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Cationic Carbon Dots for Modification-free Detection of Hyaluronidase via an Electrostatic-controlled Ratiometric Fluorescence Assay Weiqiang Yang, Jiancong Ni, Fang Luo, Wen Weng, Qiao-Hua Wei, Zhenyu Lin, and Guonan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01705 • Publication Date (Web): 21 Jul 2017 Downloaded from http://pubs.acs.org on July 23, 2017

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Analytical Chemistry

Cationic Carbon Dots for Modification-free Detection of Hyaluronidase via an Electrostatic-controlled Ratiometric Fluorescence Assay

Weiqiang Yanga, Jiancong Ni a,b, Fang Luo*a,c, Wen Wengb, Qiaohua Weia, Zhenyu Lin*a, Guonan Chena

a

MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian

Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, College of Chemistry, Fuzhou University, Fuzhou, Fujian 350116, China b

Fujian Provincial Key Laboratory of Modern Analytical Science and Separation

Technology, College of Chemistry and Environment, Minnan Normal University, Zhangzhou, 363000, China c

College of Biological Science and Engineering, Fuzhou University, Fuzhou, Fujian

350116,China

Corresponding author: Fang Luo; Zhenyu Lin E-mail: [email protected] (Fang Luo); [email protected] (Zhenyu Lin); Tel&Fax: 86-591-22866135

Address: College of Chemistry, Fuzhou University, Fuzhou, Fujian, 350116, China (ZY Lin); College of Biological Science and Engineering, Fuzhou University, Fuzhou, Fujian, 350116, China (F Luo)

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Abstract Carbon dots (CDs) emerge as excellent fluorescent nanomaterials, but the full exploitation and application of their exceptional properties in the development of fluorescence assay are still rare. In this work, cationic carbon dots (C-CDs) covered with a plenty of positive charges on the surface were synthesized through a facile ultrasonic method. Negatively charged hyaluronic acid (HA) caused the aggregation of positively charged C-CDs and neutral red (NR) along its linear chain via electrostatic adsorption, leading to a remarkable förster resonance energy transfer (FRET) from C-CDs to NR. But the presence of hyaluronidase (HAase) resulted in the enzymolysis of HA, as well as the liberation of C-CDs and NR. The corresponding change of fluorescence color from red to green-yellow afforded a reliable ratiometric assay for HAase. And the ratio of fluorescence intensity for C-CDs (I525) to that for NR (I630) was used for quantitative detection of HAase. The proposed sensing system was easily operated in aqueous media with a detection limit of 0.05 U/mL. This strategy provides a new approach for the wider application of some special CDs in detecting biomolecules.

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Introduction Carbon dots (CDs) have drawn increasing attention because of their remarkable advantages, such as outstanding fluorescence, favorable biocompatibility, convenient preparation and wide availability of raw materials.1,2 Many CDs-based fluorescent assays have been reported for the detection of metal ions, environmental pollutants, small bio-molecules and so on.3-6 However, most of reported methods often involve complicated modifications during the preparation of CDs labeled reagents, which is time-consuming and inefficient.7,8 To overcome this disadvantage, special CDs are suggested during a particular application,9 indicating that another property of CDs besides the characteristic of fluorescence is required to simplify assays. For instance, a new kind of fluorescent CDs was fabricated for the selective detection of methylmercury rather than mercury ion owing to its hydrophobic alkane coating on the surface.10 And the amphipathic CDs were skillfully employed for permittivity detection on the basis of their different solubility in different solvents.11 Although the full utilization of CDs with their special properties is promising, reports on the development of special CDs for biological assay are very rare. It has been reported that cetylpyridinium chloride (CPC) can react with sodium hydroxide and hence produces green fluorescent CDs.12-14 Such CDs are positively charged because of the residual pyridine groups, whereby it can interact with negatively charged targets through electrostatic interaction. Hyaluronic

