20 mL of HAuCl4·3H2O (2.35 mM) gradually. After that, the reaction solution was heating for another 10 min and then gradually cooled down to room temperature. The size of the GNPs synthesized by this method is around 45.18±4.22 nm. For PPi assay, 50 µL of different concentrations of PPi solutions (e.g., with a final concentration of 0, 0.007, 0.01, 0.03, 0.07, 0.14, 0.71, 1.43, 4.29 and 7.14 µM in the reaction solution) were co-incubated with 200 µL of purified GNPs and 50 µL of NADH (0.5 mM) for 30 min. Then 50 µL of CuSO4 (2 mM) was added into the reaction solution for another 2 h. The resulted samples were imaged by dark-field optical microscope at single-particle resolution.


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Analytical Chemistry To demonstrate the potential practical applicability of this method, recovery assay in artificial urine sample was performed. Firstly, the urine sample was prepared as below: NaCl (55 mM), KCl (67 mM), Ca2SO4 (2.6 mM), Mg2SO4 (3.2 mM), Na2SO4 (29.6 mM), Na2HPO4 (19.8 mM), CO(NH2)2 (310 mM), and C4H7N3O (9.8 mM).33 The PPi and GNPs samples were prepared with the diluted artificial urine solution (diluted 10× with PBS, pH=7.4). Instruments. Optical microscopic imaging experiments were performed on an inverted dark-field optical microscope (Ti-U, Nikon, Japan) equipped with a 40× objective (NA=0.75) and a true-color complementary metal oxide semiconductor camera (CMOS, Digiretina 16, Tuscen photonics Co., Ltd).34-36 All images were processed with Image J (http://rsbweb.nih.gov/ij/). To avoid the artificial error for the color-coded single-particle counting analysis, the ratio of the particles between the red and green channels in the color image was determined through splitting the color image into red, green and blue channels. All experiments were replicated three times. The UV-Vis absorption spectra of the nanoparticles were recorded by a spectrophotometer (UV-2450, Japan). The size distributions of GNPs in solution were measured with a Zetasizer Nano ZS system (Malven, U.K.). The morphology of the particles was characterized with a transmission electron microscope (JEM1230, Japan). The scattering spectra from single GNPs and GNPs@Cu nanoparticles were measured based on the procedure described before. In brief, a transmission grating beam splitter with 100 lines/mm was mounted in front of the CMOS camera. The incoming scattered light from single nanoparticles was split into zeroand first-order images. Under appropriate condition, the

undeviated zero-order and wavelength-resolved first-order images can be captured by the camera simultaneously. A laser beam with wavelength at 532 nm was used to calibrate the setup for the spectroscopic measurements.30

RESULTS AND DISCUSSION The Mechanism of Color-Coded SPD for PPi Detection with DFM. Earlier explorations have verified that NADH can be adopted as the reducing agent for the reduction of Cu2+. For example, the quantitative analysis of 1,3,5-trinitro-1,3,5triazinane (RDX) explosive residues in latent fingerprints37 and the detection of NADH-dependent intracellular metabolic enzymatic pathways.38 However, in these cases, without GNPs, Cu2+ is hardly to be reduced by NADH,39 which is essentially ascribed to the catalytic effect of GNPs according to the reac

tion of . After this reaction, a Cu shell was exactly deposited on the surface of GNPs to generate a GNPs@Cu core-shell nanostructure by the biochemical reduction of NADH. Motivated by this GNPs-dependent catalytic reaction,38 herein, we designed a color-coded SPD method for PPi detection, which is based on the refractive index change induced LSPR shift of single GNPs after the NADH-dependent catalytic deposition of copper onto the GNP surface. The thorough mechanism is delineated in Scheme 1. Since the nanoparticle with uniform morphology distribution is necessary for the color-coded single-particle assay,40 GNPs with size dimension around 40 nm were considered to be the suitable dimension for dark-field imaging which exhibit uniform green color and excellent scattering signal from individual particle in the darkfield image. In order to obtain high quality GNPs, seedmediated growth method was adopted.32 UV-Vis absorption

Figure 1. a) UV-Vis absorption spectra of GNPs (green) and GNPs@Cu (red). b) TEM image of GNPs. c) Hydrodynamic size distribution (DLS measurement) of GNPs in solution and the statistically analyzed size distribution of GNPs from the TEM image (the inset plot). d)-f) The DFM images of GNPs, GNPs in the mixture of NADH and Cu2+ solution, and GNPs in mixture of NADH, Cu2+ and PPi solution respectively. g) The single particle scattering spectra of GNPs (green) and GNPs@Cu (red). h) The TEM image of GNPs@Cu. (i) The magnified TEM image of GNPs@Cu.



