Accepted Manuscript Redox Heme-Proteins Mediated Fluorescence of CdSe/ZnS Quantum Dots Lixia Qin, Luwei He, Congcong Ji, Xiangqing Li, Shizhao Kang, Jin Mu PII: DOI: Reference:

S1011-1344(14)00062-1 http://dx.doi.org/10.1016/j.jphotobiol.2014.02.017 JPB 9680

To appear in:

Journal of Photochemistry and Photobiology B: Biology

Received Date: Revised Date: Accepted Date:

3 December 2013 18 February 2014 24 February 2014

Please cite this article as: L. Qin, L. He, C. Ji, X. Li, S. Kang, J. Mu, Redox Heme-Proteins Mediated Fluorescence of CdSe/ZnS Quantum Dots, Journal of Photochemistry and Photobiology B: Biology (2014), doi: http://dx.doi.org/ 10.1016/j.jphotobiol.2014.02.017

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Redox Heme-Proteins Mediated Fluorescence of CdSe/ZnS Quantum Dots

Lixia Qin, Luwei He, Congcong Ji, Xiangqing Li, Shizhao Kang, Jin Mu*

School of Chemical and Environmental Engineering, Shanghai Institute of Technology, 100 Haiquan Road, Shanghai 201418, China

*

Corresponding author. Tel/fax:+86-21-60873061

E-mail address: [email protected] (J. Mu) 1

Abstract The redox properties of cytochrome c (Cyt c), hemoglobin (Hb) and myoglobin (Mb) were studied based on electrostatic interactions between TGA capped CdSe/ZnS quantum dots (QDs) and proteins. Results indicated that only Cyt c quenched the fluorescence of the QDs at pH > 8.0. Under the optimized conditions, a significant fluorescence recovery of the QDs’ system was observed when the reduced form of Cyt c incubated with TGA capped QDs, however, the reduced state of Hb and Mb resulted in a more fluorescence quenching on the same size of QDs. Interestingly, the fluorescence changes of QDs-proteins could be switched by modulating the redox potentials of proteins-attached QDs. Moreover, only the oxidized Cyt c form was reduced by the generated O2•- that significantly enhanced the fluorescence of the QDs’ system, which was also demonstrated by fluorescence imaging in HeLa cells.

Keywords: Redox proteins, Quantum dots, Electrostatic interactions, Spectroelectrochemistry, Fluorescence imaging

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1. Introduction Heme proteins, such as cytochrome c (Cyt c), hemoglobin (Hb) and myoglobin (Mb) are the key members of the cellular respiration chain and play a very important role in the pathology, pharmacology and mitochondrial changes in the process of apoptosis. Biological systems also rely on them to carry out versatile biological functions essential for their survival, ranging from electron transfer, ligand binding, catalysis, oxygen transport and storage, signal transduction, and control of gene expression [1-5]. It is well known that Cyt c, Mb and Hb have similar electrochemical and spectral behavior in that they have the same porphyrin complex of iron (II)-hemein or iron (III)-hemein. Although many methods have been employed to study the properties of three heme-containing proteins, there is a great demand for systematic studying the redox properties of the three heme-containing proteins. Semiconductor quantum dots (QDs) have become a well-established photoluminescent platform for biological applications [6-9]. They are advantageous due to their strong and easily tunable luminescence, the flexibility in excitation wavelength, and their commercial availability or easily accessible synthetic routes [10]. The key to successful implementation of new uses depends on the ability to add functionality on the surface of nanoparticles and to stabilize their emission [11]. Many synthetic ways and chemical surface modifications with different capping ligands provide the possibility of effective coupling of QDs surface to biomolecules and the development of hybrid systems [12-14]. As the photoluminescence property of QDs is strongly dependent upon the nature of the surface [15], the interactions between given chemical species and the surface of QDs would have effect on QDs’ fluorescence intensity. So far, the ligand-protected QDs have been increasingly explored as optical labels for various sensing biological species, such as cells [16], proteins [17], and DNA [18]. QDs are also highly sensitive to charge transfer, which can alter their optical properties [19,20], thus generating interest in charge-transfer-based biosensing [21]. Recently, the conjugation of QDs to biological molecules has been examined as a means of modulating the behavior of QDs and improving their ability to act as fluorescent probes. For example, QD-dopamine bioconjugates could be used to stain dopamine-receptor-expressing cells for

