Single-Particle Enumeration-based Sensitive Glutathione S

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Single-Particle Enumeration-based Sensitive Glutathione STransferase Assay with Fluorescent Conjugated Polymer Nanoparticle Yameng Han, Tianyu Chen, Yiliang Li, Langxing Chen, Lin Wei, and Lehui Xiao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01849 • Publication Date (Web): 12 Aug 2019 Downloaded from pubs.acs.org on August 12, 2019

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Analytical Chemistry

Single-Particle Enumeration-based Sensitive Glutathione S-Transferase Assay with Fluorescent Conjugated Polymer Nanoparticle Yameng Han,† Tianyu Chen,† Yiliang Li,‡ Langxing Chen†, Lin Wei,§ and Lehui Xiao*,†

† State

Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and

Molecular Recognition, College of Chemistry, Nankai University, Tianjin, 300071, China. ‡ Department

of Rehabilitation Medicine, The Eighth Affiliated Hospital, Sun Yat-sen University,

Shenzhen, Guangdong, 518033, China. § Key

Laboratory of Phytochemical R&D of Hunan Province, College of Chemistry and Chemical

Engineering, Hunan Normal University, Changsha, 410081, China. *Corresponding Author: [email protected]

Abstract Glutathione S-transferase (GST) is a group of multifunctional enzyme and participates in many physiological processes, such as xenobiotic biotransformation, drug metabolism and degradation of toxic products. Herein, we demonstrate a label-free fluorescent conjugated polymer nanoparticles (FCPNPs)-based single-particle enumeration (SPE) method for the sensitive GST assay. Fluorescence resonance energy transfer (FRET) is formed between the glutathione modified FCPNPs (FCPNPs-GSH) and polyethyleneimine capped gold nanoparticles (GNPs@PEI). Therefore, the fluorescence of FCPNPs-GSH is quenched remarkably. In the presence of GST, GNPs@PEI stay away from FCPNPs-GSH due to the specific interaction between FCPNPs-GSH and GST, leading to the inhibition of FRET. As a result, the fluorescence emission of 1

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FCPNPs-GSH is restored, which is reflected as the increase of the number of fluorescent particles in the microscopic image. By statistically counting the target concentration-dependent fluorescent particle number, accurate quantification of GST is achieved. The linear range from 0.01 to 6 g/mL is obtained for GST assay and the limit-of-detection (LOD) is 1.03 ng/mL, which is much lower than the ensemble fluorescence spectra measurements in bulk solution. In urine sample assay, satisfactory recoveries in the range of 97.5-106.5.0% are achieved. Because of the high sensitivity and excellent specificity, this method can be extended to other disease-related biomolecules detection in the future.

Introduction Glutathione S-transferase (GST) is one of the most important phase Ⅱ metabolic enzymes in organisms. As a detoxification enzyme, it plays a critical role in reducing toxicity.1-3 Its main function is to catalyze reduced glutathione (GSH) to attack the electrophilic centers of target compounds such as endogenous superoxide radicals and toxic metabolites. Therefore, the tissues and cells can be protected against damage.4, 5 Moreover, due to the specific interaction with GSH, GST is also applied to tag recombinant proteins to separate and purify proteins for proteomic research.6 GST is distributed extensively in all organs of the body and has higher concentration in liver cytosol, while the level of GST in urine is extremely low under normal health condition. When the kidney-related disease occurs, the amount of GST in urine will increase. GST can be used as a biomarker for revealing the injury of renal tubular epithelial tissue.7-9 Consequently, developing effective analytical methods for GST determination is highly desired for the early discovery and diagnosis of diseases. In the past decade, conventional methods including sodium 2


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dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE),10 electrochemical assay,11,

