Silicon nanowires-based fluorescent sensor for in situ detection of hydrogen sulfide in extracellular environment

Abstract

A selective fluorescent sensor for hydrogen sulfide was realized by covalently modifying a naphthalimide derivative onto the surface of silicon nanowires (SiNWs). The as-prepared SiNW array-based sensor was successfully employed for real-time and in situ imaging of the changes in extracellular hydrogen sulfide of HeLa cells.

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Recent investigations have demonstrated that H₂S is the third endogenous gaseous transmitter existing in the human body following nitric oxide (NO) and carbon monoxide (CO), which plays important roles in regulating intracellular redox status and fundamental signaling processes related with human health and disease.^1,2 H₂S also contributes to a wide range of physiological processes, including regulation of vascular tone, myocardial contractility, neurotransmission and so on.^1,3–5 Endogenously H₂S is biosynthesized from L-cysteine by enzymes such as cystathionine-β-synthetase (CBS, EC4.2.1.22), cystathionine-γ-lyase (CSE, EC4.4.1.1), 3-mercaptopyruvate sulfur transferase (3MST),^6,7 and also produced from non-enzymatic reduction of elemental sulfur.⁸ The physiologically relevant H₂S concentration is estimated ranging from nano- to millimolar levels,^9–11 and the abnormal levels of H₂S is considered to associate with several diseases such as Alzheimer's disease,¹² Down's syndrome,¹³ diabetes,¹⁴ and liver cirrhosis.¹⁵ Despite the substantial studies on H₂S, the complex contribution of H₂S in the physiological and pathological process has not been fully elucidated, which is partly due to the limited ability in real-time and in situ monitoring of this small molecule in living biological specimens. Therefore, exploring a rational approach to monitor H₂S in living biological specimens would be great of importance to better understand the function of H₂S.

So far, several techniques such as colorimetric,^16,17 electrochemical¹⁸ and chromatographic¹⁹ etc. have been employed to detect H₂S, but these methods often require sophisticated instruments, complicated sample processing, and/or destruction of tissues or cells. Due to its high sensitivity, simplicity, non-invasiveness, and high spacial resolution when combined with microscopy, the fluorescence-based assay shows great advantages over others and has been applied to monitor H₂S in living cells and small animals.^20–23 Among the fluorescent H₂S probes reported in recent years, the ones based on the known unique reduction of an azide group by H₂S have shown an elegant performance.^20,24 By incorporating the azide moiety into a fluorophore and employing the reduction of the fluorophore azide derivatives with H₂S, these fluorescent probes have shown a specific response to H₂S.^25–30 For convenience of use, the fluorophore azide derivative could be immobilized to the surface of nanoparticles, which exhibited excellent properties.³⁰ However, such configuration would bring some inconvenience for in situ detection when the fluorescent H₂S probes based on organic molecules and the modified nanoparticles were used to stain the living cells for the fluorescent imaging and the monitoring of the biomolecule in water solution. In order to conveniently achieve the in situ detection of H₂S in living cells, anchoring the probe to the surface of appropriate carrier would be a rational strategy.

Based on their stability, nontoxicity, biocompatibility, and convenience for integration with IC, silicon nanowires (SiNWs) are excellent candidate for a carrier to fabricate various sensor devices for bioapplications.^31–36 Meanwhile, the SiNW arrays could enhance the adhesion force between the substrate and target cells and restrict the cells spreading, which would be appreciably convenient to investigate the cells.^37–39 On the basis of the advantages of SiNWs, it has been utilized as excellent biomedical materials in cellular studies, such as intracellular biomolecules delivery, gene transduction and biochemical activity measurement.^40–43 Considering the importance of hydrogen sulfide detection and the virtues of the SiNWs and azide group-based fluorescent sensor, we covalently immobilize the 4-amino-1,8-naphthalic anhydride onto the surface of SiNWs and subsequently incorporate the azide moiety into the fluorophore, a SiNWs-based fluorescent H₂S sensor was realized with high selectivity. Moreover, the as-prepared SiNW arrays-based sensor was successfully used in real time and in situ monitoring the changes of the extracellular H₂S for live HeLa cells.

