Dual-Response Detection of Oxidized Glutathione, Ascorbic Acid, and Cell Imaging Based on pH/Redox Dual-Sensitive Fluorescent Carbon Dots.

The pH/redox dual-sensitive fluorescent carbon dots (pHRCDs) with the fluorescence quantum yield of 16.97% were synthesized by the pyrolysis of l-glutamic acid (l-glu) and dopamine (DA). Compared with the quantum dot (QD)-dopamine conjugate, when the pH value of the solution was changed from neutral to alkaline, the pHRCDs exhibited unique optical phenomenon including red-shift of fluorescence peak and the fluorescence intensity first decreasing from pH 7 to 10 and then increasing from pH 10 to 13. The pHRCDs could be developed for a discriminative and highly sensitive dual-response fluorescent probe for the detection of oxidized glutathione (GSSG) and ascorbic acid (AA) activity in human blood. Under the optimized experimental conditions, the dual-response fluorescent probe can detect GSSG and AA in the linear range of 1.2-3.6 and 27-35 μM with the detection limits of 0.1 and 3.1 μM, respectively. In addition, the pHRCDs demonstrated low cytotoxicity and good biocompatibility, which can be well applied to in vitro cell imaging, and the pHRCDs/GSH fluorescence system has been successfully developed for the detection of AA in real samples.

Introduction

Biologically active molecules such as ascorbic acid (AA) and oxidized glutathione (GSSG) coexist in the serum and central nervous system and play a crucial role in human metabolic processes.[1,2] Glutathione (GSH), a tripeptide formed by cysteine, glutamic acid, and glycine, is the most important tripeptide thiol found in the human cell system and can be readily oxidized into its dimeric form in response to oxidative stress within cells.[3,4] The high concentration of GSSG is associated with asthma, human immunodeficiency virus type 1 infection, and chronicle renal failure.[5] Therefore, the change in GSH/GSSG ratio has become a key biomarker in monitoring the overall health of the cells and their resistance to oxidative damage. Ascorbic acid, a natural antioxidant, is one of the most important neuromodulators in the central nervous system.[6] It is often present in the entire physiological system as an anion and plays a key role in various physiological/pathological processes. Because of its reducing property, AA can remove free radicals generated by endogeneity and prevent cellular damage induced by free radicals and provide protection against diseases that involve oxidative stress, such as enzymatic reactions, ischemic stroke, immune system enhancement, olfactory dysfunctions, and so forth.[7,8] Considering the important roles of GSSG and AA in our daily life, it is critical to determine the concentration of GSSG and AA in the body.

In the past decades, many valuable methods such as colorimetry,[9−11] electrochemical methods,[12] liquid chromatography,[13,14] mass spectrometry,[15] and fluorescence methods[16,17] have been successfully developed for the detection of GSSG and AA. For example, Zhang and co-workers designed a method for detecting intracellular nitroxyl and GSH–GSSG based on double-site fluorescent probe NCF.[16] Shan and co-workers demonstrated the detection of cadmium(II) ions and AA based on dumbbell-shaped CQDs/AuNCs nanomixtures as an effective ratio of fluorescent probes.[17] However, most of these methods show some limitations, such as complicated processing, high cost of instruments, and time-consuming operations.[18] Carbon dots (CDs), as a new type of emerging materials, are superior to the traditional semiconductor quantum dots because of their excellent optical properties, such as biocompatibility, anti-photodegradation, and low toxicity,[19,20] and have been used in various applications such as in vitro and in vivo imaging,[21,22] disease diagnosis,[23] drug carrier,[24,25] fluorescent probe,[26] and so on.

Herein, a simple, rapid, and environmentally friendly preparation route was designed to prepare pHRCDs using DA and l-glu as raw materials, wherein l-glu is taken as a carbon source and DA provides catechol hydroxyl functional groups for pHRCDs to achieve pH/redox dual-sensitive properties. As shown in Scheme , in the presence of GSSG, catechol on the surface of pHRCDs could be oxidized by GSSG, and simultaneously generated GSH was grafted onto the surface of nonoxidized pHRCDs by the Michael addition reaction, which can significantly lead to fluorescence intensity enhancement of pHRCDs and appearance of a new fluorescence peak. When AA was added into the system, it was found that the fluorescence intensity of the pHRCDs/GSH fluorescence system gradually decreased with the increase of AA content, which may be due to the Maillard reaction between AA and GSH to produce various aromatic compounds, resulting in the fluorescence quenching of the pHRCDs/GSH fluorescence system. The above-mentioned results demonstrate that the pHRCDs/GSH fluorescence system possesses resplendent potential for application in the detection of AA in real samples.