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exposing redox-sensitive patterns and confirming the redox interactions of quinones with QDs [22]. Dopamine can behave as an electron donor that can quench or sensitize QDs through different reactive oxygen mechanisms [22,23]. Recently we have demonstrated that coupling system of QDs with redox Cyt c is capable of fluorescnece imaging of a superoxide radical with high specificity [24]. Ubiquinone-coupled QDs could also be used for the quantitative detection of Reactive Oxygen Species (ROS) in living cells [25]. Cumulatively, these results confirm a role for redox molecules in redox interactions with QDs. Moreover, the coupling of QDs with redox-active species exhibits the following advantages. First, the redox species involved in the process of electron transfer from the QDs could modulate the photoluminescence of the QDs [26,27]. Second, the redox state of redox-active ligands could be tuned by application of external potential or by introduction of oxidizing/reducing reagent [28]. Third, the photogenerated charges in the QDs could take part in reduction/oxidation reactions with the species present in the nanoparticle shell [29]. Although the possibility of such charge transfer has been established with QDs [30-32], it is still a challenge to investigate the redox properties of QDs-protein bioconjugates by spectroscopic methods. In this paper, the fluorescence enhancement/quenching of QDs-proteins will be modulated on QDs surface by employing a variety of spectroscopic methods (Scheme 1). Results indicate that a significant fluorescence recovery of the QDs’ system was observed when reduced Cyt c was incubated with TGA capped QDs; whereas the reduced Hb and reduced Mb resulted in a more fluorescence quenching on the same size of QDs. Interestingly, for three proteins, only the oxidized state Cyt c is sensitive to the concentration of O2•concentration from 0 to 1.2 ×10-6 mol·L-1 and an obvious recovery in the fluorescence intensity is observed, which are demonstrated by fluorescence imaging in HeLa cells upon PMA stimulation for the same time.

2. Experimental Section 2.1. Reagents All reagents were of analytical grade, and doubly distilled water was used throughout. Thioglycolic acid (TGA), 2-(dimethylamino) ethanethiol hydrochloride (DMAET), KBH4 (96%) and selenium powder (99.999%) were purchased from Sigma-Aldrich. Zinc sulfate

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(ZnSO4, 99%), sodium sulfide (Na2S, 99%), cadmium chloride hemi (pentahydrate) (CdCl2·2.5H2O, 99%), sodium phosphate monobasic (NaH2PO4, 99%), sodium phosphate dibasic (Na2HPO4, 99%), sodium hydroxide (NaOH, 99%), Sodium dithionite (80%) and ethanol (99%) were obtained from Aldrich (Milwaukee, WI) and used without purification. N2 (99.998%, pre-purified) was obtained from Cryogenic Gases (Detroit, MI). Cytochrome c (from horse heart), Hemoglobin, Myoglobin and Phorbol Myristate Acetate (PMA) were from Sigma-Aldrich. 2.2. Apparatus Fluorescence spectra were recorded with a Shimadzu Cary Eclipse (Varian). The pH value of a solution was measured by a PHS-3C (Switzerland Mettler Toledo Delta 320 pH meter). Desktop multi-function centrifugal ultrafilters were used as received (Eppendorf-5430, St. Co. Germany). A CHI660D electrochemical workstation (Shanghai Chenhua Co., Ltd., China) equipped with a stirring machine (CH Instruments Inc), the three-electrode system consisting of a glassy carbon working electrode, a saturated calomel electrode (SCE) as a reference electrode, and a platinum counter electrode was used for all electrochemical measurements, unless noted otherwise. Ultraviolet-visible absorption spectra were recorded on a Shimadzu UV-3600PC UV-vis-NIR spectrophotometer (Japan). Atomic force microscopy (AFM) images were acquired by using Nanoscope IIIa Multimode AFM with an extender electronics module (Veeco, Santa Barbara, CA). All fluorescence images of cellular QDs were acquired with the same parameters using wide-field inverted fluorescence microscopy (Nikon-Ti, Co. Ltd. Japan) using a 60 × 1.2 NA objective and a matched electron multiplying charge-coupled device (EMCCD) (Roper). 2.3. Synthesis of QDs Negatively capped CdSe/ZnS QDs were performed according to the method reported previously [33]. Specifically, in a three necked flask (250 mL) equipped with a reflux condenser, septa, and valves, CdCl2·2.5H2O (0.2987 g, 1.31×10 -3 mol) was dissolved in 98.0 mL degassed water. Thioglycolic acid (TGA) (0.118 mL, 1.7×10 -3 mol) was added; the solution was adjusted to pH 11.2 with aqueous NaOH solution (1 mol·L-1), and stirred under argon bubbling at room temperature for 30 minutes. Then, the clear supernatant NaHSe solution was added under argon and the molar ratio of Cd/TGA/Se was set as 1.2 : 1.3 : 0.54, 5

