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Non-invasive and Highly Selective Monitoring of Intracellular Glucose via Two-steps Recognition-based Nanokit Jianru Tang, Dandan Ma, Stevan Pecic, Caixia Huang, Jing Zheng, Jishan Li, and Ronghua Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01532 • Publication Date (Web): 14 Jul 2017 Downloaded from http://pubs.acs.org on July 18, 2017

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Analytical Chemistry

Non-invasive and Highly Selective Monitoring of Intracellular Glucose via Two-steps Recognition-based Nanokit

Jianru Tang†, Dandan Ma†, Stevan Pecic§, Caixia Huang†, Jing Zheng†,*, Jishan Li†, Ronghua Yang†,‡,*

†

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and

Chemical Engineering, Hunan University, Changsha, 410082, China; ‡School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha, 410082, China; §University Medical Center, Columbia University, New York, 10032, USA

*To whom correspondence should be addressed: E-mail: [email protected]; [email protected] Fax: +86-731-8882 2523

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ACS Paragon Plus Environment


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ABSTRACT: Accurate determination of intracellular glucose is very important for exploring its chemical and biological functions in metabolism events of living cells. In this paper, we developed a new non-invasive and highly selective nanokit for intracellular glucose monitoring via two steps recognition. The liposome-based nanokit coencapsulated the aptamer-functionalized gold nanoparticles (AuNPs) and the Shinkai’s receptor together. Upon the proposed nanokit was transfected into living cells, the Shinkai’s receptor could recognize glucose firstly and then changed its conformation to endow aptamers with binding and sensing properties which are not readily accessible otherwise. Then, the binary complexes formed by the intracellular glucose and the Shinkai’s receptor can in situ displace the complementary oligonucleotide of the aptamer on the surface of AuNPs. The fluorophore-labeled aptamer was away from the AuNPs and the fluorescent state switched from “off” to “on”. Through the secondary identification of aptamer, the selectivity of the Shinkai’s receptor could be greatly improved while the intracellular glucose level was assessed by fluorescence signal recovery of aptamer. In the follow-up application, the approach exhibits excellent selectivity and non-invasive for intracellular glucose monitoring under normoxia and hypoxia. To the best of our knowledge, this is the first time that combing advantages of organic receptors and nucleic acids and then realize highly selective monitoring of intracellular glucose via two-steps recognition. We except it will open up new possibilities to integrate devices for diagnosis of various metabolic diseases and insulin delivery.

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INTRODUCTION Intracellular glucose, as a vital bioactive substances which can induce oxidative stress and influence signal transduction, play a significant role in living cells.1-4 Specifically, the decrease of external glucose can cause the content of intracellular glucose reduced gradually and thus indicates rapid metabolism. In view of this importance, various strategies for detection of intracellular glucose have been proposed.5-12 Among these, enzyme and nonenzyme-based monitoring systems have attracted huge attention.13-18 More concretely, the former one is a kind of biological system which mainly related to the enzyme and enzymatic hydrolysate and among which the glucose oxidase (GOx) is a representative case. Though this approach is sensitive to glucose monitoring, hydrogen peroxide (H2O2), which was produced during the GOx-catalyzed oxidation of glucose sensing, could induce oxidative stress by means of biomolecular oxidation and thus resulting in cellular damage.19 The other one without involving enzyme mainly refers to designing and synthesizing organic small molecular probes.20-21 Compared with the enzymatic system, the one without enzyme possess many advantages including higher stability, lower cost and high noninvasive. Therefore, nonenzymatic-based strategy is used more and more widely in glucose sensing. Boronic acid and diol compounds which mainly based on the reversible and equilibrium-based complexation and can react with compounds containing 1,2-diols or 1,3-diols such as saccharides via a reversible ester formation with high affinity, have been successfully employed as molecular recognition elements among the nonenzymatic glucose systems for intracellular glucose monitoring.22-27 For instance, the Shinkai’s receptor is a highly desirable glucose sensor based on boronic acid proposed to overcome the natural preference of monoboronic acids for fructose over glucose.28-29 However, in spite of the promising application capability, insufficient selectivity make these boronic acid molecules-based strategies difficult to implement for glucose monitoring in a reliable and