Scheme 1

Schematic Diagram for Detecting GSSG and AA Content Based on pHRCDs and pHRCDs/GSH Fluorescence System

Experimental Section

Materials

l-Glutamic acid (l-glu), l-histidine (l-his), l-phenylalanine (l-phe), l-leucine (l-leu), and l-tryptophan (l-try) were purchased from Tianjin Institute of Fine Chemical for Guangfu (Tianjin, China). AA and Tris(hydroxymethyl)-aminomethane (Tris) were purchased from Aladdin Reagent Co., Ltd. Oxidized glutathione (GSSG) was purchased from Shanghai Shifeng Biotechnology Co., Ltd (Shanghai, China). Dopamine (DA) hydrochloride, phenethylamine, and glutathione (GSH) were purchased from Adamas Co., Ltd. Dialysis membrane (MWCO 500 Da) was purchased from Shanghai Sanggong Biotechnology Co., Ltd (Shanghai, China). Unless stated otherwise, all the chemical and biological reagents were procured from commercial sources and used without further purification.

Instruments

The morphology of pHRCDs was investigated using a JEM-2100Plus transmission electron microscopy (TEM) at 200 kV. X-ray photoelectron spectroscopy (XPS) spectra were recorded by an ESCALAB 250 surface analysis system. Fourier transform infrared (FT-IR) spectra were recorded on a Bruker Optics VERTEX 70 spectrometer. The fluorescence and absorption spectra were measured with an F-7000 fluorescence spectrophotometer and a UV-9100A spectrometer, respectively. Also, all pH measurements were obtained by a PHS-3W pH meter.

Preparation of pHRCDs

l-Glu (0.09 g) and DA hydrochloride (0.12 g) were added to a 50 mL three-necked flask, respectively. First, by heating to 230 °C under reflux for 15 min and then cooling to the room temperature, dark brown solid was obtained. Then, 10.0 mL of distilled water was added to the three-necked flask for ultrasonic treatment for 10 min and then the solution was magnetically stirred for 3 h. It is observed that the reaction mixture turned into a reddish-brown solution with black precipitation at the bottom, which is further centrifuged at 16 000 rpm for 20 min. Also, the clarified reddish-brown supernatant was collected and dialyzed against Millipore water (18.2 MΩ cm) via the dialysis membrane (MWCO 500). Finally, the resulting solution was stored at 4 °C for subsequent experiments.

Detection of GSSG by pHRCDs

For GSSG detection, different amounts of GSSG were added in a series of 1.5 mL of a solution containing 20 μL of pHRCDs and 1480 μL of Tris-HCl (10 mM, pH 7.0). Then, the solution was incubated at 25 °C for 10 min. The fluorescence spectra were recorded between 350 and 700 nm wavelength range at the excitation wavelength of 350 nm. The slit width of emission and excitation were set at 5 and 10 nm, respectively.

Detection of AA by pHRCDs/GSH Fluorescence System

For the assay of AA, different amounts of AA were added in a series of 1.5 mL of a solution containing 20 μL of pHRCDs, 1412 μL of Tris-HCl (10 mM, pH 7), and 68 μL of (0.1 mol/L) GSSG. Then, the solution was incubated at 25 °C for 10 min. The fluorescence spectra were recorded between 350 and 700 nm wavelength range at the excitation wavelength of 350 nm. The slit widths of emission and excitation were set at 5 and 10 nm, respectively.

Real Sample Assay

Aliquots of 100-fold diluted human serum with Tris-HCl (10 mM, pH 7.0) were mixed with 68 μL (0.1 mol/L) of GSSG and 20 μL of pHRCDs was added, Subsequently, different contents of AA were sequentially added to the above centrifugation tubes and diluted to 1.5 mL with Tris-HCl (10 mM, pH 7.0). Then, the mixtures were cultured in a constant temperature incubator at 25 °C for 10 min. Finally, the fluorescence spectra were collected at an excitation wavelength of 350 nm. The slit width of emission and excitation were set at 5 and 10 nm, respectively.