with an initial Cd 2+ concentration of 13.05×10 -3 mol·L-1. The mixture of the precursor materials turned from colorless to dark orange and was then refluxed to allow the growth of quantum dots. The solution composed of 40 mL of TGA and ZnSO4 (1.25×10 -3 mol·L-1) was quickly injected into 15 mL of prepared CdSe solution, and Na2S was simultaneously added under vigorous stirring, causing an immediate nucleation and growth of nanoparticles. The pH was then adjusted to 11.2 by l mol·L-1 NaOH (Zn2+ / Cd2+ / S2- / TGA =1 : 0.2 : 0.4 : 2.4). The precipitates were centrifuged, washed with water and acetone in sequence, and then dried with nitrogen gas. For the nanocrystal thin-film preparation, the resulting powders were ultrasonically dispersed in ethanol and then filtered to obtain a colloidal solution of CdSe/ZnS nanoparticles, which was kept in a refrigerator at 4 °C. 2.4. Reduction of Cyt c, Hb and Mb Stock solutions of 1.08×10-4 mol·L-1 oxidized Cyt c, Hb and Mb were incubated with sodium dithionite for 5 mins at room temperature (1 g of the salt permmolprotein). The excess salt was removed using a NAP 25 column (Amersham Biosciences). The concentrations of reduced Cyt c, Hb and Mb were determined by evaluating the absorption at 550 in UV–vis spectra nm prior to use. The solution was stored for a maximum of 24 h at 4 oC. 2.5. Fluorescence spectra of CdSe/ZnS QDs-protein bioconjugates A set of oxidized and reduced Cyt c, Hb and Mb with the constant concentration of 9.82×10-6 mol·L-1 in the presence of TGA or DMAET modified CdSe/ZnS QDs were dissolved in 2 mL of 0.2 mol·L-1 PBS (pH 8.2) solution and incubated for several minutes, then the samples were deaerated for 5 mins with nitrogen gas. The fluorescence intensities of a series of QDs adsorbed with proteins of reduced form were recorded at λex/em = 420/565 nm against a blank sample. The spectral properties of reduced protein capped QDs were tested by their reaction with superoxide radicals under a nitrogen atmosphere. 2.6. Pyrogallol Assay for Superoxide Radical Anion (O2•-) To examine the scavenging activity of radicals, a pyrogallol autoxidation assay was performed. After incubation of 2.98 mL of Tris-HCl (50×10 -3 mol·L-1, pH=8.2) in a 25 °C water bath for 20 min, the mixture was combined with 0.1 mL of pyrogallol (1×10 -3 mol·L-1), and then the reaction mixture were incubated in a 25 °C water bath shaker for 5 mins. It was immediately used for the experiment of the scavenging activity of radicals. 6