SiNWs were prepared by a typical chemical vapor deposition (CVD) method.⁴⁴ The as-prepared SiNWs have a crystalline Si core of 10–12 nm in diameter and a silicon oxide sheath of 2–3 nm in thickness determined by TEM as shown in Fig. S1.† The modification on the surface of the SiNWs are described in detail in the ESI (Fig. S2†). The SiNWs modified with 3-aminopropyl-triethoxylsilane (APTES) are abbreviated as APTES-SiNWs. After further reacting with 4-amino-1,8-naphthalic anhydride, the product was defined as 4-A-SiNWs. After incorporating the azide moiety into the 4-A-SiNWs, the finally modified SiNWs were named as MSiNWs. The high-quality SiNW arrays were fabricated according to previous work.⁴⁵ The SEM images of the SiNW arrays were shown in Fig. S3.† The diameters of the SiNWs are in the range of 150–300 nm, while the wire length is around 15 μm. The modifying procedures applied to SiNW arrays were the same as to SiNWs obtained by CVD methods.

To characterize the modifications of the SiNWs, the X-ray photoelectron spectroscopy (XPS) was used and the results were shown in Fig. S4.† It was found that no nitrogen was detected from the bare SiNWs, while the N(1s) spectrum of APTES-SiNWs shows a peak at around 399.0 eV corresponding to the C–N–H bonds from the APTES.⁴⁶ The results indicate that APTES has been modified onto the surface of the SiNWs. After the APTES-SiNWs reacted with 4-amino-1,8-naphthalic anhydride, the N(1s) spectrum changed compared with APTES-SiNWs and can be deconvoluted into three peaks: a same peak at 399.0 eV related to C–N–H bonds of the APTES, a peak at 399.6 eV related to C–N–H bonds of the amino from the fluorophore, and a peak at 400.8 eV related to N–C bonds of naphthalimide structure.⁴⁷ These results verify that the 4-amino-1,8-naphthalic anhydride has been covalently modified onto the surface of the APTES-SiNWs. Furthermore, it was found that a new broad peak around 401.2 eV related to the C–N⁻, C–N⁺, C–N bonds of azide group appeared in the N(1s) spectrum of MSiNWs, which demonstrated the azide moiety has been incorporated into the naphthalimide derivative connected to the SiNWs successfully.

To check the response of MSiNWs to hydrogen sulfide, the MSiNWs were dispersed into the HEPES-buffer (20 mM pH 7.4) to form a 150 μg mL⁻¹ suspended solution. Then 300 μM NaSH (a commonly employed H₂S donor) was added into the system. Fig. 1a shows the fluorescence spectral changes of MSiNWs as a function of H₂S incubation time. Upon addition of H₂S, the fluorescence intensity of MSiNWs increased gradually with the incubation time and 11-fold turn-on was observed after 90 min. The large change in fluorescence of MSiNWs upon exposure to H₂S could allow for the fluorescence detection of H₂S in biological samples even if the reaction with the MSiNWs has incompletely proceeded. A linear relationship is important for easy and accurate analysis. Thus, the dependence of the fluorescence enhancement of MSiNWs on the H₂S concentration was investigated. The emission spectra of MSiNWs in HEPES-buffer (20 mM pH 7.4) was scanned upon adding various concentrations of H₂S for 90 min. The calibration curve is shown in Fig. S5.† A linear relationship in the range of 0–40 μM was obtained (Fig. 1b) and the detection limit of MSiNWs for H₂S was 7.13 μM, indicating the MSiNWs have the potential to be used for quantitative analysis of H₂S. The schematic illustration of the MSiNWs's response to H₂S is shown in Scheme 1. The azide group is a strong electron withdrawing group. After it was reduced to a strong electron donor-amide group by H₂S, the MSiNWs were turned to 4-A-SiNWs which contain a D–π–A structure, leading to the fluorescence enhancement.

Fig. 1 (a) Fluorescence turn-on of MSiNWs after treatment with 300 μM H₂S. Inset: the fluorescence intensity of MSiNWs at 535 nm as a function of incubation time. (b) The linear relationship between the relative fluorescence intensity of MSiNWs and the H₂S concentration in the range of 0–40 μM. MSiNWs: 150 μg mL⁻¹ (20 mM HEPES. pH = 7.4). λ_ex = 430 nm, t = 37 °C.