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convenient way. Considering these deficiencies, a huge improvement room still exists for developing new strategy to meet the growing demand for highly selective glucose testing. Inspired by this, herein, we proposed a new liposome-based nanokit to realize high selective intracellular glucose monitoring via two steps recognition. As shown in Scheme 1, in two step recognition process, the Shinkai’s receptor could recognize glucose firstly and changed its conformation to endow aptamers with binding and sensing properties which are not readily accessible otherwise.30 Through the secondary recognition executed by aptamer, the selectivity of the Shinkai’s could be greatly improved.31 Specifically, the nanokit coencapsulated the fluorophore-labeled aptamer probe functionalized AuNPs (AuNP@ODs) and the Shinkai’s receptor together. Upon the proposed nanokit was transfected into living cells, the binary complexes formed by the intracellular glucose and the Shinkai’s receptor can in situ displace the complementary oligonucleotide of the aptamer. Thus, the fluorophore-labeled aptamer was away from the AuNPs and the fluorescent state of aptamer switched from “off” to “on”. The signal recovery of the fluorophore-labeled aptamer mainly depended on the intracellular glucose level, thus leading to a new strategy for in situ imaging and quantitative analysis of the cytoplasmic glucose level. Comparing with previously proposed methods, our strategy possess the following advantages. First and foremost, the recognition process of intracellular glucose is divided into two steps, and the introduction of aptamer can provide specific secondary glucose recognition which can greatly improve the selectivity of the Shinkai’s receptor. Then, the coencapsulating can endow simultaneously delivery of the Shinkai’s receptor and aptamer into living cells and thus ensure high efficiency of two steps glucose recognition. Additionally, the distance-dependent fluorescent quenching effect of AuNPs as well as the DNA protection function provided by liposome can provide an excellent platform for non-invasive intracellular glucose monitoring. To the best of our knowledge, this is the first time that

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combing advantages of organic receptors and nucleic acids for intracellular glucose imaging via on two steps recognition. We except that this proposed nanokit can be further applied to serving as a potential tool for diagnosis of various metabolic diseases and biomedicine research. EXPERIMENTAL SECTION Materials. All DNA sequences were purchased from Sangon Biotechnology Co. Ltd. (Shanghai, China) and the solution was prepared respectively with an 18 MΩ ultrapure water (sterile Millipore water). The concentration of the solution was calculated using UV-Vis absorption with reported sequence-specific absorption coefficients.32 Unless stated, all chemicals were used as prepared. Human cervical cancer cells (HeLa) were grown in RPMI medium supplemented with 12 % inactivated fetal bovine serum, 100 U/mL 1 % penicillin and kept in a humidified CO2 (5 %) incubator at 37°C. Preparation of AuNP@ODs. The 13 nm AuNPs were synthesized according to the previous report.33 Briefly, 100 mL, 0.01 % chloroauric acid (HAuCl4) was promoted to boiling, after that, 4.0 mL sodium citrate solution (1 %) was added quickly while stirring. Next, the color of the solution changed from pale yellow to wine red. Finally, the solution was kept boiling for extra 25 min and then cooled to 25 °C. The prepared AuNPs were stored at 4 °C for further use and the concentration was calculated by its extinction at 519 nm (ε =3.0×108 L mol-1 cm-1). The synthesized AuNPs were then modified with the capture probe (CP) to form AuNP@ODs on the basis of the reported methods.34 Briefly, the capture probe were incubated with the solution containing AuNPs for 12 h. Then, 2 M sodium chloride solution was added to the mixture solution drop by drop at every 6 h period while the final concentration was achieved to 0.25 M. Finally, the solution was centrifuged for 30 min (13000 g) and resuspended in 0.02 M phosphate buffer saline (0.125 M NaCl, pH 7.4) for three times. Then the capture probe-modified AuNPs were resuspended in 0.02 M PBS buffer. The conjugation of capture

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probe on the AuNPs surface was calculated by UV-Vis absorption measurement. The maximal absorption peak (approximately at 260 nm) of the supernatant, with free capture probe isolated from the AuNPs, was transformed to DNA molar concentrations by UV-Vis absorption. Finally, to acquire the mean number of oligonucleotides on one single particle, we divided the conjugated oligonucleotide concentration by the initial concentration of AuNPs. The concentration of the aptamer sequence on the AuNPs surface was measured by fluorescence experiment (fluorophore-labeled DNA). The supernatant’s maximal fluorescence peak, with free aptamer probes isolated from the particles, was transformed to molar concentrations of the fluorophore modifying on DNA by comparison to a standard linear calibration curve. Standard curves were measured with given concentrations of single-stranded DNA with fluorophore-labeled using uniform condition. Synthesis of the Shinkai’s Receptor. The synthetic route of the Shinkai’s receptor was shown

in

Figure

S1.30

1,1'-(anthracene-9,10-diyl)bis(N-methylmethanamine)