12

bioluminescent13, 14 and fluorescent sensors15-19 have been established for GST activity detection. Although some successes have been made, they still suffer from certain defects including time-consuming, cumbersome preparation process, large sample consumption and insufficient sensitivity. To address these issues, there is an urgent demand for developing new techniques with improved sensitivity and convenience. Recently, single-particle (or -molecule) detection based on the optical microscopic imaging techniques has received extensive attention and been used for a variety of molecules quantification, including proteins, enzymes, nucleic acids and even ions.20-25 When the targets are introduced, the features of individual particle such as the spectrum, the intensity in signal response or numbers will be changed.26-29 By measuring these variations associated with the target objects, accurate quantitative detection can be achieved. It is an ideal platform for bioassay as a result of the gorgeous advantages of high sensitivity, low sample consumption and rapid response time in comparing with common ensemble measurement. For example, the developed single-particle enumeration (SPE) assay through counting the target concentration-dependent probe numbers has been widely employed for monitoring various biomolecules.30-37 As a novel family of fluorescent nanomaterials, fluorescent conjugated polymer nanoparticles (FCPNPs) consisting of conjugated polymer monomers possess strong fluorescence emission due to the presence of abundant -electrons.38-41 In comparison with the traditional fluorescent dyes and quantum dots, they possess many interesting properties, including facile preparation, higher brightness,

non-toxicity,

exceptional

photostability,

excellent

water

solubility

and

biocompatibility. What's more, FCPNPs with multiple ligands can be obtained by 3



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copolymerization with different polymer monomers or modification with different functional molecules. As a consequence, they have been exploited in various fields, including biological sensing, photodynamic therapy, single-particle tracking, cell imaging and drug delivery.42-47 Inspired by these merits, herein we designed a novel SPE assay based on the fluorescence resonance energy transfer (FRET) between FCPNPs and gold nanoparticles (GNPs) for the sensitive determination of GST. GNPs possess a high extinction coefficient, broad ultraviolet visible absorption band and controllable size manipulation, which have been frequently used as efficient fluorescence quencher in various types of FRET sensors.48-51 On this account, the positively charged GNPs capped with polyethyleneimine (PEI) were prepared by one-pot synthesis which served as energy receptor and the FCPNPs modified with GSH through hydrogen bonding behaved as energy donor. The principle of this design is delineated in Scheme 1. The fluorescence emission of FCPNPs-GSH can be sufficiently quenched in the addition of GNPs@PEI because the negatively charged FCPNPs-GSH can combine with the positively charged

GNPs@PEI

via

electrostatic

interaction,

resulting

in

the

formation

FCPNPs-GSH+GNPs@PEI assembly for the efficient FRET. The number of fluorescent particles on the glass slide surface observed on the basis of the total internal reflection fluorescence (TIRF) microscopy imaging technique decreases significantly. When GST was introduced, FCPNPs-GSH preferentially combined with GST rather than GNPs@PEI owing to the specific interaction between GST and GSH. As a consequence, GNPs@PEI can be kept away from FCPNPs-GSH and the fluorescence of FCPNPs-GSH can be recovered, accompanying with an obvious augmentation of fluorescent particles. In the absence of GST or the presence of other interfering substances, the recovery of fluorescence intensity cannot be observed and the number of fluorescent particles will 4


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not increase accordingly. By statistically monitoring the number of fluorescent particles, the concentration of GST can be quantified accurately by the SPE method. A good linear range of 0.01-6 g/mL and low limit-of-detection (LOD) of 1.03 ng/mL are obtained for GST assay. Furthermore, the application of this approach for the determination of GST in real urine sample was performed and satisfactory recoveries (in the range of 97.5-106.5%) illustrate the promising applicability of the SPE assay for complex sample analysis in the future.

Experimental Section Materials and Instruments Poly(styrene-co-maleic

anhydride)

(PSMA,

MW

1700

poly[9,9-dioctylfluorenyl-2,7-diyl)-alt-co-1,4-benzo-{2,1′-3}-thiadiazole)]