acid

(HA),

containing

repeating

D-glucuronic

3


acid

and


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N-acetyl-D-glucosamine disaccharide units, is negatively charged and widely distributed in the extracellular matrix.15 Early studies have showed that the synthesis or degradation of this linear mucopolysaccharide is closely related to various biological processes, such as cell proliferation, differentiation, and migration.16 As its specific enzyme, hyaluronidase (HAase) is thereby relevant to many physiological and pathological processes. The overexpression of HAase is found to be associated with many malignant tumors at their early stage, so HAase is considered as a new type of tumor marker.17 Therefore, it is necessary to develop some sensitive detection techniques for HAase in serving tumor diagnosis and therapy. Several physicochemical methods are commonly used for HAase detection, such as turbidimetry18, viscosimetry19, and zymography20. However, none of them shows approving sensitivity and selectivity, and the tedious prerequisite steps hinder their rapid detection in particular. Some colorimetric methods that involve positively charged gold nanoparticles offer a fast evaluation of HAase level, but the sensitivity is insufficient.21,22 Nevertheless, such nanoprobes themselves are not stable because gold nanoparticles are apt to precipitate in biological high-salt environment. Fluorescence method has been widely utilized in many biological assays due to its high sensitivity, and some novel fluorescence probes have been designed for HAase assay23-26. A modification-free sensor for HAase detection was developed through the combination of negatively charged HA with two positively charged compounds via electrostatic adsorption.27 The change of fluorescence intensities generating from two 4


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fluorescence probes was measured simultaneously and a ratiometric fluorescence method was developed, which can effectively reduce false or unstable results caused by environmental effects, especially in the complex biological matrix.28,29 Similarly, a positively charged pyrene analog was synthesized and then applied for HAase detection, in which the ratiometric fluorescence variation depended on the electrostatic adsorption of HA towards the fluorophore as well.30 However, synthesis and purification of such well-designed fluorophore are laborious and expensive. By contrast, CDs not only exhibit excellent fluorescence, but also possess many advantages including easy preparation, low cost, and biological safety. It is promising to develop proper CDs with exceptional properties for the rapid, stable, and sensitive detection of HAsae. In this work, cationic carbon dots (C-CDs) were synthesized by a facile ultrasonic method. The plenty of positive charge on the surface endows as-synthesized CDs high affinity with negatively charged HA, which is the substrate of HAase as well. So the C-CDs have a potential in HAase detection. Positively charged neutral red (NR), whose fluorescence excitation spectrum overlaps the fluorescence emission spectrum of C-CDs, has been reported as an efficient accepter of förster resonance energy transfer (FRET).31 Herein, these two fluorescent components were unmodified and employed to develop a sensitive ratiometric fluorescence method for HAase detection, in which the FRET between C-CDs and NR was controlled by the electrostatic adsorption of HA as well as the enzymolysis of HAase towards HA. The proposed 5



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method was then applied to detect HAase activity in human serum sample.

Experimental Section Chemicals. CPC and NR were purchased from Aladdin Reagent Co., Ltd (Shanghai, China). HA (Mw = 1000 kDa), HAase (400 U/mg), glucose, glutathione (GSH), human serum albumin (HSA), alkaline phosphatase (ALP), thrombin (Thr), lysozyme (Lyz) and trypsin (Try) were ordered from Sigma-Aldrich. Enzyme-linked immunosorbent assay (ELISA) kit for HAase was purchased from Shanghai Yanxin Biological Technology Co., Ltd (Shanghai, China). All reagents were of analytical reagent grade and used as received. Ultrapure water (18.2 MΩ.cm) was prepared with a Milli-Q system (Millipore, Bedford, MA, USA) and used throughout all study. Apparatus. An ultrasonic cleaning machine (KQ-700V, Kunshan, China) with an output power of 700 W and a frequency of 40 kHz was used for the synthesis of CDs. High-resolution transmission electron microscopy (HRTEM) was performed on Tecnai G2 F20 (FEI Company, USA), with an accelerating voltage of 200 kV. Fourier transform infrared spectroscopy (FT-IR) spectra were recorded using a Magna-IR 750 Fourier transform infrared spectrometer (Nicolet, USA). Fluorescence measurements were carried out on a Hitachi F-4600 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The dynamic light scattering (DLS) and zeta potentials were measured on Zetasizer Nano ZS90 nanoparticle size and zeta potential analyzers (Malvern Instruments, Malvern, U.K.). 6