Anthracene-9,10-dicarbaldehyde (0.059 g, 0.25 mmol) was dissolved in methanol (7.5 mL) in an ice bath, followed by slow addition of 1 M methylamine in methanol (0.375 mL, 0.75 mmol). The reaction mixture was stirred for 12 h at 30 °C under an argon atmosphere. Then, the resulting mixture was cooled to 4 °C, sodium borohydride (0.048 g, 1.25 mmol) was added in one portion, and the reaction mixture was stirred for 6 h at room temperature. The resulting mixture was added into 5 mL of ice-water and the aqueous layer extracted with choroform. The combined organic extracts were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography eluting to

afford

the

title

compound

as

an

orange

powder

(0.052g,

79

%).

((((anthracene-9,10-diylbis(methylene))bis(methylazanediyl))bis(methylene))bis(2,1-phe nylene)) diboronic acid. Diamine from the previous step (0.05 g, 0.19 mmol) and 2-bromomethylphenylboronic acid pinacol ester (0.213 g, 0.75 mmol) were dissolved in 0.5

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mL of dimethyl formamide (DMF), followed by addition of N, N-diisopropylethylamine (0.10 mL, 0.565 mmol). The reaction mixture was stirred for 18 h followed by 2 h at 40 ℃. The reaction mixture was diluted with 5 mL water and the aqueous layer extracted with choroform. The organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. Yellow oil was obtained and purified by silica gel column chromatography eluting with 5 % methanol. The title compound was obtained as pale yellow powder (0.05 g, 50% yield). Preparation of the Shinkai’s Receptor-Encapsulated Liposomes. The Shinkai’s receptor infiltrated-lipid film was prepared by mixing solution of cholesterol, boronic acid and soy bean phospholipid (molar ratio of 8:1:10). The film was rehydrated at 70 °C in a 10 mM PBS buffer, pH 7, and sonicated for 6 min to form the Shinkai’s receptor-encapsulated liposomes. Then, the formed Shinkai’s receptor-encapsulated liposomes were repeated filtrated through 3000 KDa filters (Millipore) to remove free Shinkai’s receptor and resuspended it in 10 mM PBS. The concentration of the encapsulated Shinkai’s receptor was calculated by comparison to a standard linear calibration curve. Standard curves were measured via fluorescence intensity of given concentrations of the Shinkai’s receptor. Glucose Sensing by Fluorescence Detection. All measurements were performed in HEPES buffer (200 mM Na+, 5 mM Mg2+, 5 mM K+). The resulting mixtures were incubated for at least 40 minutes to establish equilibrium. The attained AuNPs@ODs was dissolved in HEPES buffer, then 50 µM Shinkai’s receptor and different concentration of glucose were added and then incubated for 60 min at room temperature. The fluorescence spectra were obtained (570 to 680 nm) by use of the maximal excitation wavelength at 560 nm. MTT Assay. The HeLa cells (1×106 cells/well) were placed in replicate 96-well microtiterplates to a final volume of 120 µL. After two days culture, the original medium was taken out. The HeLa cells were incubated with AuNPs@ODs, the Shinkai’s receptor and our constructed nanokit-encapsulated liposomes for 24 h. Subsequently, 100 µL of MTT

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(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (0.5 mg/mL in PBS buffer) was added to each well. After 4 h, the remaining MTT solution was taken out. To dissolve the formazan crystals, 120 µL of DMSO (dimethyl sulphoxide) was added to every well, then the assay plate was shaken for 5 min. The absorbance was measured at 490 nm with a RT 6000 microplate reader. RESULTS AND DISCUSSION Characterization of AuNPs, AuNP@ODs and liposome-based nanokit. The transmission electron microscopy (TEM) image of the prepared AuNPs demonstrated good distribution with an average diameter around 13 nm (Figure S2), which was further confirmed by dynamic light scattering (DLS) experiment (13.8 nm). The successful conjugation of capture sequence (CP, DNA sequences ware shown in Table S1) to AuNPs was confirmed by UV-vis spectra and zeta-potential analysis (-28 to -35 mV). As shown in Figure 1A, the UV-vis spectrum demonstrated that the characteristic peaks of AuNPs at 519 nm. Upon the conjugation between CP and AuNPs via Au-S bond, the AuNPs showed a small red shift comparing with bare AuNPs (522 nm). Based on the previous reported method, each AuNP was estimated to carry about 76 thiol-labeled CP.35 Next, the aptamer sequence specifically targeting glucose was added to the solution containing CP-conjugated AuNPs to fabricate AuNP@ODs. It worth noting that, for the aptamer sequence (all DNA sequence was shown in Table S1), the 5′-end of the aptamer probe was labeled with tetramethylrhodamine (TAMRA). As shown in Figure S3, upon the formation of the AuNP@ODs, the dye labeled on the terminal of the aptamer probe was quenched by AuNPs. Then, we successfully synthetized the Shinkai’s receptor according to the previous report,32 the characterization results was shown in Figure S4 and Figure S5. Remarkably, the characteristic emission wavelength of the Shinkai’s receptor centered around 425 nm, which did not overlapped with TAMRA and indicated non-interference signal output between these