Da) (PFBT,

and MW

140000 Da, polydispersity 3.5) were purchased from American Dye Source Inc. (Quebec, Canada). Glutathione S-transferase (GST), glutathione (GSH), polyethylene glycol (PEG, MW 3000 Da) and chloroauric acid (HAuCl4·3H2O) were purchased from Sigma-Aldrich. Tetrahydrofuran (THF, anhydrous, ≥99.9%), sodium borohydride (NaBH4), sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl2), calcium chloride (CaCl2) and zinc chloride (ZnCl2) were bought from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Polyethyleneimine (PEI, MW 2000 Da) and aminopropyltriethoxysilane (APTES) were obtained from J&K Scientific, Ltd. Chemical Company (Beijing, China). Horseradish peroxidase (HRP), bovine serum albumin (BSA), trypsin (TRY), cytochrome C (Cyt C), immunoglobulin G (IgG), transferrin (TRF), lysozyme (LYZ), cysteine (Cys), lysine (Lys) and glycine (Gly) were acquired from Aladdin Chemical Reagent Co. Ltd. (Shanghai, 5



China). All other reagents were analytical purity. The UV−vis absorption spectrum of GNPs@PEI was determined by the UV-2450 spectrophotometer (Japan). The fluorescence emission spectra from FCPNPs in bulk solution were recorded on a Hitachi F-4600 fluorescence spectrophotometer. The morphology of the nanoparticles was characterized with a JEOL JEM2100 transmission electron microscope (TEM). The size distributions and zeta potential of nanoparticles were studied with a Zetasizer Nano ZS system (Malvern, U.K.). A home-built objective-type total internal reflection fluorescence (TIRF) microscope was used for the single-particle fluorescence imaging measurements, which was equipped with a fiber coupled 473 nm cw laser as the excitation light source. The images were captured by an Andor iXon Ultra 888 electron-multiplying charge-coupled device (EMCCD) camera through a Plan Apochromat TIRF 100× oil immersion objective. All images were processed by ImageJ software. For data analysis, an imaging region of 512512 pixels was selected for the fluorescent particles counting. The average intensity of the image was obtained by stacking fifty frames and then the number of fluorescent particles was analyzed by thresholding procession. Synthesis of FCPNPs-GSH A nanoprecipitation method was employed to prepare the water soluble FCPNPs according to the reported method with minor modification.52 Briefly, the stock solution (1 mg/mL) was prepared by dissolving the fluorescent conjugated polymer PFBT in THF. Then, the stock PFBT solution was diluted in 1 mL THF containing 100 μg amphiphilic copolymer PSMA to make a homogeneous solution mixture with the final PFBT concentration of 20 g/mL. Subsequently, the mixture was quickly added into 6 mL of ultrapure water under vigorous sonication. After 90 6


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seconds of ultrasonication, the THF was removed by blowing N2 into the solution in 85 C water bath for about 1 h. Followed by cooling to room temperature, the resulting solution was filtrated through a membrane filter to remove any aggregates and further purified by ultrafiltration before use. The FCPNPs were clear and stable for months. Afterwards, the prepared FCPNPs were modified with GSH via hydrogen bonding. Specifically, 34 μL of 5% PEG and 68 μL of GSH (5 mM) were injected to 1 mL of FCPNPs solution and the mixture was sequentially stirred for 4 h at room temperature. The obtained FCPNPs-GSH solution was purified by ultrafiltration to eliminate the excess reactant and stored at 4 °C for use. Synthesis of GNPs@PEI The GNPs@PEI were prepared by one-pot synthesis. In brief, 20 μL of HAuCl4 (24.28 mM) was injected to 3 mL of ultrapure water and then 60 μL of PEI (1 mg/mL) was added to the above mixture, following by stirring for 5 min. After that, 400 μL of NaBH4 (2.63 mM) as a reducing agent was rapidly injected into the mixture and the reaction solution was stirred for 5 min at room temperature. Finally, the GNPs@PEI solution was obtained and the residual reactants were removed by ultrafiltration. GST Assay Based on SPE For GST assay, 30 μL of FCPNPs-GSH and 35 μL of GNPs@PEI were mixed in a 200 μL centrifugal tube. After incubation of 10 min, various amounts of GST were added to the mixture solution and the final volume of sample was adjusted to 100 μL with ultrapure water. The fluorescence spectra of these samples were collected at an excitation wavelength of 470 nm after 30 min. For the SPE detection, these samples were further diluted with ultrapure water. 7



