Phosphorescent Differential Sensing of Physiological Phosphates with

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Phosphorescent Differential Sensing of Physiological Phosphates with Lanthanide Ions-Modified Mn-Doped ZnCdS Quantum Dots Hengwei He, Chenghui Li, Yunfei Tian, Peng Wu, and Xiandeng Hou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00780 • Publication Date (Web): 16 May 2016 Downloaded from http://pubs.acs.org on May 18, 2016

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Analytical Chemistry

Phosphorescent Differential Sensing of Physiological Phosphates with Lanthanide Ions-Modified Mn-Doped ZnCdS Quantum Dots Hengwei He,† Chenghui Li,† Yunfei Tian,‡,* Peng Wu,‡,* Xiandeng Hou†, ‡

†

College of Chemistry, and ‡Analytical & Testing Center, Sichuan University,

Chengdu 610064, China.

E-mails: [email protected]; [email protected]

ACS Paragon Plus Environment


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ABSTRACT Phosphates, both inorganic and organic, play fundamental roles in numerous biological and chemical processes. The biological functions of phosphates connect with each other, analysis of single phosphate-containing biomolecule therefore cannot reveal the exact biological significance of phosphates. Sensor array is therefore the best choice for differentiation analysis of physiological phosphates. Lanthanide ions possess high affinity toward physiological phosphates, while lanthanide ions can also efficiently quench the luminescence of quantum dots (QDs). Taking lanthanide ions as cartridges, here we proposed a sensor array for sensing of physiological phosphates based on lanthanide ions-modified Mn-doped ZnCdS phosphorescent QDs in the manner of indicator-displacement assay. A series of lanthanide ions were selected as quencher for phosphorescent QDs. Physiological phosphates could subsequently displace the quencher and recover the phosphorescence. Depending on their varied phosphorescence restoration, a sensor array was thus developed. The photophysics of phosphorescence quenching and restoration were studied in detail for better understanding the mechanism of the sensor array. The exact contribution of each sensor element to the sensor array was evaluated. Those sensor elements with little contribution to the differentiation analysis were removed for narrowing the size of the array. The proposed sensor array was successfully explored for probing nucleotide phosphates-involved enzymatic processes and their metabolites, simulated energy charge changes, and analysis of physiological phosphates in biological samples.


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INTRODUCTION Phosphates, both inorganic and organic, play fundamental roles in numerous biological and chemical processes. In a clinical setting, serum phosphate levels are included in a routine blood analysis. Individuals with abnormally high phosphate levels are diagnosed with hyperphosphatemia, which manifests in acute or chronic renal failure. Those with low phosphate levels suffer from hypophosphatemia, which can be associated with rickets, hyperthyroidism, or Fanoci syndrome. Nucleotide phosphates, such as ATP or AMP, are well-known for their important roles in bioenergetics, metabolism, and transfer of genetic information. For example, ADP plays a central role in the fundamental biological reactions catalyzed by two classes of enzymes, namely ATPases and kinases.1 Pyrophosphate (PPi), another inorganic phosphate, is the product of ATP hydrolysis under cellular conditions. In particular, many reactions in metabolism are controlled by the energy status of the cell (energy charge). It is necessary to take into account the concentration of all three nucleotides, rather than just ATP and ADP, to account for the energy status in metabolism.2 Therefore, sensors for physiological phosphates are of vital importance for studying the metabolic processes and diseases diagnosis. A variety of sensors for phosphates have been developed.3-4 Particularly, when designing the selective receptors for PPi and nucleotide phosphates, the phosphate backbone is an important target (Scheme 1A) for these sensors. As hard acids, trivalent lanthanide ions (Ln3+) possess high affinity for oxygen-donor atoms, especially phosphates (Scheme 1B),5 which can also be evidenced from the elegant functionalization of lanthanide-based upconversion nanoparticles with DNA.6



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Considering that many Ln3+-containing complexes are luminescence-active and their luminescence is largely dependent on the energy antennas, Ln3+ complexes were frequently explored for the development of sensors for physiological phosphates and phosphate-containing biomolecules.7-10 Besides, Ln3+ can also cause perturbation to the luminescence of some optically-active nanomaterials, thereby acting as cartridges in “indicator-displacement assay” for the construction of luminescent sensors toward physiological phosphates.11-18 However, the biological functions of phosphates connect with each other (Scheme 1C), analysis of single phosphate-containing biomolecule therefore cannot reveal the exact biological significance of phosphates. (A)