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two. Next, the synthetic Shinkai’s receptor-encapsulated liposomes were also characterized using DLS, TEM and fluorescence spectrum. In DLS measurements, the liposomes demonstrated a uniform size distribution of average diameter about 51±8.4 nm (Figure 1B). The negative staining TEM images further confirmed globular liposomal structure of ca.55 nm (Figure 1C, Inset) and we could observe clear lipid bilayer. Additionally, as for the fluorescence spectrum of the synthetic Shinkai’s receptor-encapsulated liposomes, the characteristic emission peak of the liposome was demonstrated in Figure S6 which indicated the successful infiltration of the Shinkai’s receptor. Next,

two

solutions

which

containing

AuNP@ODs

and

the

Shinkai’s

receptor-encapsulated liposomes mixed and incubated together for 30 min, the AuNP@ODs and Shinkai’s receptor coencapsulated nanokit was then fabricated. After centrifugation at 5000 rpm for 10 min, a process which has since been optimized to attain the best separation effect (data not shown), the formed nanokit was also further characterized by DLS and TEM. Comparing with the blank liposome, the size distribution of mean diameter of nanokit located at 105.71±17.9 nm, which was obviously greater than the Shinkai’s receptor coencapsulated liposome (Figure 1B). In negative staining TEM image (Figure 1C), we could observe AuNPs@ODs distribution inside the lipid bilayer. We further used atomic force microscopy (AFM) images to characterize the surface features of the formed liposome-based nanokit, which exhibit a mean height between 58.76 and 82.19 nm, being largely higher than that of the free Shinkai’s receptor coencapsulated liposme and bare AuNPs (Figure 1D, Figure S7). All these collectively result confirmed that the nanokit was successfully fabricated and could be used for further application. Meanwhile, we estimated one nanokit can capsulate about ten AuNPs@ODs from DLS and AFM quantitative results. Glucose Response of AuNP@ODs Complexes with the Shinkai’s Receptor in Solution and Cell Lysate. To assess the glucose-responsive signal enhancement of AuNP@ODs with

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aid of the Shinkai’s receptor, the fluorescence intensity of the AuNP@ODs upon glucose addition with aid of the Shinkai’s receptor was observed after incubation for 1 h. The fluorescence signal demonstrated an obvious enhancement upon addition of 2 mM glucose while we could not observe obvious change without the Shinkai’s receptor or glucose addition (Figure 2A). This is mainly attributed to the conformation transition of AuNP@ODs which could be explained as the specific binding between the complex formed by glucose and the Shinkai’s receptor, and the aptamer sequence. It is worth noting that this specific binding would lead the aptamer away from AuNPs and thus result in fluorescence recovery. Then, experimental parameters, including the concentration ratio between the Shinkai’s receptor and AuNP@ODs, were optimized to attain ideal fluorescence recovery of the AuNP@ODs (Figure S8). Under optimal experimental conditions, the fluorescence spectra of AuNP@ODs upon addition of different concentrations of glucose with aid of the Shinkai’s receptor was recorded. As shown in Figure S9, we could not observe strong background fluorescence before the addition of glucose which was mainly attributed to the strong quench effect of AuNP. Upon glucose addition, glucose can complex with the Shinkai’s receptor and the complex substance could then disassemble the hybridization of AuNP@ODs followed by the signal recovery of TAMRA. The enhancement of fluorescence intensity was proportional to the addition of glucose concentration ranged from 0 to 2 mM. The calibration curve of fluorescence signal enhancement (F/F0, where F and F0 represents the fluorescence intensity of TAMRA after and before glucose addition) versus concentration of glucose was attained (inset in Figure 2A) with a linear coefficient R=0.9862. The limit of detection was calculated to be about 33 nM which was comparable with the previous report.36 Excellent selectivity of the AuNP@ODs complexes with Shinkai’s receptor is essential to implement the glucose bioanalysis since most boronic acid-based glucose sensor is not satisfactory in this respect. Focusing on this, similar interfering substance including