Subsequently, the diluted samples were dripped on the amino-silanized glass slide surface and the negatively charged FCPNPs-GSH were immobilized relying on the electrostatic interaction. After adsorption completely, the single-particle fluorescence images were attained by TIRF microscope. Next, some interfering substances including relevant proteins (BSA, TRY, Cyt C, TRF, IgG, HRP and LYZ), ions (K+, Na+, Ca2+, Mg2+ and Zn2+) and amino acids (Cys, Lys and Gly) were selected to evaluate the selectivity of the SPE detection. GST or the interfering substance was added to the FCPNPs-GSH and GNPs@PEI mixture solution, respectively. Additionally, the mixture containing GST and interfering substance was also explored to verify the anti-interference ability. These samples were detected by the SPE method as described above. The practical applicability of this SPE platform was further investigated by the standard recovery experiment in real samples. The urine sample from a healthy volunteer was firstly diluted with ultrapure water and then different amounts of GST were added to the prediluted urine samples. Then, the spiked urine samples were mixed with the FCPNPs-GSH+GNPs@PEI assembly solution. After incubating 30 min, the samples were analyzed according to the SPE method.

Results and Discussion Fabrication and Characterization of FCPNPs-GSH and GNPs@PEI for SPE Assay Water-soluble FCPNPs were prepared by a nanoprecipitation method. An amphiphilic polymer PSMA was utilized to improve the hydrophilicity of FCPNPs. During the nanoprecipitation process, the hydrophobic polystyrene backbone of PSMA was embedded inside FCPNPs while the maleic anhydride units were extended out to the water environment and further 8


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hydrolyzed to carboxyl groups on the surface of FCPNPs. As a consequence, the obtained FCPNPs show prominent colloidal stability and water dispersibility. What’s more, the carboxyl groups facilitate the functionalization of nanoparticles and GSH can be modified onto the surface of FCPNPs through hydrogen bonding. Thus, FCPNPs-GSH as specific single-particle probes were attained for GST assay. The TEM image of FCPNPs-GSH is shown in Figure 1a. The particles exhibit excellent monodispersity and spherical morphology. The dynamic light scattering (DLS) measurement of FCPNPs-GSH in Figure 1e indicates that the particles possess a diameter of approximately 27 nm, which is in good agreement with the TEM result. The GNPs@PEI were prepared by one-pot reduction method, where PEI served as stabilizer and NaBH4 was used as a reducing agent. HAuCl4 was rapidly reduced as a result of the strong reducibility of NaBH4 and PEI was capped on the surface of GNPs, endowing the particles superior dispersity in water. As a consequence, positively charged and small size GNPs@PEI were obtained. The TEM image in Figure 1b shows that the particles are spherical in shape and dispersed without aggregation, which is further confirmed by DLS characterization. A hydrodynamic diameter with approximate 12 nm is depicted in Figure 1f. Subsequently, the zeta potential of nanoparticles was measured to verify the successful modification. As illustrated in Table 1, the GNPs@PEI display a positive potential (11.5 mV), which is attributed to the great deal of amino groups on the particles surface. The carboxyl groups functionalized FCPNPs show negative charge (-22.4 mV) whereas the zeta potential of FCPNPs-GSH (-12.8 mV) has an obvious increase due to the cross-linking of GSH to FCPNPs via hydrogen bonding which partly shields the carboxyl groups. These characterizations confirmed that GSH was assembled on the FCPNPs surface successfully. Then, the negatively charged FCPNPs-GSH can combine with the positively charged GNPs@PEI based on the 9