Cell Cytotoxicity of pHRCDs

Hela cells were inoculated into 96-well plates and cultured under 5% CO2 at 37 °C. Subsequently, different concentrations of pHRCDs (0–1000 μg/mL) were added to each well for 48 h and then 50 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (1 mg/mL) was added to each well for 4 h. The supernatant was removed and 100 μL of dimethyl sulfoxide (DMSO) was added to each well for 10 min. Finally, the absorbance was measured using a microplate reader (Bio-Rad, Hercules) at 490 nm.

Cell Imaging

To observe the influence of pHRCDs on cell imaging, Hela cells were inoculated on six-well plates and cultured under 5% CO2 at 37 °C for 24 h. Next, different concentrations of pHRCDs (100 and 500 μg/mL) were added to each well. After incubation for 4 h, the supernatant was removed by washing the cells with PBS and then adding 4% polyformaldehyde. Fluorescent images of living cells were acquired on an inverted fluorescence microscope (Leica DMI8, Germany).

Results and Discussion

Characterization of pHRCDs

The pHRCDs were prepared by high-temperature pyrolysis using l-glu and DA hydrochloride as raw material. The morphology and size of pHRCDs were characterized by means of TEM. As can be seen in Figure A and the inset, the average diameter of spherical particles was calculated to be 6.45 nm, with the relative narrow size distribution ranging from 5 to 9.5 nm, and lattice fringes with a spacing of 0.21 nm were clearly observed from the HRTEM image (Figure B), which are ascribed to the (100) diffraction facets of graphite.[27]

Figure 1

(A) TEM image and histogram of the size distribution of pHRCDs. (B) HRTEM image of pHRCDs.

The chemical structure information of pHRCDs was known from the FT-IR spectra. Compared with the FT-IR spectra of pure l-glu, the pHRCDs exhibit strong broad-band absorption at 1687 cm–1 (stretching vibration of C=O) (Figure S1), which is the characteristic performance of dehydration and carbonization. We further performed X-ray photoelectron spectroscopy (XPS) measurements to determine the doping content and chemical state of nitrogen and oxygen in the pHRCDs. The full XPS spectrum presented in Figure A shows three typical peaks: C 1s (283.6 eV), N 1s (401.6 eV), and O 1s (532.3 eV) giving contents of 67.02 atom % (C), 10.96 atom % (N), and 22.02 atom % (O), respectively. The N/C and O/C atomic ratios are 16.35 and 32.86%, respectively, indicating that the pHRCDs were highly doped with N and O. In the high-resolution C 1s spectrum (Figure B), the band can be deconvoluted into three peaks, corresponding to C–C/C=C (284.55 eV), C–N (285.64 eV), and C–OH (288.12 eV). The N 1s band can be deconvoluted into two peaks at 399.91 and 401.25 eV, representing pyrrole N (54.08 atom %) and graphitic N (45.92 atom %), respectively (Figure C). The O 1s band can be deconvolved into a peak at 531.9 eV, representing C–OH (Figure D). The emission peak of pHRCDs was red-shifted with the increase of excitation wavelength in the range of 320–410 nm. The pHRCDs exhibit obvious excitation-dependent fluorescence emission behavior, which was the hallmark nature of CDs.[28] The strongest emission peak was obtained at 350 nm excitation wavelength (Figure S2).

Figure 2

(A) Survey XPS spectrum. (B) High-resolution C 1s spectrum. (C) High-resolution N 1s spectrum. (D) High-resolution O 1s spectrum of pHRCDs.

Mechanism of Fluorescence Color Change of pHRCDs

Because DA is usually characterized by two inherent redox properties: a Nernstian dependence of formal potential on pH and oxidation of hydroquinone to quinone by O2 at basic pH, the synthesized CDs using DA as a raw material have usually pH-sensitive properties.[29,30] It has been reported that QD–dopamine conjugates exhibit pH-dependent quenching of photoluminescence,[31] which can be used as a pH sensor to detect changes in cytoplasmic pH when cells underwent drug-induced alkalosis. Hence, according to these previous reports, we infer that the pHRCDs would possess similar pH-dependent characteristics induced by redox reactions on DA.