spectroscopy measurement illustrates that the as-synthesized GNPs show a characteristic absorption peak at 529.5 nm (the green curve in Figure 1a). The morphology was characterized with transmission electron microscopy (TEM) as shown in Figure 1b. Evidently, in the TEM image, the GNPs were well dispersed and exhibit uniform size distribution with diameter around 45.18±4.22 nm (the inset of Figure 1c) which was further confirmed by dynamic light scattering measurements (DLS) as shown in Figure 1c. Then single-particle dark-field imaging experiment was further performed to evaluate the color purity of these GNPs. As shown in Figure 1d, all of the GNPs adsorbed on the cover glass surface exhibit uniform green color, which is suitable for the subsequent color-based assay. Before the PPi sensing experiment, the gold-catalyzed color change experiment was firstly explored in the bulk solution. Upon the addition of Cu2+ (2 mM, 50 µL) and NADH (0.5 mM, 50 µL) to 200 µL GNPs solution, the color of the solution was red-shift. This can be ascribed to the reduction of Cu layer onto the GNPs surface by NADH as descried above. Around 100 nm red-shift in the UV-vis absorption spectra was observed as shown in Figure 1a. From the single-particle darkfield image (Figure 1e), the majority of the particles exhibit yellow color on the cover glass surface, which is also confirmed by the single-particle scattering spectra, where noticeable red-shift (~100 nm) of the scattering spectrum (Figure 1g) was observed after the deposition of Cu layer. To further confirm the color change of these particles indeed results from the copper deposition process rather than from the salt-induced aggregation between the GNPs, TEM imaging experiment was performed (Figure 1h). A thin layer of copper was readily observed after the reaction as shown in Figure 1i, and the enlarged image in Figure 1h. It is worth to point out that besides the red-shift of the LSPR spectrum, the scattering inten-

sity from individual particles was also increased after the coating of Cu layer. However, when PPi (14 µM, 50 µL) was injected into the reaction solution, no obvious color change was observed even NADH was added subsequently. In the dark-field image, all of the particles were still exhibiting green color as shown in Figure 1f. This should be ascribed to the higher coordination capability between Cu2+ and PPi, where a complex was formed.41-43 Owing to the formation of stable Cu(P2O7)26− complex, Cu2+ could not be reduced by NADH. As a result, the core-shell GNPs@Cu nanostructure cannot be formed and the color from those individual particles will not be changed accordingly. Real-time Monitoring Catalytic Deposition of Copper onto GNPs Surface. To further verify the practicability of this strategy for SPD-based PPi sensing, we monitored the NADHdependent catalytic deposition of copper on GNPs surface insitu with dark-field optical microscopy. In this experiment, the GNPs were firstly immobilized on the amino group functionalized glass slide surface. Prior to the catalytic reaction, all of the particles displayed green color in the dark-field image. A mixed solution of Cu2+ and NADH was then gradually injected into the flow channel. The reaction was monitored real-time through taking the time-dependent dark-field image within a fixed observation window. As shown in Figure 2a, as time goes on, the color of the particles adsorbed on the glass slide was gradually changed into yellow, indicating the gradual formation of Cu layer on the GNPs surface. Around 2 h later, noticeable yellow color was observed. It is worth to point out that not only the red-shift of the scattering spectrum from the GNPs, the scattering intensity from individual particles was also gradually increased as shown in Figure 2c. These observations are in good consistent with the results from the bulk solution measurements. However, when PPi was introduced to

Figure 2. a) and b) In situ single-particle observation of the time-dependent GNPs-catalyzed deposition of Cu shell onto GNPs without and with PPi in the reaction solution respectively. The representative time-dependent single-particle intensity tracks from these two samples are shown in c) and d) respectively.


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Analytical Chemistry the reaction solution, the color of the particles still maintains green. As shown in Figure 2b, even 2 h later, the color of the particles remains the same. The unchanged scattering intensity from individual particles further confirms this argument (Figure 2d). Quantitative Detection of PPi based on Color-Coded SPD in Aqueous Solution. Given the fact that PPi has strong affinity toward Cu2+ as confirmed from the bulk solution and single-particle spectroscopic measurements,44,45 one can expect that a concentration dependent color-change can be readily observed at single-particle level from the dark-field image. Ideally, through counting the concentration dependent yellow particles in the dark-field image, a quantitative relationship can be established between the number of yellow particle and PPi concentration. By naked eye counting, artificial error will be introduced due to the different color sensitivity from altered people as well as the non-homogeneous adsorption of particles on the cover glass surface. To address these issues, the color dark-field image was split into three channels, blue, green and red respectively. The corresponding spectral responses of the camera from these three channels are shown in Figure 3a with different color bands. Prior to the Cu coating process, the scattering spectrum of GNPs matches well with the spectrum response from the green channel, where green color is observed. Under this condition, in the color-coded dark-field image, the signal from the GNPs is basically resulted from the green channel. No particle was observed in the red channel. However, after the Cu coating process, the LSPR of the GNPs@Cu was red shift and associated with increased scattering intensity. The resulted scattering spectrum of the nanoparticle overlapped well with that from

