Hydrogen Sulfide Detection Based on Reflection: From a Poison Test

Jul 2, 2014 - The parameters were set as 900 of Heat, 5 of Fil., 50 of Vel., 128 of Del., and 55 of Pul. Then, quartz needles were ultrasonically clea...
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Hydrogen Sulfide Detection Based on Reflection: From a Poison Test Approach of Ancient China to Single-Cell Accurate Localization Hao Kong, Zhuoran Ma, Song Wang, Xiaoyun Gong, Sichun Zhang, and Xinrong Zhang* Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Department of Chemistry, Center for Atomic and Molecular Nanosciences, Tsinghua University, Beijing 100084, P. R. China S Supporting Information *

ABSTRACT: With the inspiration of an ancient Chinese poison test approach, we report a rapid hydrogen sulfide detection strategy in specific areas of live cells using silver needles with good spatial resolution of 2 × 2 μm2. Besides the accuratelocalization ability, this reflection-based strategy also has attractive merits of convenience and robust response when free pretreatment and short detection time are concerned. The success of endogenous H2S level evaluation in cellular cytoplasm and nuclear of human A549 cells promises the application potential of our strategy in scientific research and medical diagnosis.

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light reflection, has been demonstrated to be more sensitive than fluorescence.12 For example, a single 60 nm gold nanoparticle has a scattering intensity that is equivalent to the fluorescence intensity of 105 fluorescein molecules.13 In 2013, He’s group reported a sulfide mapping strategy based on the surface plasmon resonance scattering of single Au−Ag core−shell nanoparticles.14 They achieved very high sensitivity and time-dependent linear logarithmic responses through mapping the Ag2S formation with a single-particle spectral dark-field microscope. The sensitivity is about 1000 times higher than ordinary fluorescent probes, but this methodology still suffers two limitations. First, nanoparticles would interfere with each other unless their dispersion in cells is good enough. Second, positions of nanoparticles in live cells are unpredictable, and only areas with well-dispersed nanoparticles can be evaluated so that accurate analysis in targeted areas remains unsolved. However, the reflection-based strategy might offer a solution to accurate-localization single-cell analysis due to the controllability of silver needles. In addition, the synthesis and modification of silver needles can be much easier, offering the advantages of convenience and versatility.

n the Song Dynasty of China (around the 13th century), the world’s remaining first book on forensic science, named XiYuanLu (Witness of a Prosecution), reported a poison test approach based on observations of silver needles turning black after reaction with the sulfide residue in poisonous white arsenic (As2O3). The reflection light, as the crucial output signal, is actually the magic hand behind this interesting approach. However, the reflection light barely draws attention in modern analytical chemistry with the exception of application in optical path adjusting.1−3 What’s worse, it is considered as a “bad guy” sometimes, and we have to avoid it for background suppression in fluorescence or scattering detection.4 Although a few analytical approaches based on the output signal of reflection light, such as X-ray reflectivity (XRR),5 attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR),6 near-infrared diffuse reflectance spectroscopy (NIRDRS),7−9 and confocal reflection microscopy,10,11 succeed in material characterization, substance discrimination, and biomedical imaging, the application of visible light reflection signal still remains an open field in biological analysis in specific areas of cells. Hopefully, reflection signals might be sensitive enough to meet the requirements for biological analysis. Taking silver needles as an example, the mirror reflection of microtips can be easily detected and sensitively related to formation of black Ag2S, promising convenient and sensitive intracellular microarea analysis. Actually, light scattering, as a similar case with © 2014 American Chemical Society

Received: May 6, 2014 Accepted: July 2, 2014 Published: July 2, 2014 7734

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Figure 1. Schematic of H2S evaluation in live cells using silver needles.