2.7. Electrochemical measurements A standard jacketed three-electrode cell was used for all electrochemical experiments. A Pt electrode and an SCE electrode were used as counter electrode and reference electrode, respectively. Electrochemistry measurements of approximately 1.08×10-4 mol·L-1 Cyt c, Hb and Mb were carried out at the ITO electrode in 0.2 mol·L-1 PBS (pH 8.2). The temperature was maintained at 25 °C using a circulating water bath. All reported potentials were referenced to the formal potential of the ferrocene/ferrocenium (Fc/Fc+) couple. 2.8. Cellular Imaging HeLa cells were cultured in Rosewell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated bovine serum, penicillin (100 U/mL) and streptomycin (100 U/mL). Cells were incubated at 37 °C in humidified atmosphere of 95% air and 5% CO2. Cells were plated into a 24-well culture plate (200–300 cells/well) and allowed to adhere for 10 h before treatment. Culture medium containing 200 µg/mL QDs, CdSe/ZnS QDs-oxidized Cyt c bioconjugate, CdSe/ZnS QDs-oxidized Hb bioconjugate and CdSe/ZnS QDs-oxidized Mb bioconjugate with the constant concentration of proteins (9.82×10 -6 mol·L-1) were added, and cells were incubated for 10 h. Next, the growth medium was removed, and the cells were washed several times with PBS. Then, the cells were fixed using a 10% methanol solution at room temperature for 20 mins, followed by washing with PBS. The cover glass was then mounted on a microscopic glass slide and studied under a microscope. The conjugated QDs-loaded cells were cultured for 4 h with and without the presence of PMA stimulation (400 ng/mL; a stimulator of cell respiratory burst to give rise to ROS) at 37 °C. Cells were washed three times with 0.2 mol·L-1 PBS buffer before imaging (λex = 420 nm). The images were taken by using an inverted fluorescence scanning microscope with an objective lens (×60). All background parameters (laser intensity, exposure time and objective lens) were kept constant when the different fluorescence images were captured.

3. Results and Discussion 3.1. Fluorescence characteristics of TGA capped CdSe/ZnS QDs with three oxidized heme-containing proteins

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Oxidized Cyt c, Hb and Mb can act as electron acceptor for the photoexcited QDs which could show varying degrees of quenching on fluorescence intensity dependent on the pH value. To the best of our knowledge, at pH > 8.0, only Cyt c quenched the fluorescence of the QDs, and no significant fluorescence changes were observed for Hb or Mb [34]. Based on these results, we tested the interactions of TGA capped QDs with oxidized Cyt c, Hb and Mb at pH 8.2. As shown in Figure 1, we found that addition of Cyt c to the TGA capped CdSe/ZnS QDs solution resulted in an obvious reduction of the fluorescence intensity on QDs (Figure 1, curve d, fluorescence quenching: 88%), while Hb and Mb exhibited 8.7% (Figure 1, curve b) and 15% (Figure 1, curve c) of fluorescence quenching efficiency on QDs at the constant concentration (9.82×10 -6 mol·L-1). It is worthwhile to note that the matching degree is of crucial importance for the essence of quenching mechanism between the isoelectronic points of three redox proteins and the external environment. It is known that the isoelectronic points of Cyt c, Hb and Mb are 10.83, 7.07 and 6.99, respectively. While pH>8.0, Hb and Mb would take less positive charge or negative charge while the TGA capped QDs would take negative charge. The electrostatic repulsive interaction makes it difficult for the proteins to approach the QDs surface, reducing the electron-transfer rate. On the contrary, however, the pH value is still lower than the isoelectric point of Cyt c. Thus, the strong electrostatic affinity between Cyt c and TGA capped QDs would favor the photoinduced electron transfer process, resulting in the fluorescence quenching on the TGA capped QDs. Moreover, in order to evaluate the stability of conjugates, time-dependent fluorescence spectra of QDs-proteins were obtained carefully in the air-saturated atmosphere (Fig. S1, A, B and C). Obviously, the fluorescence intensity of QDs-proteins slightly decreased with increasing time (a-f), indicating that the conjugates are stable under near physiological conditions, which also provides a good foundation for studying the fluorescence imaging of QDs-proteins in living cells. 3.2. Spectral properties of oxidized and reduced forms of Cyt c, Hb and Mb on QDs surface Figure 2A and B illustrate the absorption spectra of reduced and oxidized Cyt c, Hb and Mb incubated with QDs. Clearly, in contrast to the oxidized Cyt c (Figure 2A, curve a), the slight bathochromic shifts of the Soret-band (i.e., ~410 nm) and the development of α- (i.e., 8