the red channel. Therefore, all of the GNPs@Cu particles could be readily counted in the red channel. Due to the strong scattering intensity from the GNPs@Cu particles, under the same threshold, those particles can still be counted in the green channel. That is in the green channel, all of the nanoparticles (GNPs and GNPs@Cu) could be detected. Therefore, to avoid the artificial error for the data analysis, the ratio of the counted particles between the red and green channels was determined and applied for the quantification of PPi in the solution. As shown in Figure 3b, different concentrations of PPi from 0-7.14 µM was added into the reaction solution (2 mM Cu2+ and 0.5 mM NADH). Initially, in the control, all of the GNPs exhibit green color with the counted ratio close 0. Introducing NADH alone to the solution still results in a low ratio value, indicating no trace amount of metal ion in the GNPs solution (i.e., HAuCl4 and so on). On the simultaneous addition of Cu2+, the majority of the particles exhibit a yellow color with a statistically counted red/green ratio of 0.7. With the increasing of PPi in the reaction solution, the numbers of green particles gradually increase when the PPi concentration was elevated from 0 to 4.29 µM as shown in the dark-field images (Figure 3b). A dynamic range from 0-4.29 µM was readily achieved as illustrated in the plot in Figure 3c. It is worth to point out that using the ratio rather than directly counting the number of green or yellow particles in the darkfield image greatly improves the quantification accuracy. This is because the error due to the nonhomogeneous particle adsorption on the cover-glass surface can be effectively avoided. A good linear relationship between color ratio and the concentration of PPi was observed from 0 to 140 nM with regres-

Figure 3. a) Schematic diagram of the method for the digital image analysis. The color image was firstly split into three channels (Red, Green and Blue). The spectral responses of these three channels are labelled with red, green and blue color respectively in the middle plots. Representative single-particle scattering spectra from GNPs and GNPs@Cu are shown in green and red dotted lines respectively in the figure. b) Typical dark-field images of GNPs, GNPs in NADH solution only, and GNPs in the mixture of NADH, Cu2+ and PPi solution with different concentrations. c) The fraction of yellow particles as a function of PPi concentrations from 0 to 4.29 µM. d) Expanded linear region cure from 0 to 140 nM in the plot of figure c).



Figure 4. The selectivity assay of PPi detection with color-coded SPD. The counted fraction of yellow particles in the reaction solution with the components of GNPs, NADH and Cu2+ maintained while the anion compound PPi was replaced with the same concentration of ATP, AMP, ADP, HPO42-, H2PO4-, PO43-, CO32-, HCO3-, Cl-, NO3- and SO42- respectively.

sion equation of y 0.0022x 0.70 (correlation coefficient R2=0.99), Figure 3d. The LOD was calculated to be 1.49 nM (3σb/slope, σb is the standard deviation of the blank samples), which was much lower than the recently reported organic dye-based fluorescent probe10 and GNP-based colorimetric sensor.46 A summarization of the detection dynamic range and LOD from the recently reported methods are summarized in Table 1. In comparison with other methods, this color-coded SPD method is more sensitive, which is essentially ascribed to the high sensitivity of LSPR based SPD. The Selectivity for PPi Detection with Color-Coded SPD. Besides the sensitivity assay, the selectivity of this strategy was further explored. On this account, different kinds of anion were tested, including ATP, AMP, ADP, HPO42-, H2PO4-, PO43-, CO32-, HCO3-, Cl-, NO3- and SO42-. These aforementioned anions were added into the reaction solution instead of PPi. Representative dark-field images from these reaction solutions are shown in Figure 4. Evidently, many yellow particles could be observed in the dark-field images in contrast to the control with PPi. The calculated ratios from these compounds are close to 0.6. These results demonstrated that inorganic orthophosphate anions, including HPO42-, H2PO4- and PO43- (0.71 µM) have no interference for PPi sensing (Figure 4). Other anions, including CO32-, HCO3-, Cl-, NO3- and SO42(0.71 µM) could not disturb the identification process by PPi (Figure 4). According to the previous explorations, PPi could preferentially bind to Cu2+ with much stronger stability constant (K) for Cu(P2O7)26− complex (logK=12.45) over other ions as noted above.46 This well explains the excellent antiinterference of this sensor for PPi detection. Furthermore, the interference effect from those phosphate-related anions, such as ATP, AMP and ADP (0.71 µM), was also explored. From the experimental results, it was found that the presence of ATP exhibited minor interference on PPi detection, and the impact from AMP and ADP was negligible. These observations are satisfactory because PPi was formed by the hydrolysis of nucleoside triphosphates such as ATP hydrolysis. Detection of PPi in Urine Sample. The level of PPi in urine denotes a key inhibitor of urinary crystallization and participates in the construction of calcigerous stones. In order to explore the robustness of this method for biological sample