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Synthesis of C-CDs from CPC. The C-CDs were synthesized according to the reported method13,14 with an improvement that using ultrasonic treatment to accelerate the reaction process. Typically, 9.0 mL NaOH (2.0 M) was mixed well with CPC aqueous solution (15 mM, 100 mL), followed by an ultrasonic treatment. The color of solution changed from clear to light yellow and then to dark yellow, which indicated the formation of CDs. After 30 min, the reaction was terminated by adjusting pH of the solution to neutral (pH = 7.0) with HCl. Then the product was purified by dialysis (the cutoff of the dialysis membrane was equivalent to Mw≈3500) and vacuum dried at 65 ℃. The as-prepared C-CDs were re-dispersed in ultrapure water (0.5 mg/mL) and stored at 4 ℃ before use. Analytical Procedures for HAase Detection. The sensing system used for HAase detection was prepared by mixing C-CDs, NR, and HA in PBS buffer solution (10 mM, pH = 6.0) on an appropriate concentration (e.g. 50 µg/mL C-CDs, 5 µg/mL NR, and 0.05 mg/mL HA). Then various levels of HAase were added to such solution, which was then incubated at 37 ℃ for 100 min. Fluorescence emission spectra (λex = 390 nm) were finally recorded for the quantitative analysis. To prepare the spiked samples for standard addition recovery assays, the blood sample obtained from a local hospital (Fuzhou, China) was centrifuged for 20 min to separate the clear serum, which was mixed with various levels of HAase for further determination.25,27 The assay of HAase by ELISA method followed its instructions enclosed in the kits.

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Results and Discussion Characterization of the Prepared C-CDs. As shown in Figure 1A, the fluorescent C-CDs, prepared by a pathway of ultrasonic treatment, had a rather uniform spherical shape and an average diameter of 3 nm. Its FT-IR spectrum in Figure 1B also exhibited similar absorptions to the N-doped carbon dots, such as the peaks of aromatic stretching vibrations (1639 cm−1 for C=C, 1432 cm−1 for C=N), and the broad absorption band (around 3436 cm−1 for the stretching vibrations of O–H/N– H).32 Besides, the existence of pyridine residue on the surface of C-CDs has already been confirmed by previous reports where CPC acted as the precursor of CDs.12,13 Therefore, this kind of CDs was speculated to be positively charged, which was verified by the following zeta-potential measurement. Figure 1C shows that the zeta potential of C-CDs was as high as +39.2 mV. Meanwhile, NR is known as a weak alkaline dye that presents on cationic when the aqueous pH is under 6.8 (pKa of NR), its zeta potential was measured as +16.6 mV. On the contrary, the zeta potential of HA was measured as -66.1 mV under the same conditions. These results indicate the two fluorescent components can be simultaneously electrostatic absorbed by negatively charged HA. On the other aspect, spectra of these two fluorescent components were recorded to investigate the feasibility of FRET between them. As shown in Figure 1D, the fluorescent C-CDs had a strong emission around 525 nm under an excitation of 390 nm, and the NR had its maximum emission at 630 nm under an excitation of 538 nm. The fine overlap of fluorescence spectra between the emission of C-CDs and the 8


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excitation of NR suggests a potential FRET between them. All the characterization results demonstrate the applicability of the employed two fluorescent components in the sensing system. Principle of the Sensing System. Scheme 1 depicts the principle of the electrostatic-controlled ratiometric fluorescent assay for HAase. Two positively charged components of C-CDs and NR were mixed with negatively charged HA firstly. Because of the intense electrostatic absorption of HA towards C-CDs as well as NR, these two fluorescent components gradually aggregated along the linear chain of HA to form stable nanoaggregates. The distance between C-CDs and NR thereby decreased, resulting in a remarkable FRET from C-CDs to NR. In this case, a red fluorescence appeared and this phenomenon was regarded as the initial state for HAase detection. In the presence of HAase, the linear HA was enzymatically degraded into low molecular weight fragments, indicating a scaffolding disassembly of the nanoaggregates too. Because the small HA fragments no longer provide sufficient restraining force to offset the electrostatic repulsion between C-CDs and NR, the absorbed fluorescent components were gradually dissociated from the nanoaggregates and repelled away from each other into a separated situation. So the FRET between C-CDs and NR became weak, and the fluorescence emission at 630 nm decreased while the fluorescence emission at 525 nm increased; meanwhile, the fluorescence color of the detecting system changed from red to yellow-green, which could even be observed by naked eyes under an UV light irradiation. Because this variation depends 9