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monosaccharides (fructose, galactose, maltose, etc.) and other biomolecules (amino acids, proteins, metal ion, Cholesterol ,etc.) that present in living cells or cell culture were employed to evaluate the selectivity of our proposed system. We next investigated the fluorescence signal responses of 1 mM glucose and interferential biomolecules with different concentrations (Figure S10). As we could see from Figure 2B, as for the most common interfering substances, such as GSH, negligible signal enhancement could be attained even at high concentration. Most importantly, as for these similar carbohydrate such as fructose and galactose, significant difference of TAMRA signal response could be observed (Figure 2B). On the contrary, we also investigated the response of the Shinkai’s receptor upon addition of monosaccharide which including fructose and galactose, the results demonstrated that obvious F/F0 could all be attained as for fructose or galactose addition (Figure 2B, Figure S11). All these results strongly confirmed the improved specificity of our proposed strategy. The AuNP@ODs complex with the Shinkai’s receptor was then used to determinate the glucose level in cell lysate. Considering the linear response range of our proposed strategy, the attained cell lysate samples were diluted 100 times with PBS buffer to ensure appropriate the concentration of glucose. The concentration of glucose in the cell lysate was calculated by the standard addition method according to the previous report and the results are shown in Table S2. The content of glucose in HeLa cells was calculated to be 16.9 fmol/cell, which was consistent with the previous report.36 All these results demonstrate the feasibility and veracity of our proposed AuNP@ODs complex for glucose testing in practical applications. Imaging of Glucose in Living Cells. To assess the biocompatibility of the proposed system, we firstly conducted a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay in HeLa cells. The absorbance of MTT at 490 nm is correlated with the viability of HeLa cells. The results showed that the AuNP@ODs, the Shinkai’s receptor and our proposed liposome-based nanokit demonstrated almost no cytotoxicity or side-effects in living cells

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within 12 h when the appropriate concentration of AuNP@ODs, the Shinkai’s receptor and liposome were added (Figure 3A). This result inspired that the nanokit hold potential for intracellular glucose imaging. In order to efficiently deliver the AuNP@ODs and the Shinkai’s receptor into cells, we constructed the liposome-based nanokit and the details of synthesis and characterization have described above. The obtained nanokit could be considered as “a smart vesicle” for monitoring the intracellular glucose level by fluorescence imaging. We firstly investigated the performance of nanokit on glucose detection in buffer solution. Since the AuNPs@OD could not exude from the nanokit because of the lipid bilayer, Triton-100 was added the nanokit solution to destroy the phospholipid bilayer structure of liposomes. 37 Then, the fluorescence spectra of nanokit upon addition of different concentrations of glucose was recorded. As shown in Figure S12, the result was similar with Figure 2A which confirmed the good performance of the nanokit on glucose detection. Next, we optimized the concentration of nanokit on living cell imaging and the results was shown in Figure S13. Under the optimum conditions, the dynamic change of the TAMRA fluorescence was monitored to confirm the feasibility of our proposed nanokit for in situ imaging of intracellular glucose imaging. After the nanokit absorbed onto the cell surface, liposome can fuse with the cell membrane and then release their contents into the cell cytoplasm.

38

As shown in Figure 3B, negligible

fluorescence signal was observed within 0.5 h. It is worth noting that the fluorescence intensity enhanced with the extension of incubation time (0.5 h to 4 h), which confirmed the distribution of glucose in the cytoplasm. In order to highlight the high intracellular delivery efficiency of liposome, a control experiment was conducted by employing AuNP@ODs and the Shinkai’s receptor, respectively (Figure 3C). Without co-encapsulation of liposome, we could not observe obvious fluorescence signal within a short period of incubation time even after 2 h which was largely due to the lower internalization efficiency of AuNP@ODs and the