550 nm) and β-bands (i.e., 500 nm) were observed, which are the characteristics of the reduced Cyt c (Figure 2A, curve b) in the TGA capped QDs solution. However, the absorption spectra of reduced and oxidized forms of Hb (Figure 2B, curve a and b) and Mb (Figure 2B, curve c and d) have negligible differences in QDs solution, without the distinct characteristic spectra of α- and β-bands except the bathochromic shifts of the Soret-band (i.e., ~410 nm). Thus, the absorption spectra of reduced and oxidized forms of Cyt c, Hb and Mb exhibit the different redox characteristics on the QDs surface. Based on above results, the representative fluorescence spectra of reduced and oxidized Cyt c, Hb and Mb in the presence of CdSe/ZnS QDs solution were studied under the identical conditions. Clearly, the oxidized states Cyt c, Hb and Mb (9.82×10-6 mol·L-1) (Figure 3, A-a, B-a and C-a) present quenching on fluorescence intensity of QDs in varying degrees (pH=8.2). Interestingly, the fluorescence of QDs enhanced gradually (∆Fmax = 300) when TGA capped QDs were incubated with the different concentrations of reduced state Cyt c (Figure 3A, from a ~ e, 4.91×10-7 mol·L-1 ~ 9.82×10-6 mol·L-1) for several minutes. However, the same concentrations of reduced state Hb and Mb resulted in a more quenching on the same size of QDs (Figure 3B and C, from a ~ e, 4.91×10-7 mol·L-1 ~ 9.82×10 -6 mol·L-1). Thus, according to the absorption spectra of redox proteins, the contrast in fluorescence spectrum behavior of reduced and oxidized Cyt c, Hb and Mb indicated that the three mitochondrial redox proteins maybe have the possible differences in the heterogeneous rate constant of the redox process for proteins centers around the photoexcited QDs. Moreover, we performed atomic force microscopy (AFM) of TGA capped QDs, QDs-reduced Cyt c, QDs-reduced Hb and QDs-reduced Mb to estimate the interaction of the QDs-protein bioconjugates. As shown in Figure 4A, AFM amplitude scans revealed well-dispersed and uniform CdSe/ZnS QDs particles with the size of 3 ~ 4 nm. Obviously, the QDs-reduced Cyt c bioconjugates also exhibited the discrete and uniform particles, and the vertical height of QDs-Cyt c was about 7 ~ 8 nm, indicating that the reduced Cyt c as a better capping layer was formed on QDs surface (Figure 4B). However, when reduced Hb and Mb were conjugated to TGA capped QDs, an increase in particle density was observed, while clustered particles on the mica surface appeared (Figure 4C and Figure 4D). The possible explanation for large differences is coupling of QDs by reduced Cyt c which could form a 9

better passivation layer on the surface of the QDs than Hb and Mb that helps to overcome potential surface defects, and maintain higher luminescence efficiency under nitrogen atmosphere. 3.3. Fluorescence spectroelectrochemistry of Cyt c, Hb and Mb in the presence of negatively capped QDs Based on above investigations, the fluorescence of QDs-redox proteins changes with the potential were conducted by in situ fluorescence spectroelectrochemistry. Figure 5A shows the fluorescence spectra of oxidized Cyt c in the presence of CdSe/ZnS QDs change with the potential from -1.0 V to 0.3 V. At an applied potential of -0.47 V vs SCE, the fluorescence intensity of QDs-oxidized Cyt c (Figure 5A-a), increased gradually when operated for 60 s (curve a to d). It should be noted that when the potential was negative, the QDs-oxidized Cyt c biocongujates were almost completely reduced on the electrode surface. When operated at a constant applied potential of -0.15 V vs. SCE for 90 s (curve d to g), the decrease in fluorescence intensity indicated that the capping layer of oxidized Cyt c was regenerated. Moreover, for the QDs-oxidized Hb and QDs-oxidized Mb systems (Fig. S2A and B), the fluorescence intensity all quenched at given -0.46 V and -0.48 V vs. SCE for 60 s (curve a to d); surprisingly, the fluorescence intensity of d continually quenched when operated at a constant applied potential of -0.27 V and -0.26 V vs. SCE for 90 s (curve d to g). Thus, the spectroelectrochemical results indicated that only fluorescence quenching of the QDs-oxidized Hb and QDs-oxidized Mb systems was observed whether the positive potential or reverse potential applied on the system, which was in good agreement with the above fluorescence properties. In order to confirm the presence of QDs having a reduced Cyt c layer, changes in the QDs absorption spectra were recorded at a constant applied potential in a optically transparent thin-layer electrochemical cell (optical path length is 0.4 ± 0.1 mm; PBS at pH = 8.2). The QDs-oxidized Cyt c exhibited a characteristic of absorption band at 410 nm, as shown in Figure 5B (curve b). Applying a potential of -0.47 V vs. SCE for 60 s, the main absorption band at 410 nm showed the obvious bathochromic shift of the Soret-band and new α- (i.e., 550 nm) and β- (i.e., 500 nm) absorption bands appeared, as shown in Figure 5B (curve c). When operated at a constant applied potential of -0.15 V vs. SCE for 90 s, the salient 10