We first studied the influence of pH of the medium on the fluorescence of pHRCDs in the open air. As shown in Figure S3, the color of pHRCDs solutions gradually change from colorless transparent to yellow under sunlight (up), and under irradiation of a UV lamp, the pHRCDs show a unique first blue-shifted and subsequently red-shifted optical phenomenon with increasing pH (down). It is further verified by their fluorescence spectra, as shown in Figure A, that the fluorescence emission peak first blue-shifted and subsequently red-shifted with the increase of pH value, which exhibited the pH-dependence of the pHRCDs over the pH range 1–10, consistent with the pH-dependence of QD–dopamine conjugates.[31,32] A slight difference is that the fluorescence intensity gradually increased when the pH exceeds 10, and no fluorescence quenching occurs under strong alkali conditions (Figure B). To explore the fluorescence conversion mechanism, CoOOH nanosheets were used as an oxidant to study the fluorescence changes of pHRCDs solutions. It is found that with the increase of the CoOOH content, the fluorescence intensity of pHRCDs gradually decreases and fluorescence quenching occurs. The main reason is that CoOOH oxidizes catechol hydroxyl on the surface of pHRCDs to o-quinone, resulting in fluorescence quenching of pHRCDs (Figure S4).[33] However, the CDs synthesized by phenethylamine and l-glu have no pH dependence (Figure S5), and the fluorescence color has no blue-shifted or red-shifted phenomenon with the change of pH of the medium (Figure S6).

Figure 3

(A) Fluorescence spectra and (B) the linear relationship between the fluorescence intensity of pHRCDs and pH value. (C) Absorption spectra of pHRCDs (inset: photographs of pHRCDs at different pH values under visible (up) and UV light (down)). (D) FT-IR spectra of pHRCDs.

At the same time, by analyzing the UV–vis absorption spectrum as shown in Figure C, four distinct absorption peaks at 223, 246, 278, and 342 nm may be attributed to the n−π* transition of sp2 aromatic conjugate domains, the n−π conjugation on phenol, the π–π conjugation on benzene ring, and the n−π* broadened absorption bands of C=O transitions at pH 3,[34,35] respectively. However, when the pH increased to 12, the p−π conjugation on phenol and the π–π conjugation on the benzene ring disappeared and a new absorption peak appeared at 256 nm. It may be due to the decomposition of hydrogen peroxide (H2O2) generated by oxygen at a strongly alkaline, resulting in the production of a large number of hydroxyl radicals (•OH), which can cause oxidative o-quinone cleavage (Figure ).[30,36] The composition and structure of the pHRCDs at different pH values were characterized by FT-IR spectroscopy. As shown in Figure D, the carboxyl group was clearly recognized through the very broad O–H stretching absorption at 3408 cm–1 and the C=O stretching vibration at 1672 cm–1, the characteristic peak of the benzene ring disappears when the pH value is 12, which further verifies the formation mechanism of carboxyl groups produced by the oxidative pyrolysis of o-quinone. Also, compared with the CDs synthesized using phenethylamine and l-glu as raw materials, it was found that the UV–vis absorption peak (Figure S7) and FT-IR spectrum (Figure S8) of the CDs did not change with the change of the pH value.

Figure 4

Conversion mechanism of the change of the pH value leading to the change of the fluorescence color of the pHRCDs.

Optimization of Experimental Condition

To optimize the detection conditions for GSSG and AA, we studied the effects of incubation time, pH, and temperature on the fluorescence intensity of pHRCDs. As shown in Figure B, when the pH was less than 7.0, the fluorescence intensity of pHRCDs gradually increased with the increase of pH. However, when the pH was between 7.0 and 10, the fluorescence intensity decreased linearly with the increase of pH. Therefore, pH 7.0 of the Tris-HCl buffer solution was used as the optimal experimental condition. The optimum quenching efficiency is 35.8% at 25 °C. Therefore, 25 °C was chosen as the best experimental condition for the pHRCDs/GSH fluorescence system (Figure S9). Moreover, the CDs have good optical stability (Figures S10 and S11).

Based on the above optimization, the effects of different biomolecules on the fluorescence intensity of pHRCDs were studied. Figure S12 shows the relationship between different biomolecules and fluorescence intensity of pHRCDs. It was found that AA had little effect on the fluorescence intensity of pHRCDs, and the fluorescence efficiencies of GSSG and GSH were 21.1 and 8.96%, respectively. However, the fluorescence quenching efficiencies of the simultaneous detection of AA/GSSG and AA/GSH were 13.4 and 21.2%, respectively. Therefore, it can be used to detect the content of GSSG and AA by fluorescence change.