(B) Ln3+ Eu

25.75

Nd3+

25.95

Ce3+

23.00

Sm

(C)

pKsp of phosphate

3+

3+

25.99

Pr3+

26.06

Tm3+

26.05

3+

Er

25.78

Ho3+

25.57

3+

25.39

Lu

Scheme 1. (A) Structures of phosphate (Pi), pyrophosphate (PPi), and adenosine-based nucleotide phosphates (ATP, ADP, and AMP); (B) solubility (pKsp) of several lanthanide phosphates (data from ref. 5); and (C) several typical biological transformations of physiological phosphates.

Sensor arrays or differential sensing protocols, inspired from the working mechanisms of mammalian olfaction and gustation, allow high-throughput detection of multiple analytes of similar structures,19-20 for example, proteins,21-23 metal ions,2426

and saccharides.27-28 Sensor arrays for physiological phosphates have also been

reported,29-32 but receptors for phosphates needed to be synthesized first. Exploring


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the high affinity of Ln3+ toward varied phosphates has never been reported for sensor array fabrication.

Phosphorescence intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Phosphate 2

Phosphate 1

Phosphate 3

Scheme 2. Schematic principle of the differential sensing of phosphates with Ln3+modified phosphorescent Mn-doped ZnCdS@ZnS QDs.

Therefore, in this work, we proposed the use of Ln3+-modifed phosphorescent Mn-doped ZnCdS@ZnS QDs33-36 for differential sensing of physiological phosphates (Scheme 2). In the proposed sensor array, nine Ln3+ ions (Scheme 1B) were randomly selected as the quencher for QDs,37 and carboxyl-terminated phosphorescent QDs were employed as the indicator. Ln3+ ions could coordinate with the carboxyl groups on the surface of QDs and thereby quench the phosphorescence. In the presence of physiological phosphates, Ln3+ could be displaced, resulting in phosphorescence restoration. Varied affinity of Ln3+ toward phosphates provides cross-reactivity for the sensor array and differential sensing of phosphates was thus achieved.



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EXPERIMENTAL SECTION Materials. All reagents used were of at least analytical grade. Zn(NO3)2·6H2O, Cd(NO3)2·6H2O, MnAc2·4H2O, and Na2S·9H2O were supplied by Sinopharm Chemical Reagent Co., Ltd, Shanghai, China. 3-Mercaptopropionic acid (MPA, 99%) was purchased from Alfa Aesar. Lanthanide nitrate salts were obtained via acidification of the Lanthanide oxides with nitric acid. Adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) were purchased from Alfa Aesar. Apparatus. Fluorescence and phosphorescence measurements were performed on an Hitachi F-7000 spectrofluorometer equipped with a plotter unit and a quartz cell (1 cm × 1 cm). Absorption spectra were recorded on a Shimadzu UV-1750 UV-vis spectrophotometer. Phosphorescence lifetime measurements were performed on a Fluorolog-3 spectrofluorometer (Horiba Jobin Yvon) with a SpectraLED (280 nm, S280, Horiba Scientific) as the excitation source and a picosecond photon detection module (PPD-850, Horiba Scientific) as the detector. For measurements, the instrument was set at the phosphorescence lifetime mode and the time range was set at 44 ms. The average lifetime was calculated using the equation below: τ =∑fiτi = f1τ1 + f2τ2 where τi is the lifetime and fi is the contribution factor of τi to τ, which can be collected from the phosphorescence lifetime measurements after proper fitting. Here, the data are then fitted with the 2nd order exponential decay.


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Procedures for array sensing of phosphates. In a 48-well plate, Mn:ZnCdS@ZnS (500 µL, 10 mgL-1), Tris-HCl buffer (200 µL, 0.05 M), and desired amounts Ln3+ ions were pipetted into the 5 columns (for five phosphates) and 6 rows (6 replicates). Then, phosphates were added to their corresponding columns and the mixture was brought to 1 mL with ultrapure water. The solutions were equilibrated for 10 min before measuring the phosphorescence intensity one by one (Figure S1). The raw date was performed by [Ph-Ph0]. Then the data were subjected to PCA (Principal Component Analysis) and LDA (Linear Discriminant Analysis). All multivariate analysis were processed on the platform of ORIGIN8.6. The confidence ellipses were processed by using Matlab (The MathWorks Inc., U.S.).