Scheme 1 The schematic illustration of MSiNWs's response to H₂S.

To study the selectivity, the fluorescence responses of the MSiNWs toward other biologically reactive sulfur, oxygen, and nitrogen species (RSONS) were investigated. Each RSONS was added to the system and the fluorescence response was monitored over time. As shown in Fig. 2, addition of 300 μM SO₃²⁻, HOCl, S₂O₃²⁻, H₂O₂, KSCN, NO₂⁻, KO₂, ALA, ^tBuOOH and 5 mM GSH, cysteine (Cys) had little effect on the fluorescence emission from the sensing system. In contrast, addition of 300 μM H₂S to the sensing system could result in the obvious fluorescence enhancement. Furthermore, the fluorescence response of MSiNWs towards H₂S in the presence of these RSONS were also investigated (Fig. S6†). It can be observed that these competing species have ignorable influences on the detection of H₂S. These results demonstrate that the present MSiNWs could bear the interference from these coexist competing species which usually existed in biological milieu in a very low concentration.

Fig. 2 Fluorescence responses of MSiNWs (150 μg mL⁻¹) to biologically relevant RSS, RNS, and ROS. Bars represent relative responses at 535 nm at 30, 60, and 90 min after addition of RSS, RNS, or ROS. Data shown are for 5 mM GSH, 5 mM Cys, and 300 μM for other RSS, RNS, and ROS (20 mM HEPES, pH 7.4, λ_ex = 430 nm).

To verify the cellular application of the MSiNW arrays to H₂S, the as-prepared MSiNW arrays were utilized to monitor the extracellular H₂S changes for live HeLa cells (Fig. 3). The HeLa cells (blue dots in Fig. 3a and d) were captured by the MSiNW arrays (the preparation details of the cell-captured MSiNW arrays can be found in ESI†) and the fluorescence of the cell-captured MSiNW arrays before and after treated with the buffer containing 250 μM H₂S, a concentration of H₂S comparable with physiological H₂S levels, was recorded. Fig. 3 shows the fluorescence images of cell-captured MSiNW arrays before (a–c) and after (d–f) incubation with H₂S. Fig. 3a and d show the blue fluorescence images of the HeLa cells captured by the substrate with Hoechst staining. Fig. 3b and e show the green fluorescence images of cell-captured MSiNW arrays. Fig. 3c and f show the overlay images of blue fluorescence from the stained cells and the green fluorescence from SiNWs. As expected, the addition of H₂S resulted in the obvious enhancement of the green fluorescence from substrate, however, little change of the blue fluorescence from the stained nuclei of HeLa cells was observed. This phenomenon implies the potential application of MSiNW arrays in real-time and in situ monitoring of H₂S in biosystems.

Fig. 3 Fluorescence images of the substrate for in situ monitoring of extracellular H₂S changes for live HeLa cells. Fluorescence images of cell-captured MSiNW arrays before (a–c) and after (d–f) incubation with 250 μM H₂S at 37 °C for 30 min. Left (a and d): the images of the HeLa cells captured by the substrate with Hoechst staining. Middle (b and e): the fluorescence images of cell-captured MSiNW arrays; right (c and f): overlaid with left and middle.

In summary, we have designed and configured a fluorescent turn-on sensor for H₂S by covalently immobilizing the naphthalimide azide derivative onto the surface of SiNWs. The present sensor is highly selective for detection of H₂S and displays a good linear relationship between the fluorescence intensity and H₂S concentration from 0 to 40 μM. Moreover, the modified SiNW array-based sensor was successfully used for real-time and in situ monitoring of extracellular H₂S changes for live HeLa cells.

Acknowledgements

This work was supported by Chinese Academy of Sciences (Grant KGZD-EW-T02), NSFC (Grants 51272302, 51272258, and 91333119), National Basic Research Program of China (973 Program) (Grant 2012CB932400) and Youth Innovation Promotion Association CAS.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: General materials and methods, experimental details, TEM, SEM, XPS of SiNWs, and the fluorescence response of MSiNWs. See DOI: 10.1039/c5ra08728g

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