Detection of GSSG by pHRCDs

Under the above-mentioned optimal conditions, the sensing performance of the pHRCDs system to GSSG was studied. As shown in Figure A, at 350 nm excitation wavelength, the emission peak of pHRCDs at 430 nm was gradually enhanced, and simultaneously a new emission peak appeared at 504 nm with the addition of GSSG. The dual-emission fluorescence intensity was gradually enhanced with the increase of GSSG concentration. As seen from the inset of Figure A, the fluorescent color changes from cyan to green with the increase of the GSSG content, and the inset of Figure B shows that there was a good linear relationship between ΔF (ΔF = F – F0, F and F0 were the fluorescence intensities of the sensing system in the presence and absence of GSSG, respectively) and the concentration of GSSG was in the range of 1.2–3.6 μM (the equation is y = 74.5032C – 54.0337, R2 = 0.9968) with a detection limit of 0.1 μM. Figure C illustration also shows a good linear relation in the range of 0–4 μM (the equation is y = 89.9290C + 7.6808, R2 = 0.9972), with a detection limit of 50 nM. The detection limit was based on the equation LOD = 3σ/s, where σ is the standard deviation of the corrected blank signals of the pHRCDs and s is the slope of the calibration curve. Therefore, the pHRCDs can perform a dual-response detection of GSSG in the range of 1.2–3.6 μM, with a detection limit of 0.1 μM. Compared with the detection methods of GSSG reported in different literatures (Table S1), these results show that the pHRCDs have great potential as a highly sensitive platform for GSSG sensing. It can be seen that the pHRCDs have better selectivity, as shown in Figure S13. In addition, AA was added to the pHRCDs/GSH fluorescence sensing system, which showed obvious fluorescence quenching. Therefore, the pHRCDs/GSH fluorescence sensing system can be further applied to the detection of AA.

Figure 5

(A) Fluorescence dual response of the pHRCDs upon the addition of different concentrations of GSSG at 350 nm excitation wavelength. Illustration: the addition of 68 μL (0.1 mol/L) GSSG results in a change in fluorescence color. (B) Plot of the fluorescence intensity against the GSSG concentration within the range of 0–6.8 μM. Inset: a linear correlation of ΔF value versus the concentration of GSSG over the range from 1.2 to 3.6 μM (the emission peak is located at 430 nm). (C) Plot of the fluorescence intensity against the GSSG concentration within the range of 0–6.8 μM. Inset: a linear correlation of ΔF value versus the concentration of GSSG over the range from 0 to 4.0 μM (the emission peak is located at 504 nm).

Detection of AA by pHRCDs/GSH Fluorescence System

Under optimal conditions, the sensing performance of the pHRCDs/GSH fluorescence system against AA was studied. As shown in Figure A, the fluorescence intensity of the pHRCDs/GSH fluorescence system gradually decreased with the increase of AA concentration, and it can be seen from the inset of Figure A that the fluorescence color quenched with the increase of the AA content. The main reason may be the Maillard reaction between AA and GSH in solution, which produces a variety of aromatic compounds and quenches the fluorescence of the pHRCDs/GSH fluorescence system.[37] Also, Figure B inset shows that there was a good linear relationship between ΔF (ΔF = F0 – F, F0 and F are the fluorescence intensities of the sensing system in the absence and presence of AA, respectively) and the concentration of AA was in the range of 27–47 μM (the equation is y = 3.3686C + 223.1288, R2 = 0.9955) with a detection limit of 3.1 μM, and Figure C also shows a good linear range of 12–35 μM (the equation is y = 6.5971C + 150.7681, R2 = 0.9926), with a detection limit of 0.51 μM. Therefore, the pHRCDs/GSH fluorescent probe can perform a dual-response detection of AA in the range of 27–35 μM, with a detection limit of 3.1 μM. Compared with the detection methods of AA reported in different literatures (Table S2), these results show that the pHRCDs/GSH fluorescence system has great potential as a highly sensitive platform for AA sensing.