solution (1 μg in 100 μL water) was added. After 30 s vortexing and 30 s sitting at room temperature, quartz needles were dipped into the solution for 1 min and then pulled out for 30 s. The procedure was repeated three times. The color of the mixture solution turned from yellow-greenish to yellow-brown, and the color of the needle tips became bright gray (see Figure S1 in the Supporting Information for the silver needle photo). Finally, silver needles were rinsed with water, blow-dried by nitrogen gently, and ready for use. All the reagent solutions were freshly made. All the chemicals were purchased from the Sinopharm Chemical Reagent Co., Ltd., except sodium sulfide anhydrous (Na2S, >92%), which was from the Shanghai Sangon Biotech Co., Ltd., and sodium nitroprusside dehydrate (SNP, 99%), which was from the Sigma-Aldrich (Wuxi) Life Science & Technology Co., Ltd. Water was deionized and further purified (>18.3 MΩ) by a Milli-Q water purification system (Millipore, Milford, MA) before begin used in this work. General Procedure for Light Reflection Measurements. The microscopic images of silver needles were taken by confocal laser scanning microscopy (CLSM, Olympus Fluoview FV1000, 20× objective lens, N.A. 0.75). The light reflection intensity was recorded when the focal distance was adjusted to make the reflection of the front of silver needles strongest. Also, the average reflection intensity was obtained through Gaussian fitting of the intensity of each pixel of the 50 μm front area of silver needles. The laser of 488 and 559 nm was used (power: 1.5−2.5%), and the band-pass was set as 495−595 and 565−665 nm, respectively. The scanning speed was 40 μs/pixel, and the voltage of photomultiplier tube (PMT) was 550 V. Cell Culture and Intracellular H2S Detection. The cell lines of HepG2 (human hepatocellular carcinoma) and A549 (human nonsmall cell lung cancer, adenocarcinoma) were both purchased from the Cell Resource Center, Institute of Basic Medical Science at the Chinese Academy of Medical Sciences (Beijing, China). They were cultured at 37 °C in 5% CO2, in DMEM medium (Corning), which was added with 10% fetal bovine serum (FBS, HyClone) and 100 units/mL penicillinstreptomycin (Sigma). All the mediums and buffers for cell experiments were sterilized. For the intracellular H2S detection, cells were washed with the Dulbecco’s phosphate-buffered saline (1× DPBS, pH = 7.4, Corning) twice, and then trypsinized with 1× trypsin EDTA (Corning). After being resuspended in culture medium, 100 μL

Herein, we plan to detect H2S in specific areas of live cells using the reflected light of silver needles to examine our hypothesis. As we know, H2S plays an important role in many physiological and pathological processes,15,16 including angiogenesis,17 apoptosis,18 anti-inflammation,19 and energy production in mitochondria under oxidative stress conditions,20 as well as mental deficiency like Down’s syndrome21 and Alzheimer’s disease.22 Many efforts, such as fluorescent imaging,23−28 chemiluminescent detection,29 and above-mentioned singleparticle light scattering,14,30 have been made to detect this endogenous signaling gaseous transmitter and neuromodulator in recent years. However, great challenges still remain when it comes to the accurate localization to specific cellular or subcellular locales, while in this study, with the help of the three-dimensional manipulator and microscope, we are able to target specific areas in live cells with the spatial resolution of 2 × 2 μm2 (Figure 1). To the best of our knowledge, this is the first time that the light reflection signals are used to detect H2S in specific areas of live cells. Combined with acceptable sensitivity and robust response, this strategy holds the promise of real-world application in biological research and medical diagnosis. Remarkably, with the development of microelectrode pulling technology, we expect to make silver needles with tips that are hundreds of nanometers and even smaller in the future. Then, this “proof-of-concept” strategy will further improve the spatial resolution, and more interesting phenomena like diffraction and surface plasmon might take place and give us more information if the size of needle tips reduces to 200 nm.

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EXPERIMENTAL SECTION Silver Needles Fabrication. We deposited silver onto quartz needles via silver-mirror reaction to fabricate silver needles. Generally, we pulled quartz tubes (o.d. 1.5 mm, i.d. 0.7 mm, 7.5 cm of length) into quartz needles (o.d. ∼1 μm) using a microelectrode puller (P-2000, Sutter Instrument Company). The parameters were set as 900 of Heat, 5 of Fil., 50 of Vel., 128 of Del., and 55 of Pul. Then, quartz needles were ultrasonically cleaned by water, ethanol, and water, in order. After blow-dried by nitrogen, quartz needles were dipped into silver-mirror reaction solutions, which were made according to our previous report.31 In a typical experiment, 100 μL of 0.1 M NaOH solution was added to an aqueous silver nitrate solution (1 mL, 0.1 M). Subsequently, the resulting dark-brown precipitate was redissolved by slowly adding 100 μL of 50% (v/v) ammonia−water with gentle vortexing. Then, a D-glucose 7735

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Figure 2. Microscopic images of silver needles and the reflection spectra of quartz needles, silver needles, and silver needles reacted with 1 μM Na2S for 3 min. The light sources are 488 nm (A and B) and 559 nm (C and D).