absorption bands at 550 nm and 500 nm completely disappeared while the absorption band at 410 nm was kept. This absorption profile indicated that the oxidized capping layer accepted electrons and converted to its reduced form on the electrode surface. Figure 5A (insert b) displays the time-dependent fluorescence changes of the QDs-oxidized Cyt c upon the change of potential from -1.0 V to 0.3 V. The fluorescence intensity of QDs-oxidized Cyt c is restored with an increase in the incubation time (0~60 s). These results indicate that reduction of the capping layer takes place, yielding the enhanced luminescence of QDs. The gradual decrease of fluorescence intensity of the QDs with time is consistent with the transformation of the reduced Cyt c to the quenched oxidized Cyt c. In addition, the process is reversible and there is a small loss of fluorescence efficiency in QDs in the next cycle. Overall, these differences enable us to “fine-tune” the luminescence of the QDs. 3.4. Fluorescence properties of QDs-Cyt c, QDs-Hb and QDs-Mb systems in the presence of O2•As discussed above, the oxidized Cyt c efficiently quenches the fluorescence of the QDs under nitrogen atmosphere, whereas the reduced form Cyt c incubated with TGA capped QDs would lead to the obvious recovery of QDs’ fluorescence intensity. However, no obvious fluorescence changes were observed when the reduced state Hb and Mb incubated with QDs. It should be noted that the oxidized Cyt c form could be reduced by O2•- [25,35].Thus, the fluorescence properties of QDs-oxidized Cyt c, QDs-oxidized Hb and QDs-oxidized Mb systems were investigated in the presence of O2•-. As shown in Figure 6A (from curve a to i), the fluorescence intensity of QDs was enhanced significantly (∆Fmax = 170) when the O2•- was added to the QDs-oxidized Cyt c system. This indicated that the oxidized Cyt c could be reduced by O2•- generated in solution. A linear relationship between F-F0/F0 and the concentration of O2•- was obtained over a range from 0 µM to 1.2 µM. However, the fluorescence intensity of QDs-Hb (Figure 6B, from curve a to i) and QDs-Mb (Figure 6C, from curve a to i) systems was quenched weakly (∆Fmax = 30), when the concentrations of O2•- from 0 µM to 1.2 µM were generated in solution. The rate of fluorescence intensity increasing as the generation of radicals is plotted against the steady-state O2•- concentration (Figure 6, inserts). Moreover, control experiments also showed 11

the slight decrease in QDs’ fluorescence when the same concentration of O2•- was added into the only QD solution, which demonstrated that the generated O2•- resulted no effect on the oxidized Hb and Mb. Based on above investigations, These results suggest that the oxidized Cyt c could be reduced by O2•-, and the QDs-Cyt c system shows promise for the specific quantification of O2•-, with a wide linear concentration range. 3.5. Fluorescence imaging of QDs-bioconjugates in HeLa cells Extensive studies demonstrate that tumor occurrence is related to reactive oxygen species [36] and an antioxidant sensors have been designed to study their properties of radical scavenger [37-39]. Therefore, we test the application of the three redox proteins modified QDs for fluorescence imaging in HeLa cells in order to investigate whether there are significant differences in the fluorescence imaging of QDs upon stimulation with PMA for 4 h (Figure 7). As shown in Figure 8a, 8d and 8g, the bright-field measurements clearly show that the cells are viable throughout the imaging experiments. Obviously, the QDs-oxidized Cyt c loaded HeLa cells (Figure 7b-c) displayed a striking bright fluorescence, whereas, the QDs-oxidized Hb (Figure 7e-f) and QDs-oxidized Mb probe loaded HeLa cells (Figure 7h-i) showed only a faint fluorescence upon PMA stimulation with the same time. However, control experiments indicated that fluorescence was weak for Hb-modified QDs and Mb-modified QDs and there were no fluorescence changes of non-stimulated HeLa cells loaded by QDs-oxidized Cyt c after an equivalent time of 4 h (data not shown). These results demonstrate that the only capping layer of oxidized Cyt c is reduced presumably via the generation of O2•- under PMA stimulation, yielding the enhanced layer, which is consistent with our fluorescence and spectroelectrochemical studies on these systems. Moreover, fluorescence imaging of QDs-oxidized Cyt c loaded with HeLa cells with and without PMA stimulation for 6 h were collected (Fig. S3). Interestingly, images revealed that HeLa cells (Fig. S3, a and b) still showed stronger fluorescence. In addition, fluorescence was weak and there were no fluorescence changes of non-stimulated HeLa cells loaded by oxidized Cyt c-modified QDs after an equivalent time of 6 h (Fig. S3, c and d). These results indicated that the structures of QDs-proteins are stable in HeLa cells. Since this probe responds to changes in the O2•- concentrations in living cells, QDs carrying the oxidized Cyt c capping layer should be broadly applicable to the quantitative detection of O2•- in biological systems. 12