RESULTS AND DISCUSSIONS Phosphorescence quenching of QDs by Ln3+. Highly phosphorescent and carboxylterminated Mn-doped ZnCdS@ZnS QDs was synthesized in aqueous phase. Spectroscopic characterizations of the as-prepared Mn-doped ZnCdS@ZnS QDs were shown in Figure S2. The absorption edge of the as-prepared QDs located at about 350 nm. Emission spectra revealed that the QDs exhibited the characteristic Mn2+ dopant emission centered at 580 nm originated from the Mn2+ 4T1 → 6A1 d-d triplet transition, which could also be visualized in the phosphorescence model (with lifetime of about 4.3

ms).

Such

long

lifetime

phosphorescence

can

efficiently

eliminate

autofluorescence background and light scattering of biological samples. All these spectroscopic features agreed well with the previously reported characteristics of Mndoped QDs.33-35,38



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In the presence of Ln3+ ions, gradual phosphorescence quenching of QDs was received upon increasing the concentrations of Ln3+ (exemplified with Eu3+ in Figure 1A and 1B, the rest were given in Figure S3), which agreed well with previous reports.12 Varied phosphorescence quenching of QDs by different Ln3+ ions were observed in the order of Eu3+ > Nd3+ > others (Figure 1C, at the concentration level of 1 µM). Due to energy difference between the conduction band of QDs (about -2 V)39 and the redox potential of Ln3+ (for example, Eu3+ + e- → Eu2+, -0.35 V; Sm3+ + e- → Sm2+, -1.55 V), electron transfer from the conduction band of QDs to the partially occupied orbit of Ln3+ is expected. Besides, the phosphorescence lifetime of QDs was shortened in the presence of Ln3+ (Figure 1D), confirming potential electron transfer quenching pathway. Probably, the different phosphorescence quenching by Ln3+ lies in their different redox potentials. Proximity of Ln3+ ions with QDs was expected through coordination of Ln3+ ions with the carboxyl groups on the surface of QDs. Such weak interaction permitted facile removal of the quencher by phosphates with higher affinity for Ln3+.


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2500

12

(B) 10

2000

0 8

3+

1500

[Eu ] 40 µM

1000

Ph0 / Ph


(A)

6 4

500 2 0 500

550

600

650

700

0

10

20

30

3 (C)

(D)

QDs QDs + Nd3+ QDs + Sm3+

1000

Itensity

2

1

0

40

[Eu3+] / µM

Wavelength / nm

Ph0/Ph

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100

10

Eu3+ Nd3+ Ce3+ Sm3+ Pr3+ Er3+ Tm3+ Ho3+ Lu3+

1 10

20

30

40

50

60

Time / ms

Figure 1. Phosphorescence quenching of QDs by Ln3+ ions: (A) phosphorescence spectra of QDs in the presence of increased amounts of Eu3+; (B) Stern-Volmer plot of the quenching by Eu3+; (C) varied phosphorescence quenching of QDs by Ln3+ at the concentration level of 1 µM, here Ph0 and Ph represent the phosphorescence of QDs in the absence and presence of Ln3+; and (D) phosphorescence decay curves of QDs in the absence and presence of Sm3+ and Nd3+. The decay curves for the rest Ln3+ were similar and therefore not given out. Experimental conditions: concentration of QDs, 5 mg/L; and pH, 7.4 Tris-HCl buffer.

Phosphorescence turn on of Ln3+-QDs by phosphates. In the presence of physiological phosphates, the previously quenched phosphorescence of QDs could be restored, as can be evidenced in Figure 2A and 2C. Without phosphates, the quenched phosphorescence by Ln3+ ions remained stable for at least 4 h (Figure S4). Interestingly, at the same concentration levels, the recovered phosphorescence by different physiological phosphates was varied, which provides the required crossreactivity for subsequent sensor array development (Figure S5). Moreover, the phosphorescence recovery was specific toward phosphates (Figure S6), again demonstrating the specificity of Ln3+ ions toward phosphates. Together with the



steady-state intensity restore, the phosphorescence lifetime was also recovered (Figure 2B and 2D, Table S1), again confirming the electron transfer-involved modulation of phosphorescence of QDs. The overall kinetics of phosphorescence quenching and subsequent restoration were very fast that both processes were finished in about 8 min (Figure S7). QDs 3+ QDs + Eu 3+ QDs + Eu + ATP 3+ QDs + Eu + Pi