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Shinkai’s receptor compared with the smart nanokit. To further confirm the highly selectivity of our proposed nanokit for intracellular glucose sensing, the Shinkai’s receptor alone was employed as control while the HeLa cells were chose as model cells. After cultured for about 24 h, the cells were harvested and equally divided into 4 new groups containing glucose-free fresh cell medium for 12 h. Within that 2 h, the intracellular glucose was regarded as completely metabolized. Then, the 4 new group was treated with blank RPMI medium, glucose-, lactose-, galactose-contained RMPI medium, respectively. Among which, glucose could be transported into the HeLa cells through glucose transporters, and the lactose would be transported into HeLa cells and then metabolized to galactose.39-40 Subsequently, as for these 4 groups, each group was divided into 2 new dishes again and incubated with our constructed nanokit or the only Shinkai’s receptor, respectively. As seen from Figure 4A, obvious fluorescence recovery of glucose-treated Hela cells could be observed upon the nanokit addition. As for the lactose and galactose-treated Hela cells, almost no fluorescence signal could be observed upon the nanokit addition (Figure 4B). On the contrary, as for the Shinkai’s receptor-treated group, obvious fluorescence signal of monosaccharide (including glucose, lactose and galactose)-treated HeLa cells were demonstrated upon the only Shinkai’s receptor addition (Figure 4C, 4D). It is worth noting that there was almost no difference between these 3 dishes which indicated the unsatisfactory specificity of the Shinkai’s receptor for intracellular glucose imaging. Imaging of Glucose Consumption in Living Cells under Normoxic and Hypoxia Conditions. To assess the practical application of our proposed nanokit, we next investigated the fluorescence monitoring of glucose consumption in HeLa cells. Specifically, the HeLa cells were initially cultured in glucose-contained RPMI 1640 medium for about 48 h and then incubated with glucose-free RPMI 1640 medium for 0, 4, 8, 12 and 24 h. It is worth noting that we incubated HeLa cells with 0.5 % CTAB to enhance the membrane permeability. Next,

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the nanokit was added into the glucose-free culture medium and incubated with HeLa cells for about 30 min, and then we used the confocal imaging to record the average fluorescence intensity of cells. In Figure 5A, as the very beginning (the time point when the HeLa cells just be treated by glucose-free medium represented as 0 h), HeLa cells demonstrated bright fluorescence of TAMRA which indicated high intracellular glucose level. As time went on, as the HeLa cell treated with glucose-free culture for 0 to 24 h, the fluorescence signal became weak which may be explained by the fact that the absence of external glucose would influence the glucose uptake normally and thus the concentration of glucose was reduced by the continuous metabolism in cells. The observable fluorescence in Figure 5B (in which the HeLa cells were treated with glucose-free medium for more than 12 h) shown that our proposed nanokit could monitor the dynamic change of intracellular glucose concentration even at low level under normoxic. Considering the complex mechanism of glycolysis under hypoxic conditions(Figure 5C), our proposed nanokit was further used for in situ monitoring the intracellular glucose level under different oxygen levels in view of the ability of the nanokit. Hypoxia is of high probability to induce the excessive glycolytic activity, and thus result in reduction of glucose level.39-40 Therefore, an attempt was made to testify our proposed nanokit’s ability in mapping the fluctuation of intracellular glucose induced by hypoxia. Specifically, HeLa cells were cultured in glucose-contained RPMI medium and kept under different hypoxia conditions for 2 h. Then, the nanokit was added and prepared for confocal imaging. The results were satisfactory and shown in Figure 5D. As can be seen, the fluorescence signal of HeLa cells under hypoxia was apparently weaker than that under normoxia (Figure 5D). Meanwhile, with the passage of incubation time under hypoxia (10 %), fluorescence intensity became weaker (8 h vs 4 h, Figure S14). This suggested that the intracellular glucose level was downregulated with hypoxia stress and there is positive correlation between the glucose level and hypoxia

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aggravation. To consolidate the conclusion, 2-deoxy-D-glucose (2-DG) was employed in our experiment.