Moreover, these systems should enable the construction of probes that could target the specific regions and organelles in living cells and provide information about their redox state.

4. Conclusion In summary, the redox properties of Cyt c, Hb and Mb were studied by employing a variety of spectroscopic methods. For three redox proteins, only Cyt c quenched effectively the fluorescence of the QDs at pH=8.2, and also the absorption spectra of reduced Cyt c exhibited distinct characteristic spectra of α- and β-bands compared with the reduced Hb and Mb. Under the optimized conditions, a significant fluorescence recovery of the QDs’ system was observed when the reduced Cyt c incubated with TGA capped QDs, however, the reduced Hb and Mb resulted in a more fluorescence quenching on the same size of QDs. Interestingly, for three proteins, only the oxidized state Cyt c is sensitive to the concentration of O2•concentration from 0 µM to 1.2 µM and an obvious recovery in the fluorescence intensity is observed, which are demonstrated by fluorescence imaging in HeLa cells upon PMA stimulation for the same time. The platform described here might be extended to study the redox characteristics of other redox proteins by using different promoter molecules for modification of the nanoparticles, thus providing the basis for use in a protein array. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant No. 21305092, 21301118 and 20933007), and the Shanghai Municipal Natural Science Foundation (No. 13ZR1461800).

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Scheme

1.

Schematic

diagram

of

the

reduced

and

oxidized

proteins

by

spectroelectrochemistry modulation and fluorescence change of the CdSe/ZnS QDs-oxidized protein system in the presence of O2•-.

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Fig. 1. Emission spectra of (a) TGA capped QDs; (b) oxidized Hb; (c) oxidized Mb and (d) oxidized Cyt c in the presence of TGA capped QDs with the constant concentration of proteins (9.82×10 -6 mol·L-1) in PBS (pH=8.2).

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Fig. 2. (A) Absorption spectra of oxidized Cyt c (a) and corresponding spectra of reduced Cyt c with the constant concentration (9.82×10 -6 mol·L-1) in the presence of TGA capped QDs (b). (B) Absorption spectra of oxidized Mb (9.82×10 -6 mol·L-1) (a); reduced Mb (b); oxidized Hb (c) and reduced Hb (9.82×10 -6 mol·L-1) in the presence of TGA capped QDs in PBS (pH=8.2).

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Fig. 3. Emission spectra of A: (a) oxidized Cyt c (9.82×10-6 mol·L-1); (b-e) TGA capped QDs incubated with the different concentrations of reduced Cyt c (4.91×10-7 mol·L-1, 9.82×10 -7 mol·L-1, 4.91×10 -6 mol·L-1 and 9.82×10-6 mol·L-1); B: (a) oxidized Hb (9.82×10 -6 mol·L-1); (b) TGA capped QDs incubated with the different concentration of reduced Hb (4.91×10 -7 mol·L-1, 9.82×10-7 mol·L-1, 4.91×10-6 mol·L-1 and 9.82×10 -6 mol·L-1); C: (a) oxidized Mb (9.82×10-6 mol·L-1); (b) TGA capped QDs incubated with the different concentrations of reduced Mb (4.91×10-7 mol·L-1, 9.82×10-7 mol·L-1, 4.91×10-6 mol·L-1 and 9.82×10-6 mol·L-1).