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on the enzymolysis of HAase towards HA, the ratio of fluorescence intensity for C-CDs (I525) to that for NR (I630) can be applied to represent the level of HAase. To confirm the feasibility of the proposed sensing system, DLS and TEM measurements were employed to investigate the nanoaggregates formation and disassembly triggered by enzymatic reaction firstly. The size distributions of nanoaggregates measured by DLS were recorded every 20 min in the presence of HAase, and the results were displayed in Figure 2A. Clearly, the sizes of nanoaggregates gradually decreased from 200 nm to 10 nm with extension of the enzymatic time, indicating the degradation of HA as well as the disassembly of nanoaggregates. Additionally, the situations of the nanoaggregates before and after the addition of HAase were characterized by TEM. Images in Figure 2B show the nanoaggregates changed from an aggregate to disperse situation eventually. These results demonstrate that undegraded HA plays as an adhesion in gathering the positively charged fluorescent components to form nanoaggregates, but its specific enzyme disassembles the nanoaggregates well. So the degree of FRET between the C-CDs and NR depends on the enzymolysis of HAase towards HA. Optimization of the Sensing System towards HAase. To establish the ratiometric fluorescent assay responding towards HAase, relevant conditions were investigated in detail. Firstly, an appropriate pH of the sensing system was determined by overall consideration of its effect on C-CDs, NR, and the stability of prepared composite including HA. Figure 3A shows that the fluorescence of C-CDs exhibited an excellent 10


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stability towards pH variations. But the fluorescence intensity of NR at 630 nm decreased with the increasing pH values, especially when the pH values surpassed 6.8 (pKa of NR). The values of I525/I630 caused by FRET from C-CDs to NR were measured to investigate the stability of as-prepared detecting system. From the results in Figure 3A, I525/I630 reached the lowest at pH 6.0 but increased when the pH decreased or increased, indicating that overly basic or acidic condition leads to the instability of composite.15 For another aspect, the measurements of zeta potential revealed that both C-CDs and NR had a trend of positive charge decreasing or even negative charge increasing with the increasing pH values (Figure 3B). To ensure the two fluorescent components can be electrostatic absorbed by HA, the proposed assay was carried out in a PBS buffer solution whose pH value was restricted to 6.0. The ratio of two fluorescent components has a significant effect on the ratiometric fluorescence sensing system. The C-CDs/NR ratio was optimized by varying their mass concentrations while keeping the concentrations of HA and HAase invariable. As shown in Figure 4, when the C-CDs/NR ratio changed from 50:1 to 50:20, the fluorescence intensities at 525 nm gradually decreased but increased at 630 nm, as an enhancement of FRET from C-CDs to NR. Only when the C-CDs/NR ratio was set at 50:5, the fluorescence intensities at 525 nm and 630 nm were nearly equal, suggesting a more flexible scope of ratiometric variation. By contrast, when the C-CDs/NR ratio deviated from this value, a negligible peak at 525 nm or 630 nm was observed, probably because too much C-CDs or NR could submerge the fluorescence from each 11



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other. Thus a medium ratio of 50:5 was adopted in the following study. Figure 5A displays the time-dependent fluorescence spectra, the fluorescence intensities at 525 nm gradually increased while those at 630 nm decreased. And fluorescence color of the sensing system correspondingly changed from red to yellow-green under a UV lamp (inset in Figure 5A). This phenomenon suggests that the enzymatic time is another indispensable factor during the HAase detection. Therefore, the fluorescence kinetic curves of HAase at different levels were plotted by measuring the fluorescent ratio of I525/I630. Figure 5B reveals that almost no change of I525/I630 was observed in the absence of HAase (blue curve). By contrast, the fluorescent ratio of I525/I630 gradually increased to a plateau with extension of enzymatic time when different levels of HAase were present. Particularly, a higher concentration of HAase resulted in a larger variation of the I525/I630. To ensure the enzymolysis was completed fully, the incubation time of 100 min was employed in further study. Performance of the Proposed Sensing System. Various levels of HAase (0.1−8.0 U/mL) were added to the sensing system (50 µg/mL C-CDs, 5 µg/mL NR, and 0.03 mg/mL HA), and the responding fluorescence spectra were measured. As shown in Figure 6A, the fluorescence intensities at 525 nm increased while those at 630 nm decreased with the increasing levels of HAase. And the fluorescence color under a UV lamp gradually changed from red to yellow-green too (inset in Figure 6A). In addition, the fluorescent ratio of I525/I630 was linearly related to the HAase concentrations in 12


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the range of 0.1−8.0 U/mL (Figure 6B). The regression equation can be described as follows: Ratio (I525/I630) = 0.41 + 1.14 [HAase] U/mL

(1)