assay, PPi detection in artificial urine samples was performed. Firstly, 5 µM of PPi (with a final concentration of 0.71 µM) was spiked into deionized (DI) water and artificial urine sample respectively. The samples without PPi were used as the control. Representative dark-field images from these experiments were shown in Figure 5a. In DI water, without addition of PPi, the majority of the particles display yellow color, indicative of the selective deposition of copper layer on to the GNPs surface as demonstrated above. When PPi was added, noticeable green particles were observed. Analogous phenomena were found in the artificial urine sample. To be a more quantitative evaluation of the performance of this probe, the fraction of yellow particles from these samples were determined. As shown in Figure 5b, no difference was found between the samples in DI water and artificial urine solution, demonstrating the excellent reliability of the results in urine sample assay. Then the recovery efficiency for urine sample assay was further explored. Different concentrations of PPi (with a final concentration of 14, 71 and 143 nM) were spiked in the artificial urine. Typical dark-field images are shown in Figure 5c. The concentration dependent fractions of yellow particles under these solutions were then determined respectively (Figure 5d). Satisfactory recoveries between 93% and 103% were obtained. The detailed results are shown in Table 2.

CONCLUSIONS In summary, a convenient, sensitive and selective color-coded SPD method for the quantification of PPi by using single GNP as the probe was demonstrated. In the absence of PPi, a coreshell GNPs@Cu structure was formed through the goldcatalyzed reduction of NADH, leading to the color change of GNPs before and after the coating of the Cu atoms on GNPs surface. However, in the presence of PPi, Cu2+ cannot be reduced to Cu0 because PPi preferentially and strongly coordinates with Cu2+ to form a Cu(P2O7)26− complex. The selective coating of Cu shell onto the GNPs surface results in the extraordinary red shift of LSPR from single GNPs. As a consequence, PPi could be readily quantified by statistically count-

Figure 5. a) Representative DFM images of the sample for PPi assay in DI water and artificial urine sample with and without PPi in the solution. b) The counted fraction of yellow particles in these samples. c) Representative DFM images of the recovery assay in the artificial urine sample under different PPi concentrations. d) The corresponding counted fraction of yellow particles in these samples.


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Analytical Chemistry Table 1. Comparison of the performance of the methods for PPi assay. Method

Linear range

LOD

Reference

Fluorescence

0-60 µM

60 nM

10

SERS

0-160 µM

0.5 µM

6

SPD

0-4.29 µM

1.49 nM

This work

Colorimetric sensing

0.02-3 µM

3.1 nM

8

Aggregation-induced emission

0-320 µM

1.1 µM

7

Enzymatic methods

0-50 mM

0.2 µM

5

Chemiluminescence

6.6-13.3 µM

4 µM

4

100 nM-10 mM

10 nM

9

Electrochemical sensing

Table 2. Determination of PPi concentration in artificial urine samples. Samples

PPi Added (nM)

Measured (nM)

Recovery (%)

RSD (%)

1

14

13

93

0.70

2

71

67

94

2.00

3

143

147

103

1.86

ing the concentration dependent fraction of yellow particles in the DFM image. This SPD-based method exhibited excellent sensitivity and selectivity for PPi detection in aqueous solution with a remarkable LOD of 1.49 nM, which is far below the commonly reported fluorescence or colorimetric spectroscopic measurements. Furthermore, satisfactory recoveries between 93% and 103% in artificial urine samples were obtained, indicating the promising application of this approach for biological sample quantification. Further applications with this colorcoded SPD method can be envisioned in the detection and quantification of target objects under complex biological milieu, such as kinase and polymerase catalytic reaction assay.47,48

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Fax: +86-022-23500201.

ORCID Lehui Xiao: 0000-0003-0522-2342

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by national natural science foundation of China (NSFC, Project no. 21522502), the Fundamental Research Funds for the Central Universities, the Excellent Youth Scholars of Hunan Provincial Education Department (17B155) and the Opening Fund of Key Laboratory of Chemical Biology and Tradition Chinese Medicine Research (Ministry of Education of China), Hunan Normal University.

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Color-Coded Single-Particle Pyrophosphate Assay with Dark-Field

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