(A) 2000 1500 1000

1000

(B)

QDs QDs + Eu3+ QDs + Eu3++ATP

100

Itensity

Phosphorescence Itensity

2500

10

500 0 500

550

600

650

700

10

20

Wavelength / nm QDs QDs + Nd3+ 3+ QDs + Nd + ADP QDs + Nd3+ + PPi

(C)

1500

1000

(D)

40

50

60

QDs 3+ QDs + Nd 3+ QDs + Nd + ADP

1000

500

0 500

30

Time / ms

Intensity

2000

Phoshporescence Itensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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100

10

550

600

650

Wavelength / nm

700

10

20

30

40

50

60

Time / ms

Figure 2. Phosphorescence turn-on of Ln3+-modified QDs by phosphates (exemplified with Eu3+- and Nd3+-modified QDs): (A) phosphorescence spectra of Eu3+-modified QDs in the presence of ATP and Pi (5 µM); (B) phosphorescence decay curves of QDs in the absence and presence of Eu3+, and in the presence of Eu3+ + ATP; (C) phosphorescence spectra of Nd3+-modified QDs in the presence of ADP and PPi (5 µM); and (D) phosphorescence decay curves of QDs in the absence and presence of Nd3+, and in the presence of Nd3+ + ADP. Experimental conditions: concentration of QDs, 5 mg/L; and pH, 7.4 Tris-HCl buffer.

To maximize the phosphorescence restoration and increasing the detection sensitivity, the concentrations of Ln3+ ions were optimized and the results were summarized in Figure S8. With ATP as the model analyte, maximum phosphorescence restoration was observed with Ln3+ concentration of Eu3+, 1 µM;


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Nd3+, 1 µM; Ce3+, 1 µM; Sm3+, 2 µM; Pr3+, 5 µM; Lu3+, 10 µM; Tm3+, 10 µM; Ho3+, 6 µM; Er3+, 7 µM. To permit sensitive phosphate analysis, these Ln3+ concentrations were employed for subsequent sensor array fabrication. Generation of sensor array for physiological phosphates. Once the optimal concentrations of Ln3+ were determined, a phosphorescent sensor array with nine sensor elements (nine Ln3+-modified QDs) was established. We first tested the feasibility of the sensor array to detect physiological phosphates in Tris-HCl buffer. As shown in Figure 3A, the presence of the analyte phosphates resulted in varied phosphorescence restoration of Ln3+-modified QDs, i.e., the fingerprints of these phosphates. By linear discriminant analysis (LDA), these phosphates were successfully clustered into five groups that correspond to each specific phosphatecontaining species (95% confidence ellipses). LDA converts the patterns of the training matrix (9 Ln3+-modified QDs × 5 physiological phosphates × 9 replicates) to four canonical scores and the first two most significant discrimination factors (80.57% and 15.34%) were used to generate a 2D canonical score plot. Excellent discrimination of the 5 physiological phosphates (Figure 3B, 95% confidence ellipses) was achieved, i.e., 100% accuracy according to the jack-knifed classification matrix that removes and replaces one case at a time (cross-validation routine, Table S2).



1600 (A)

Eu3+ Nd3+ Sm3+ Pr3+ Ho3+

∆Ph

1200

Er3+ Lu3+ Tm3+ Ce3+

800 400 0

ATP

ADP

AMP

PPi

Pi

12 (B)

PPi Factor 2 (15.34%)

8 4

ADP

ATP

0 -4

Pi

-8

AMP

-12 -30

-20

-10

0

10

20

30

Factor 1 (80.57%) 12 (C)

PPi

8

Factor 2 (16.15%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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4

ADP

ATP

0 -4

Pi -8

AMP

-12 -30

-20

-10

0

10

20

30

Factor 1 (83.12%)

Figure 3. Generation of the phosphorescent sensor array for physiological phosphates based on Ln3+-modified QDs: (A) phosphorescence response (∆Ph) patterns of nine Ln3+modified QDs in the presence of five physiological phosphates at 5 µM concentration level; (B) canonical score plot for the phosphorescence patterns as obtained from LDA against five physiological phosphates at a fixed concentration of 5 µM, with 95% confidence ellipses; and (C) LDA canonical score plot after narrowing the array with the protocol developed by Anzenbacher et al.26,40 Experimental conditions: concentration of QDs, 5 mg/L; and pH, 7.4 Tris-HCl buffer.