Its corresponding regression coefficient was 0.9987, and the limit of detection (LOD) was determined to be as low as 0.05 U/mL. Given that HAase level in normal human serum is 0.26 ± 0.20 U/mL,27 these results demonstrate that this sensing system is sensitive to HAase activity. Compared with other methods for HAase detection (Table 1), this sensing system not only shows sufficient sensitivity towards HAase, but also avoids tedious operations. This advantage might be attributed to the excellent fluorescence of C-CDs as well as its unique property of positive charge. More importantly, the ratiometric fluorescence mechanism endows the detecting system colorful results which are more sensitive to naked eyes. It is beneficial for semi-quantitative measurement when the detecting instrument is limited. To test the selectivity of this sensing system during HAase detection, some potential interferences including inorganic salts (NaCl, KCl, MgCl2, and CaCl2), glucose, GSH, HSA, ALP, Thr, Lyz and Try were examined under the same conditions. As shown in Figure 7, only HAase induced a distinct change of I525/I630, whereas the addition of other species led to almost no fluorescence change. These results confirm that the sensing system has an outstanding selectivity towards HAase and a prominent capacity of resisting disturbance. These superiorities would be contributed to the enzymatic specificity of HAase towards HA as well as the ratiometric 13



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fluorescence mechanism. Determination of HAase in Human Serum Samples. The proposed sensing system was then applied to evaluate HAase in biological samples. Different levels of HAase were added to the serum samples to prepare the spiked samples and then detected by the proposed method. Table 2 summarizes the results obtained by the standard addition method, and the accuracy of the proposed method is evaluated by the recovery rate. The recovery rates were in the range of 93.8−97.0% and the relative standard deviations (RSDs) were not over 7.2%, suggesting an accurate and reliable determination for HAase. Additionally, a commercial ELISA kit was used to detect HAase in the same serum samples for a comparison. And the deviations between the two methods were less than 15%, indicating a fine agreement of the results. Overall, the proposed method is demonstrated practicable for quantitative determination of HAase in biological samples.

Conclusion In summary, an electrostatic-controlled ratiometric fluorescence assay for HAase detection has been developed based on the electrostatic interaction between negatively charged HA and positively charged fluorescent components. Unmodified HA was employed as both the scaffold of nanoaggregates and the substrate of enzymatic reaction, while the fluorescent components of C-CDs and NR were used to develop the ratiometric fluorescence method. Compared with other methods for HAase 14


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determination, this method shows favorable performance but avoids tedious operations or complicated modifications. Moreover, owing to the ratiometric sensing mechanism, higher sensitivity and reliability are achieved. In particular, the fluorescence color change is easily observed by naked eyes, which is beneficial for semi-quantitative measurement with limited instruments. The proposed method has been employed to detect HAase spiked in serum samples and showed approving results. It skillfully combines the excellent fluorescence of CDs with the unique property of positive charge in the sensing system, which illustrates a new approach for a wider application of some special CDs in detecting biomolecules.

Acknowledgements This project was financially supported by NSFC (21575025, 21575027), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT15R11), and the Foundation for Scholars of Fuzhou University (No. XRC-1671, XRC-17007).

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Figures and Captions Scheme 1 Schematic illustration for the electrostatic-controlled ratiometric fluorescence assay in response to HAase.

Figure 1 (A) Representative TEM image of the C-CDs synthesized from CPC, inset is the corresponding particle size distribution histograms. (B) FT-IR spectrum of the synthetic C-CDs. (C) Zeta potentials measurements of C-CDs, NR and HA in PBS buffer solution (10 mM, pH = 6.0). (D) The excitation, emission fluorescence spectra of C-CDs and NR.

Figure 2 (A) Size distributions of the nanoaggregates measured by DLS every 20 minutes (0, 20, 40, 60, 80 and 100 minutes) in the presence of HAase (1.0 U/mL). (B) TEM images of the assay systems before (left) and after (right) addition of HAase (1.0 U/mL).

Figure 3 (A) Effect of pH on the fluorescence intensity of C-CDs, NR and the stability of prepared composite including HA. (B) Zeta potentials of C-CDs and NR vary with the increasing pH values. Experiments were carried out in a PBS buffer solution (10 mM) containing C-CDs (50 µg/mL), NR (5 µg/mL) and HA (0.05 mg/mL).

Figure 4 Effect of C-CDs/NR ratio on the fluorescence intensity at 525 nm and 630 nm. 18


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Experiments were carried out in a PBS buffer solution (10 mM, pH = 6.0) with HA (0.05 mg/mL) and HAase (1.0 U/mL).