2-DG

is

a

glucose

analogue,

which

can

inhibit

glycolysis

since

glucose-6-phosphate inhibits phosphoglucose isomerase.41 The results demonstrated that the nanokit could be successfully applied to estimation of glucose fluctuations associated with different stimuli under hypoxia, and also confirmed that the glucose fluctuations is caused by intracellular glycolytic activity which has a potential application for studying the pathway of cell metabolism under hypoxia. CONCLUSIONS In conclusion, we have proposed a unique intracellular glucose monitoring nanokit via two steps recognition based on the Shinkai’s receptor and aptamer. The nanokit coencapsulated the aptamer-functionalized gold nanoparticles and the Shinkai’s receptor together. Upon the proposed nanokit was transfected into living cells, the releasing Shinkai’s receptor could complex with glucose and changed its conformation which demonstrate a different spatially arrangement of epitopes to aptamers. Through the secondary identification functionalized aptamer, the selectivity of the Shinkai’s could be greatly improved. Besides the high biocompatibility and cellular uptake ability of liposome, the approach demonstrates excellent selectivity toward glucose over other monosaccharides present in living cells. In a follow-up application, dynamic imaging of intracellular glucose consumption under normoxia and hypoxia was successfully achieved in human cervical cancer cells. We except that this proposed nanokit can be further applied to serving as a potential tool for diagnosis of various metabolic diseases and biomedicine research. Acknowledgment. This work is supported by the National Natural Science Foundation of China (21405038, 21575018, 21475036), the Fundamental Research Funds for the Central Universities, China Postdoctoral Science Foundation funded project and the Hunan Provincial Natural Science Foundation (2016JJ1005).

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Supporting Information Available: Instruments, DNA sequences and the synthesis and characterization of the Shinkai’s receptor. The spectroscopic data also shown in supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure Captions Scheme 1. (A) Two-steps recognition of glucose based on the Shinkai’s receptors and the aptamers-functionalized gold nanoparticles. (B) Non-invasive and high selective monitoring of intracellular glucose via a smart nanokit based on gold nanoparticles-functionalized aptamers and the Shinkai’s receptor- coencapsulated liposome. Figure 1. (A) Absorption spectra of AuNPs before (black curve) and after (red curve) the capture probe conjugated. Green curve represented the capture probe before conjugated the AuNPs. (B) Hydrodynamic size distribution characterized by DLS of AuNPs (blue columns), the synthetic Shinkai’s receptor-encapsulated liposomes (purple columns) and the fabricated nanokit (bluish green columns). (C) Representative TEM image of the fabricated nanokit. Inset: Representative TEM image of the synthetic Shinkai’s receptor-encapsulated liposome. (D) Topography AFM images of the fabricated nanokit (the above image) and the corresponding height profiles of the fabricated nanokit. Figure 2. (A) Fluorescence spectra of the AuNP@ODs (10 nM) in the presence of 2 mM glucose with (red curve) and without (green curve) aid of the Shinkai’s receptor (50 µM). Pink curve represent the fluorescence spectra of the AuNP@ODs in the absence of glucose with aid of the Shinkai’s receptor while the black cure represent the fluorescence spectra of the bare AuNP@ODs. Inset: The calibration curve of fluorescence signal enhancement (F/F0, where F and F0 represents the fluorescence intensity of TAMRA after and before glucose addition) versus concentration of glucose. (B) Selectivity of AuNP@ODs for glucose over other monosaccharides (fructose, galactose, etc.) at a concentration of 1 mM, biological species at a concentration of 10 mM with aid of the Shinkai’s receptor (50 µM). The error bars signify the standard error obtained from three repetitive measurements. Inset: the response of the AuNP@ODs with aid of the Shinkai’s receptor (the first three columns, F/F0, where F and F0 represents the fluorescence intensity of TAMRA after and before glucose

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addition) and the Shinkai’s receptor (50 µM) alone upon addition of at a concentration of 1 mM monosaccharide (the three columns behind, F/F0, SR represent the Shinkai’s receptor, where F and F0 represents the fluorescence intensity of the Shinkai’s receptor after and before glucose addition) which including fructose and galactose, glucose. Figure 3. (A) Cell relative viability of HeLa cells after treated with AuNPs@ODs (grass green bars), Shinkai’s receptor (blue bars) and the fabricated nanokit (purple bar). 1-7 represent the concentration of AuNPs@ODs:0.5, 1, 1.5, 2, 2.5, 3, 3.5 nM respectively , while represent the concentration of the Shinkai’s receptor: 10, 20, 30, 40, 50, 60, 70 µM, and the fabricated the nanokit: 0.5, 1, 1.5, 2, 2.5, 3, 3.5 nM (The concentration of nanokit was defined by AuNP@ODs). (B) Confocal microscopy images of HeLa cells which were cultured in glucose-contained RPMI medium for 24 h. After 24 h, all HeLa cells were washed with PBS buffer for three times and then treated with the fabricated nanokit (2 nM) for different time (0.5 h, 1 h, 2 h, 4 h). Scale bar: 100 µm. (C) Confocal microscopy images of HeLa cells which were cultured in glucose-contained RPMI medium for 24 h, respectively. After 24 h, all HeLa cells were washed with PBS buffer for three times and then treated with the Shinkai’s receptor (30 µM) and AuNP@ODs (2 nM) separately for different time (0.5 h, 1 h, 2 h, 4 h). Scale bar: 100 µm. (D) Histogram of the relative TAMRA fluorescence intensity which attained from the above nanokit-treated HeLa cells (the purple columns), the Shinkai’s receptor and AuNP@ODs separately treated-HeLa cells (the bluish green columns) as function of time (0.5 h, 1 h, 2 h, 4 h). The maximum fluorescence intensity of TAMRA was normalized to 100. Figure 4. Selective intracellular imaging of monosaccharide under CLSM. (A) Confocal imaging of HeLa cells which were pre-treated with blank RPMI medium, glucose-, lactose-, galactose-contained RMPI medium (10 mM), respectively, and then incubated with the fabricated nanokit (2 nM). Scale bars: 100 µm. (B) Histogram of the relative fluorescence intensity of the above HeLa cells (A). The intracellular monosaccharide were recorded by