Figure 6

(A) Fluorescence dual response of the pHRCDs/GSH fluorescence system upon the addition of different concentrations of AA at 350 nm excitation wavelength. Illustration: the addition of 69 μM AA results in a change in fluorescence color. (B) Plot of the fluorescence intensity against AA concentration within the range of 0–69 μM. Inset: a linear correlation of ΔF value versus the concentration of AA over the range from 27 to 47 μM (the emission peak is located at 430 nm). (C) Plot of the fluorescence intensity against AA concentration within the range of 0–69 μM. Inset: a linear correlation of ΔF value versus the concentration of AA over the range from 12 to 35 μM (the emission peak is located at 504 nm).

Selectivity Study

The selectivity of the developed pHRCDs/GSH fluorescent system was evaluated prior to the actual testing in serum. In detail, two times of different biomolecules, including GSH, AA, l-his, l-phe, l-val, l-leu, and l-try, were added to the pHRCDs/GSH fluorescent system, respectively. Also, fluorescence intensity ratios (ΔF/F0) of pHRCDs at 350 nm were recorded in the presence of different biomolecules. From Figure , it can be seen that the fluorescence intensity of the pHRCDs/GSH fluorescent system does not show an obvious change with the introduction of various interfering biomolecules. Furthermore, the response of the pHRCDs/GSH fluorescent system to AA was not disturbed by all of the interference. Only AA exhibits a noticeable response, indicating that pHRCDs/GSH have good selectivity to AA.

Figure 7

Selectivity of pHRCDs/GSH fluorescence system to AA in the presence of other interference.

Detection of AA in Serum

To evaluate the practicability of the process of detecting AA, the concentration of AA in bovine serum was detected. Based on the comparison between the results of three repeated tests and the content of AA added, as shown in Table , the recovery rate of the sample was between 97.8 and 103.7%, and the relative standard deviation is between 1.1 and 4.5%. The results in Table show that the detection method has good reliability, accuracy, and repeatability, indicating that the pHRCDs/GSH sensing system has great application potential. The results in Table S1 show that the detection method has good reliability, accuracy, and repeatability, indicating that the pHRCDs/GSH sensing system has great application potential.

Table 1

pHRCDs/GSH System Used to Detect AA with Different Contents in Serum

samples	added (μM)	measurement (μM)	recovery (%)	RSD (%, n = 3)
1	12	11.74	97.8	3.2
2	13	13.17	101.31	1.1
3	14	14.52	103.7	4.5
4	16	16.31	101.94	1.6

In Vitro Imaging Using pHRCDs

To study the cytotoxicity of pHRCDs on living cells, Hela cells were selected as a simulation system for detecting pHRCDs. By detecting the cell survival rate at different concentrations of pHRCDs, it was found that the cell survival rate was over 90% when the concentration of pHRCDs is as high as 1000 μg/mL (Figure S14). The higher cell survival rate proved that pHRCDs had low toxicity and significant biocompatibility. Then, the fluorescence of Hela cells at different concentrations was determined (Figure ), it can be clearly seen that cell imaging becomes more and more obvious with the increase of material content. The first column is bright-field image, the second and third columns show the image obtained by the green channel and the combined image of the first and second columns, respectively. These results indicate that the intensity of fluorescence image is closely related to the concentration of pHRCDs in the culture medium, which indicates that the probe has a wide range of applications in biological imaging and biomolecular detection.

Figure 8

Inverted fluorescence images of Hela cells incubated with different concentrations of pHRCDs at 37 °C for 4 h: (a) 100 μg/mL pHRCDs and (b) 500 μg/mL pHRCDs.

Conclusions

In summary, we developed a novel biocompatible pHRCDs based on DA, which could sensitively detect GSSG by one-excitation and dual-emission method. Also, AA was selectively detected based on the constructed pHRCDs/GSH fluorescent system. Under optimized experimental conditions, the dual-response fluorescent probe can detect GSSG and AA in the linear range of 1.2–3.6 and 27–35 μM with detection limits of 0.1 and 3.1 μM. Moreover, the pHRCDs/GSH fluorescence system was successfully applied to the detection of AA in the serum samples with satisfactory results. The pHRCDs and pHRCDs/GSH fluorescence systems have significant biocompatibility, low toxicity, high sensitivity, and good selectivity, demonstrating the great application potential in biological imaging and molecular detection.