electrostatic interaction, which is further confirmed by the TEM in Figure 1c. Moreover, the broad UV-vis absorption spectrum of GNPs@PEI overlaps largely with the fluorescence emission spectrum of FCPNPs-GSH as shown in Figure 1g. As a result, the FRET between FCPNPs-GSH and GNPs@PEI can take place and the fluorescence intensity of FCPNPs-GSH can be quenched effectively. After the addition of GST, due to the specific interaction of FCPNPs-GSH with GST, the number of GNPs@PEI attached on the FCPNPs-GSH surface decreases greatly in Figure 1d, resulting in the fluorescence recovery from FCPNPs-GSH. The fluorescence stability of individual particle is a critical characteristic for SPE assay, which can be verified by the time-dependent fluorescence intensity track. It is evident that the fluorescence intensity of FCPNPs-GSH is quite stable and no photobleaching occurs under this case (Figure 1h and i). These results imply that the FCPNPs-GSH have an extraordinary photostability and are suitable for SPE detection. Design of the FRET Pair for GST Assay The detection platform was constructed based on the FRET between FCPNPs-GSH and GNPs@PEI. The GST concentration dependent fluorescence emission from FCPNPs-GSH was firstly interrogated. It is obvious that the change of fluorescence intensity from FCPNPs-GSH was few or negligible when different concentrations of GST were introduced (Figure 2a). However, when the GNPs@PEI were added to the FCPNPs-GSH solution, the fluorescence emission of FCPNPs-GSH was gradually quenched as the increase of GNPs@PEI concentrations, suggesting the occurrence of FRET between FCPNPs-GSH and GNPs@PEI (Figure 2b). Meanwhile, the FRET efficiency was estimated on the basis of the definition of E = 1 ― FDA/FD, where FDA and FD are the fluorescence intensities of the donor in the presence and absence of acceptor, respectively. As shown in Figure 2c, the quenching efficiency gradually grows up with the 10


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concentrations of GNPs@PEI increasing accordingly and reaches a plateau of ~80% when the concentration of GNPs@PEI is more than 0.72 nM. There are two main reasons for this high FRET efficiency. On the one hand, FCPNPs-GSH and GNPs@PEI are staying close enough through the electrostatic interaction because the FRET efficiency is inversely proportional to the distance between the donor and acceptor. On the other hand, GNPs have strong optical absorption capability and broad UV-vis absorption band, which can improve the quenching effect. In order to attain the optimal sensitivity, 0.72 nM was selected as the final concentration of GNPs@PEI for the subsequent experiments. When GST was added to the reaction solution containing FCPNPs-GSH and GNPs@PEI, FCPNPs-GSH preferred to bond with GST due to the specific interaction between GSH and GST. As a result, GNPs@PEI stayed away from FCPNPs-GSH and the fluorescence of FCPNPs-GSH was restored. As depicted in Figure 2d, the fluorescence of FCPNPs-GSH gradually enhances along with the increase of GST concentrations. A good linear relationship was obtained by plotting the (F ― F0)/F0 against the concentration of GST in the range of 0.5-10 g/mL (Figure 2e). The calculated LOD was 0.13 g/mL according to the 3/slope ( is the standard deviation of blank sample). Moreover, control experiments were performed to examine the specific recognition capability of GST assay. FCPNPs without GSH modification were used to replace FCPNPs-GSH. As shown in Figure 2f, in the presence of GNPs@PEI, the fluorescence intensity of FCPNPs decreases greatly because the strong electrostatic interaction between the negatively charged FCPNPs and positively charged GNPs@PEI. However, when different concentrations of GST were added to the FCPNPs and GNPs@PEI mixture solution, no enhancement was noted in the fluorescence intensity of FCPNPs, indicating that there is no specific interaction between FCPNPs and GST. Only when the surface 11



of FCPNPs is modified with GSH, the fluorescence recovery of FCPNPs-GSH can be observed based on the specific combination of GSH-GST. Encouraged by the high sensitivity of SPE assay, we tried to detect GST based on the above principle at single-particle level. First of all, the feasibility of SPE method was explored. As shown in Figure 3a, very few FCPNPs can be observed on the glass slide surface as determined by the TIRF microscope in the presence of GNPs@PEI due to the fluorescence quenching caused by electrostatic interaction. When GST is added to the FCPNPs and GNPs@PEI system, the number of FCPNPs doesn’t change evidently, which is consistent with the fluorescence spectroscopic measurements in bulk solution (Figure 3b). Likewise, due to the FRET effect, the number of FCPNPs-GSH is also few on the glass slide surface in Figure 3c. Nevertheless, in the presence of GST, an obvious increment of the fluorescent particle can be observed in Figure 3d. GSH on the surface of FCPNPs has stronger combination with GST than the electrostatic interaction with GNPs@PEI and the FRET process from FCPNPs-GSH to GNPs@PEI is blocked, resulting in the fluorescence recovery of FCPNPs-GSH. These results give clear evidence that the SPE measurements coincide with the ensemble fluorescence spectra response and can be employed for the sensitive target analysis. Quantitative Detection of GST Based on SPE Next, the accurate quantification of GST based on the SPE assay was carried out. When different concentrations of GST are introduced to the reaction solution, it is observed that the number of FCPNPs-GSH on the glass slide surface displays a sequential enhancement with growing concentrations of GST (Figure 4a). In order to quantify the target object accurately, a ratio was adopted to obtain the quantitative response instead of the absolute numbers, which can 12