Figure 5 (A) Time-dependent fluorescence spectra of the sensing system (50 µg/mL C-CDs, 5 µg/mL NR and 0.05 mg/mL HA) respond to HAase (4.0 U/mL) in PBS buffer solution (10 mM, pH = 6.0). Inset is the fluorescence color change under a UV lamp (λex=365 nm). (B) Plot of I525/I630 versus enzymatic time in the presence of HAase at different levels: (a) 0, (b) 1.0, (c) 2.0, (d) 4.0, and (e) 8.0 U/mL.

Figure 6 (A) Fluorescence spectra of the sensing system (50 µg/mL C-CDs, 5 µg/mL NR and 0.05 mg/mL HA) respond to different levels of HAase in PBS buffer solution (10 mM, pH = 6.0). (B) Linear relationship between the fluorescent ratio of I525/I630 and the HAase concentrations.

Figure 7 The fluorescence sensing system responses to various species: NaCl (100 mM), KCl (100 mM), MgCl2 (10 mM), CaCl2 (10 mM), Glucose (10 mM), GSH (10 mM), HSA (10 nM), ALP (10 nM), Thr (10 nM), Lyz (10 nM), Try (10 nM), and HAase (1 U/mL). Results are the mean ± standard deviation of three separate measurements.

Table 1 Comparison of different methods for HAase detection.

19



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Table 2 Determination of HAase spiked in serum samples by the proposed and ELISA methods respectively. Results are the mean ± standard deviation of three separate measurements.

20


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Scheme 1

21



Figure 1

(B) Transmittance (%)

100 80 60 1432 C=N

40 1639 C=C

20

3436 O-H/N-H

0 4000 3500 3000 2500 2000 1500 1000

Wavenumber (cm-1)

(C)

(D)

60 40 20

HA 0 -20 -40

C-CDs

NR

HA

C-CDs Em

Normalized Intensity

Zeta potential (mV)

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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NR Em

C-CDs Ex NR Ex

-60 300

400

500

600

Wavelength (nm)

22


700

500

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Figure 2

(B)

(A) 25

Before 100 min

20

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


After

0 min

15 10 5 0 1

10

100

1000

100 nm

Diameter (nm)

23


100 nm


Figure 3

(A)

C-CDs NR

I 525 / I 630

8

600

6 400 4 200

Zeta potential (mV)

Intensity (a.u.)

(B) 60

10

800

I 525 / I 630

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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2 0

0 4

5

6

7

8

9

C-CDs NR

40

20

0

-20

10

4.0

5.0

pH

6.0

7.0

pH

24


8.0

9.0

10.0

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Figure 4 1000

I 525 I 630

800

Intensity (a.u.)

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


600 400 200 0 50:1

50:2

50:5

50:10

50:20

CCDs / NR ratio

25



Figure 5

(B)10

(A)1000

600 400 200 0 500

600

0 U/mL 1.0 U/mL 2.0 U/mL 4.0 U/mL 8.0 U/mL

8

Time

800

I525 / I630

Intensity (a.u.)

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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700

e

6 d

4 c

2

b

0

a

0

20

Wavelength (nm)

40

60

Time (min)

26


80

100

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Figure 6

(A)1000

(B) 10

800

8

HAase

I525 / I630

Intensity (a.u.)

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


600 400 200

6 4 2 0

0 500

600

0

700

Wavelength (nm)

2

4

6

HAase (U/mL)

27


8


Figure 7 3.0

2.5

2.0

1.5

1.0

0.5

l K C l M gC l C 2 a G Cl lu 2 co se G SH H SA A LP Th r Ly z Tr H y A as e

0.0

N aC

I525 / I 630

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

28


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Table 1 Comparison of different methods for HAase detection. LOD

Linear Range

Detection Time

(U/mL)

(U/mL)

(h)

Zymography

0.625

0.625-5

48

20

Colorimetry

2.4

0-150

2

21

Fluorescence

0.625

1.25-50

3

24

Fluorescence

0.05

0.1-8

1.6

This work

Method

Reference

29



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Page 30 of 31

Table 2 Determination of HAase spiked in serum samples by the proposed and ELISA methods respectively. Results are the mean ± standard deviation of three separate measurements. HAase

Detected by

Detected by Recovery

Sample

Deviation

spiked

current method

ELISA method

(U/mL)

(U/mL)

Blank

/

0.28±0.02

/

0.25±0.02

11.5

1

1.0

1.24±0.05

96.0

1.28±0.04

3.2

2

2.0

2.22±0.08

97.0

2.13±0.06

4.2

3

4.0

4.03±0.13

93.8

4.21±0.11

4.4

rate (%)

(%) (U/mL)

30


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For TOC only:

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