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Fig. 4. (A) AFM images of TGA capped CdSe/ZnS QDs; (B) TGA capped CdSe/ZnS-Cyt c bioconjugates; (C) TGA

capped

CdSe/ZnS-Hb bioconjugates; (D)

TGA capped

CdSe/ZnS-Mb bioconjugates.

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Fig. 5. (A) Fluorescence emission spectrum changes with a constant applied potential. Fluorescence intensity of QDs-oxidized Cyt c (a, red) enhanced at given -0.38 V voltage and stabilized for 60 s (a-d, red); Fluorescence intensity of d quenched at given -0.25 V potential and stabilized for 90 s (d-g, black); Insert a: Cyclic voltammetry of QDs-Cyt c on ITO electrode in PBS (pH = 8.0), Insert b: Time-dependent fluorescence intensity changes of oxidized Cyt c in the presence of TGA capped QDs with a constant applied potential; (B) Absorption spectra change of three nanoparticles with constant applied potentials of -0.47 V and -0.15 V, respectively. (a) TGA capped CdSe/ZnS QDs, (b) oxidized Cyt c (9.82×10 -6 mol·L-1) and (c) reduced Cyt c (9.82×10-6 mol·L-1) in the presence of TGA capped QDs.

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Fig. 6. Fluorescence intensity measurement in the TGA capped CdSe/ZnS QDs incubated with Cyt c (A), Hb (B), and Mb (C) for 8 mins when the O2•- generates in solution. Insets: the relationship between F-F0/F0 of TGA capped QDs-Cyt c, QDs-Hb and QDs-Mb systems with CO2•- from 0 to 1.2 µM.

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Fig. 7. Fluorescence microscopic images of HeLa cells incubated with QDs-redox proteins bioconjugates with the constant concentration of proteins (9.82×10-6 mol·L-1). (a) Bright-field image of QDs-Cyt c loaded with HeLa cells, (b) Fluorescence image of QDs-Cyt c loaded with HeLa cells upon stimulation with PMA for 4 h, (c) Merged images of QDs-Cyt c loaded with HeLa cells; (d) Bright-field image of QDs-Hb loaded with HeLa cells, (e) Fluorescence image of QDs-Hb loaded with HeLa cells upon stimulation with PMA for 4 h, (f) Merged images of QDs-Hb loaded with HeLa cells, (g) Bright-field image of QDs-Mb loaded with HeLa cells, (h) Fluorescence image of QDs-Mb loaded with HeLa cells upon stimulation with PMA for 4 h, (i) Merged images of QDs-Mb loaded with HeLa cells.

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The table of contents The redox properties of heme-proteins have been reported based on electrostatic interactions between quantum dots (QDs) and proteins. Interestingly, the fluorescence I and II could be switched by modulating the redox potential of three proteins-attached QDs, which opens up the possibility to “read-out” the redox state of biological species of interest. Lixia Qin, Luwei He, Congcong Ji, Xiangqing Li, Shizhao Kang, Jin Mu* School of Chemical and Environmental Engineering, Shanghai Institute of Technology, 100 Haiquan Road, Shanghai 201418, China

Redox Heme-Proteins Mediated Fluorescence of CdSe/ZnS Quantum Dots

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Redox Heme-Proteins Mediated Fluorescence of CdSe/ZnS Quantum Dots Lixia Qin, Luwei He, Congcong Ji, Xiangqing Li, Shizhao Kang, Jin Mu* School of Chemical and Environmental Engineering, Shanghai Institute of Technology, 100 Haiquan Road, Shanghai 201418, China *

Corresponding author. Tel/fax:+86-21-60873061

E-mail address: [email protected] (J. Mu)

Graphical abstracts

The fluorescence I and II could be switched by modulating the redox potential of three proteins-attached QDs, which opens up the possibility to “read-out” the redox state of biological species of interest. Interestingly, only the oxidized Cyt c form was reduced by the generated O2•- that significantly enhanced the fluorescence of the QDs’ system, which was also demonstrated by fluorescence imaging in HeLa cells.

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Highlights ●Three proteins have different redox properties on QDs surface. ●Fluorescence recovery was observed when reduced Cyt c incubated with QDs. ● Fluorescence changes of QDs-proteins were modulated by spectroelectrochemistry. ● Fluorescence imaging of QDs-proteins was demonstrated in HeLa cells. ● This platform might be extended to study other redox proteins.

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