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avoid the error from different batches of samples. The definition of the ratio is as follows: R =

(Ni ― N0)/N0, where N0 and Ni are the counted numbers of FCPNPs-GSH in the absence and presence of GST respectively. As shown in Figure 4b, when the concentrations of GST increase from 0 to 6000 ng/mL, the ratio gradually grows up until reaching a saturation. It was found that the ratio was linearly associated with the logarithm of GST concentrations ranging from 10 to 6000 ng/mL (Figure 4c). The linear regression equation is y = 0.83x ― 0.35 (R2 = 0.99) and the LOD is determined to be 1.03 ng/mL (3/slope) at single-particle level, which is 2 order of magnitude lower than the fluorescence spectroscopic detection in bulk solution. It is worth noting that the SPE method can improve the signal-to-noise ratio and cut down the LOD greatly in comparing with the previous reported methods in Table 2. Selectivity Performance of SPE for GST Detection In order to evaluate the selectivity of this SPE assay for GST detection, the interfering effect from different substances was investigated. As presented in Figure 5a, when each of substance was added to the mixture of FCPNPs-GSH and GNPs@PEI respectively, the number of fluorescent particles nearly kept constant, which was almost the same as that from the blank control. By contrast, there are many fluorescent particles on the glass slide surface in the presence of GST in Figure 5b. Furthermore, in the coexistence of other interference substance and GST, the number of fluorescent particles still increase significantly, indicating that these tested interfering substances don't affect the target GST activity and the selective recognition could be achieved. The concrete result is obtained in Figure 5c. Therefore, the FCPNPs-based SPE method possesses excellent selectivity and the resisting interference ability for GST assay. Detection of GST in Urine Sample 13



It has been reported that excessive GST in urine sample is associated with the appearance of kidney disease.9 To further elucidate the potential application of this bioassay platform, the real urine sample from a healthy volunteer was monitored based on the standard addition method. None of GST was found in diluted urine samples. As illustrated in Figure 6a, when different concentrations of GST were added to the simulated urine samples as that from the kidney injury patient, the number of fluorescent particles on the glass slide surface gradually grew up with the increase of spiked GST concentrations. The accurate quantitative relation between the (Ni ― N0)/ N0 and GST concentrations is presented in Figure 6b. The calibration curve (y = 0.82x ― 0.32, R2 = 0.99) for GST determination in urine samples is attained by plotting the ratio versus the logarithm of GST concentrations (Figure 6c). In addition, the spiked recovery efficiencies between 97.5 and 106.5% are acquired with RSD from 1.7 to 5.5 in Table 3, indicating the satisfactory accuracy of the proposed method. These results disclose that the developed SPE approach possesses promising capability for GST determination in complex biological media.

Conclusions In summary, we have rationally designed a new SPE method for GST determination based on the FRET effect between the FCPNPs-GSH and GNPs@PEI. The FRET is switched on from FCPNPs-GSH to GNPs@PEI, leading to the quenching of FCPNPs-GSH fluorescence and the decline of fluorescent particle counts. In virtue of the specific interaction between GSH and GST, the FRET was inhibited when GST was introduced, resulting in the fluorescence recovery of FCPNPs-GSH. Therefore, the GST concentration dependent fluorescent particle number can be monitored on the glass slide surface through TIRF microscope. By counting the number of 14


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FCPNPs-GSH probe, high sensitivity with LOD of 1.03 ng/mL and excellent selectivity can be achieved. Furthermore, the proposed SPE assay has been successfully applied for GST detection in real urine samples with a satisfactory recovery in the range of 97.5-106.5%. As a consequence, this label-free FCPNPs-based SPE assay offers an invaluable platform for various targets quantification in the future and holds great potential applicability under complex biological environments. AUTHOR INFORMATION Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Conflicts of Interest The authors declare no competing financial interest. Acknowledgements 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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Scheme 1. Schematic diagram of the light path for single-particle imaging and the principle of SPE assay for GST detection.