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TAMRA with 568 nm excitation. The maximum fluorescence intensity of TAMRA was normalized to 100. (C) Confocal imaging of HeLa cells which were pre-treated blank RPMI medium, glucose-, lactose-, galactose-contained RMPI medium, the concentration was 10 mM respectively, and then incubated with the Shinkai’s receptor (30 µM) for 1.5 h at 37 °C. Scale bars: 100 µm. (D) Histogram of the relative fluorescence intensity of the above HeLa cells(C). The intracellular monosaccharide were recorded by Shinkai’s receptor with 405 nm excitation. The maximum fluorescence intensity of the Shinkai’s receptor was normalized to 100. Figure 5. (A) Dynamic monitoring intracellular glucose consumption under normoxia as functions of time. The intracellular carbohydrate were recorded by TAMRA with 568 nm. (B) Histogram of the relative fluorescence intensity of the above HeLa cells (A). The maximum fluorescence intensity of TAMRA was normalized to 100. (C)Schematic representation of the mechanisms of glucose consumption under hypoxia. (D) Fluorescence images of glucose in HeLa cells under different oxygen concentration (20%, 10%, 5%, 1% O2) and upon 2-DG addition (5 mM). The concentration of the added nanokit was 2 nM. Scale bars: 100 µm.

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Scheme 1

A

B

23



Figure 1 2.0

A Number percence

1.5 1.0 0.5

B

24 18 12

0.0

6 0

3 00 4 00 5 00 6 00 W avelength/n m

700

0

50

100 S ize/nm

DD D

C

150

200 53.2 nm

-22.8 nm 80 60 40 20

nm

Absorbance

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Figure 2

2 .4

2 .4

A

3 .0

3 .0

B

1 .2

1 .5

0

Gal

2 .0 1 .5

1 .6

1 .0

1 .0

500 1000 1500 2000 G lu c o se /µ M

A uN P @ O D s+S R

S R o n ly

1 .2

600 630 660 W a ve le n g th /n m

Gl u Ga l Fru ma M n n al os a ll e os e GS H BS A T y lysin r ch os e ole ine s te ro l C a 2+ Z n 2+ Al 3 + b la nk

0 .6 570

F/F0

2 .0

F/F0

1 .8

2 .0

G lu F ru

2 .5

2 .5 F/F 0

5 Fluorescence intensity ( x10 )

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


690

25



Figure 3 0.5 h

A

100

TAMRA

2h

4h

75 50 25

Overlay

0

1 0.5 h

TAMRA

C

1h

B

2

3

4 1h

5

6 2h

B

7 4h

Normalized Fluorescence Intensity

Cell viability (%)

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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C

Overlay

100

D

75 50 25 0 0 .5 h

1h

2h

T im e

26


4h

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Blank

+Glu

+Fru

+Gal

A TAMRA

Overlay

Blank

C SR

Overlay

+Glu

+Fru

+Gal


Figure 4


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100

B

75 50 25 0

100

b la n k

G lu

F ru

G al

G lu

F ru

G al

D

75 50 25 0

b la n k

27



Figure 5 0h

4h

8h

12 h

24h

A TAMRA

Overlay


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

B

75 50 25 0

0h

4h

8h

12h

24h

T im e

Glucose 20 % O2

20 % O2

10 % O2

5 % O2

1% O2

D

C TAMRA Glycolysis Free Glucose

Overlay CO2 1% O2

H2O

ATP ATP ATP ATP

Glucose

28


Low

1 % O2+2-DG

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For TOC Only

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Noninvasive and Highly Selective Monitoring of ... - ACS Publications

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