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Subsequently, we tried to decrease the number of sensor elements to simplify the phosphorescent sensor array with the protocol developed by Anzenbacher et al.26,40 The basic idea of such attempt is to calculate the exact contribution of each sensor element based on principal component analysis (PCA). Then, the sensor element having the smallest contribution was removed. The PCA score plots presented here utilized the first three principal components (PCs) that represented at least 95% of variance. As shown in Figure S9, Tm3+, Lu3+, Ho3+, and Er3+ were stepwise removed. Meanwhile, the sensor array still kept efficient discriminatory capacity. Interestingly, the result of sensor array narrowing is coincide with their quenching ability to QDs (Figure 1C) and subsequent phosphorescence restoration by phosphates (Figure 3A). Eventually, a sensor array with five sensor elements were generated, which could still efficiently discriminate the five physiological phosphates (Figure 3C, 95% confidence ellipses, 100% accuracy according to the jack-knifed classification matrix, Table S3). At present, the minimal concentration of physiological phosphates that can be successfully differentiated was about 2 µM. The detection and discrimination efficiency were validated through identification of 30 unknown physiological phosphates samples randomly taken from the training set, with 100% accuracy achieved (Table S4 and S5).

Probing the nucleotide phosphates-involved enzymatic processes and their metabolites with the phosphorescent sensor array. Nucleotide phosphates are wellknown for transporting chemical energy within cells for metabolism. Particularly,



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ATP can be hydrolysed into ADP and Pi in the presence of ATPase.1 To show the potential usefulness of the proposed array for differentiation of phosphates, we first monitored the ATPase-catalysed ATP hydrolysis and compared with the results from the standard molybdenum blue photometric method. ATP was subjected to hydrolysis in the presence of ATPase (Supporting Information), and the enzymatic assay samples at different hydrolysis intervals (0 h, 1 h, 3 h, and 5 h) were collected. As shown in Figure 4A, these mixtures of ATP and hydrolysis products could be successfully clustered into four groups (100% accuracy according to the jack-knifed classification matrix, Table S6), indicating that the proposed array can be successfully used for analysis of physiological phosphates mixtures. Notably, the first canonical score was higher than 96%. The projection of each group on the x-axis of the score plot (factor 1) correlated well with the results by standard enzymatic assay (Figure S10), indicating that the discrimination was majorly ascribed to Pi concentration increase.


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10 8

(A)

ATPase

ATP

ADP + Pi

Factor 2 (2.64%)

6 3h 70%

4 2 0

0h

1h 50%

-2 5h 96%

-4 -6 -8 -20

-10

0

10

20

Factor 1 (96.98%) 8 (B) 0h

6 Factor 2 (10.60%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4h

4 2 0 -2

6h 2h 8h

ATP

-4 -6 -10

-5

0

5

10

Factor 1 (87.38%)

Figure 4. Monitoring the ATP hydrolysis and metabolic process with the proposed sensor array: (A) LDA canonical score plot for the phosphorescence patterns of the sensor array for analysis of time-dependent ATP hydrolysis; and (B) LDA canonical score plot for the phosphorescence patterns of urine samples collected after pre-medication of ATP.

ATP hydrolysis is the major metabolic process, we also employed the proposed array to monitor the ATP metabolism. A healthy volunteer was pre-medicated with 1000 mg ATP, and the urine was collected over 8 h at various time slot. Urine samples have no phosphorescence background, although the fluorescence background from urine (also serum) was significant (Figure S11). Phosphorescence detection can



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therefore eliminate the background interference from biological samples. As shown in Figure 4B, urine samples collected at 2 h, 4 h, 6 h, and 8 h after oral uptake of ATP could be clearly discriminated from that of 0 h (Table S7). Besides, these groups approach that of 0 h as the metabolic time increased. Control experiments showed that no such trend existed in spite of successful discrimination (Figure S12). The 6 h and 8 h urine samples overlapped with each other, indicating the pre-medicated ATP was almost completely metabolized, which agreed well with the above study on enzymatic process of ATP. These results clearly demonstrated the successfulness of the proposed array for studying of the physiological phosphate-involved biochemical processes.