23



Figure 1. The TEM images of a) FCPNPs-GSH, b) GNPs@PEI, c) the mixture solution of FCPNPs-GSH and GNPs@PEI before and d) after binding with GST. The size distributions of e) FCPNPs-GSH and f) GNPs@PEI. g) The UV-vis absorption spectrum of GNPs@PEI (black line) and the fluorescence emission spectrum of FCPNPs-GSH (red line). h) Representative single-particle fluorescence image of FCPNPs-GSH with long exposure time. i) The time-dependent fluorescence tracks from individual particles as noted in panel h.

24


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Figure 2. a) Fluorescence intensity of FCPNPs-GSH in the presence of various concentrations of GST. b) Fluorescence spectra of FCPNPs-GSH in different concentrations of GNPs@PEI. c) Fluorescence quenching efficiency versus GNPs@PEI concentrations. d) Fluorescence spectra of FCPNPs-GSH and GNPs@PEI mixture at various concentrations of GST. e) The linear relationship between the (F ― F0)/F0 and the GST concentrations in solution. f) Control experiments for GST assay using FCPNPs. F and F0 are the fluorescence intensities in the presence and absence of GST, respectively.

25



Figure

3.

Representative

fluorescence

FCPNPs+GNPs@PEI+GST,

c)

images

Page 26 of 33

of

a)

FCPNPs+GNPs@PEI,

FCPNPs-GSH+GNPs@PEI

FCPNPs-GSH+GNPs@PEI+GST.

26


and

b) d)

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Figure 4. a) Fluorescence images of FCPNPs-GSH on the glass slide surface at various concentrations of GST. b) The relationship between the concentrations. c) The linear range for the quantitative assay.

27


(Ni ― N0)/N0 and the GST


Figure 5. a) Fluorescence images of FCPNPs-GSH with different interfering substances without GST. b) Fluorescence images of FCPNPs-GSH in the presence of GST and interfering substances. c) The specificity assay of GST detection. The concentration of GST is 6 g/mL, whereas the concentrations of relevant proteins (BSA, TRY, Cyt C, TRF, IgG, HRP and LYZ) and amino acids (Cys, Lys and Gly) are 50 g/mL, those of ions (K+, Na+, Ca2+, Mg2+ and Zn2+) are 1M.

28


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Figure 6. GST assay in urine samples. a) Fluorescence images of FCPNPs-GSH with various concentrations of GST spiked in urine samples. b) The relationship between the (Ni ― N0)/N0 and the GST concentrations. c) The calibration curve in urine samples.

29



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Table 1. Zeta potential measurements of the nanoparticles with different functionalizations. Name

FCPNPs

FCPNPs-GSH

GNPs@PEI

Zeta/mV

-22.4

-12.8

11.5

30


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Table 2. Comparison of the detection performance of the methods for GST assay. Method

Linear range

LOD

Ref.

Fluorescence

-

250 nM

15

2-100 nM

1.5 nM

17

0-10 nM

0.21 nM

19

0-12 g/mL (0-460 nM)

30 ng/mL (1.15 nM)

18

0.01-6 g/mL (0.4-230 nM)

1.03 ng/mL (0.04 nM)

this work

SPE

31



Page 32 of 33

Table 3. Recovery for GST detection in urine samples. Samples

Spiked

Detected

Recovery

RSD

(μg/mL)

(μg/mL)

(%)

(%)

1

0.02

0.02

100.0

5.5

2

0.40

0.39

97.5

4.3

3

2.00

2.13

106.5

1.7

32


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For Table of Contents Only

33


Single-Particle Enumeration-based Sensitive Glutathione S

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