Detection of phosphates in serum samples. Biological samples, especially blood serum, have abundant protein and electrolyte content and therefore serve as excellent matrix for testing the performances of the proposed sensor array. Besides, due to the metabolic process of nucleotide phosphates, blood serum also contains appreciable amounts of phosphates. In the next array, we applied buffer (control), serum, and serum samples with spiked physiological phosphates. As shown in Figure 5, the serum created unique phosphorescence pattern as compared to that of control sample (buffer). With the serum matrix, most of these physiological phosphates could still be differentiated (Table S8), demonstrating the usefulness of the proposed array for analysis of phosphates in biological samples. The only except is AMP, probably because AMP caused little phosphorescence restoration in the sensor array as compared to serum sample only. Also, the serum proteins are expected to potentially influence the detecting phosphates in serum samples,40 but such effect can be largely


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eliminated by proper dilution since the proposed sensor array possessed high sensitivity toward phosphates. 6

Serum

4

Factor 2 (2.74%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Serum + AMP

Serum + ADP

2

Serum + Pi

0 -2 Serum + PPi

Buffer

-4

Serum + ATP

-6 -8 -40

-20

0

20

40

Factor 1 (95.51%)

Figure 5. LDA canonical score plot for the phosphorescence patterns of the sensor array for analysis of human blood serum and serum with spiked phosphates (5 µM). Experimental conditions: concentration of QDs, 5 mg/L; and pH, 7.4 Tris-HCl buffer.

Application of the sensor array for energy charge evaluation. In cellular activities, many reactions in metabolism are controlled by the energy status of the cell. ATP is the major energy currency molecule of the cell, which transports chemical energy within cells for supporting enormous amount of activity that occurs inside cells. On the other hand, ADP and AMP can also regulate the ATP concentration through a series of enzymatic processes. Therefore, it is necessary to take into account the concentrations of all three nucleotides. One index to reveal the energy status is called energy charge (EC), which is defined by the following ratio:41 EC =

[ATP] + 0.5[ADP] [ATP] + [ADP] + [AMP]

To simulate the EC change, five mixtures of ATP, ADP, and AMP (total



concentration of 5 µM, but different ratio of the three nucleotide phosphates) were prepared. These mixtures were subjected to array analysis. As shown in Figure 6, these five simulated mixtures of different energy charge could be clustered into five independent groups (Table S9). Besides, the alignment of the projection for each confidence ellipse on the x-axis was in good accordance with their energy charge values, probably because the first canonical score was higher than 96%. These results demonstrate the usefulness of the proposed array for energy charge evaluations. 12 10 8 Factor 2 (2.82%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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6 EC 1

4 2 0 EC 0.2

EC 0

-2

EC 0.8

-4

EC 0.5

-6 -40

-30

-20

-10

0

10

20

30

Factor 1 (96.85%)

Figure 6. LDA canonical score plot for the phosphorescence patterns obtained from simulated mixtures of different energy charge. The total concentration of ATP, ADP, and AMP was set at 5 µM. Experimental conditions: concentration of QDs, 5 mg/L; and pH, 7.4 Tris-HCl buffer.

CONCLUSION In summary, a phosphorescent sensor array for differentiation analysis of physiological phosphates was developed based on lanthanide ion-modified Mn-doped ZnCdS QDs. The working mechanism of the sensor array was based on competitive


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binding of lanthanide ions between carboxyl-terminated QDs and physiological phosphates. In the manner of indicator-displacement assay, a phosphorescence fingerprint for physiological phosphates was generated via removal of lanthanide ions (quencher) from the surface of QDs and restoring the phosphorescence. The proposed sensor array was successfully explored for probing of nucleotide phosphates-involved enzymatic processes and their metabolites, energy charge change of cellular processes, and analysis of physiological phosphates in biological samples.

ACKNOWLEDGEMENT We gratefully acknowledge the financial support from the National Natural Science Foundation of China (Nos. 21475090 and 21522505) and the Youth Science Foundation of Sichuan Province (Grant 2016JQ0019).

Supporting Information Available: Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Notes The authors declare no competing financial interest.

REFERENCE (1)

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For TOC only:


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Phosphate 2

Phosphate 1


Phosphate 3

Phosphorescent Differential Sensing of Physiological Phosphates with

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