Figure 3. (A) Reflection intensity change plots with the increase of reaction time in Na2S solutions of different concentrations. (B) The reflection intensity changes (1 − I/I0) of silver needles as a function of Na2S concentration from 0.1 to 20 μM at various time points. Each error bar represents the standard deviation of 3 replicates.

of suspension (∼1 × 105 cells/mL) was added with 200 μL culture medium and cultured for 24 h. Then, cells were washed by 1× DPBS twice and cultured in Na2S or SNP added medium for other different periods. Before sampling with silver needles, cells were washed by 1× DPBS twice and 5 mM phosphate buffer (PB, pH = 7.4, homemade) with 0.1 M NaCl in it once, and then were added with 200 μL of 5 mM PB with 0.1 M NaCl. Silver needles were controlled by a three-dimensional manipulator (MP-225, Sutter Instrument Company) to precisely insert into specific areas of targeted cells. The smallest microstep size of the manipulator was 65 nm. All the operations were observed with a microscope (Olympus IX81, 40× objective lens, N.A. 0.95). After insertion, silver needles were kept in cells for 3 min to react with the intracellular H2S. Then silver needles were taken away from cells gently and rinsed in water for 30 s before reflection measurements.

then used a CLSM to characterize the reflection. As shown in Figure 2A,C, silver needles (o.d. 1−2 μm) could reflect the incident light (both 488 and 559 nm) effectively. This signal was light reflection due to the zero wavelength-shift of the light spectra (Figure 2B,D) taken by a fluorophotometer (F-7000, Hitachi). Optimized Dipping Times and Tip Size. Notably, the dipping times of needles into reaction solutions played a crucial role in the reflection intensity. Due to the round surface and refractive index difference between air and quartz,4 quartz needles could reflect light weakly while the reflection intensity increased significantly after silver deposition as shown in Supporting Information Figure S2. We obtained the optimized reflection signal when the dipping times reached three. So, we speculated that the smoothest silver deposition created the best reflection signal just as a smoother mirror proves to be a better mirror. In addition, we studied the interference of reflection profiles by the size of silver needles. As shown in Supporting Information Figure S3, the reflection position changed with the increase of the outside diameters (o.d.) of silver needle tips, indicating that the strongest reflection happened on the inner

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RESULTS AND DISCUSSION Silver Needle Design and Reflected Light Obtained. We readily fabricated silver needles by dip-coating silver growth on micron-sized quartz tips through silver-mirror reaction31 and 7736

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and outer walls of silver needles. Although the commercialized silver needles (medium NanoES spray capillaries, Thermo) gave the finest reflection signal with the sharpest tip (Supporting Information Figure S3A), they were too fragile to insert human cells, and the price (∼$40/needle) was much higher. In order to obtain the best sampling resolution in live cells, we chose the smallest silver needles with the tip o.d. of 1− 2 μm we could achieve for the following experiments. In-Vitro H2S Detection. It could be expected that H2S would reduce the reflection intensity of silver needles just like silverware turns black after reaction with sulfide. To demonstrate the analytical performance of silver needles toward H2S, we tested the reflection intensity changes of silver needles after reaction with Na2S aqueous solutions32 of varying concentrations (0.1−50 μM). As shown in Supporting Information Figure S5, the reflection intensity of silver needles reduced over time with the increase of fraction of Ag2S. A common explanation of reflection reduction was that black Ag2S covered the surface of bright silver and absorbed some part of incident light like painting black on a mirror. We also speculated that the destruction of the smooth structure of silver deposition after reaction with sulfide would lead to reflection reduction according to the scanning electron microscopic (SEM) images of the surface of silver needles (Supporting Information Figure S4). Also, the detachment of silver after reaction with 50 μM Na2S (Supporting Information Figure S5H) supported this speculation. To further investigate the quantitative performance of our approach, we studied the relationship between the reflection intensity decay rate and concentration of sulfide. As illustrated in Figure 3A, the reflection intensity reduced with reaction time, and the decay rate increased with rising of sulfide concentration. According to the previous report based on the same reaction,14,30 we charted the reflection change-concentration plots at the same reaction time (Figure 3B) and found that the reflection changes were very linearly dependent on the natural logarithm of applied Na2S concentrations at every time point except 50 μM Na2S, which made the silver deposition detach from the quartz tip (Supporting Information Figure S5H). As shown in Figure 3B, we obtained the best working curve with a LOD (limit of detection) of 50 nM and a linear logarithmic range 0.1−20 μM when the reaction time reached 3 min. So, our approach possessed comparable sensitivity with conventional fluorescent probes23,24 and short detection time (3 min). Combined with the micron localization ability, silver needles were considered as a novel candidate for endogenous H2S detection. Selectivity Study. We studied the selectivity of our approach by examining various species in parallel under the same condition, including reduced glutathione (GSH), oxidized glutathione (GSSG), L-cysteine (L-Cys), hydrogen peroxide, sodium nitrite, carbonates, sulfur-containing inorganic salts, and so on. As shown in Figure 4, the reflection intensity reduced significantly toward Na2S, whereas the reflection change toward other analytes at higher concentration was less obvious. Among the thiols at millimolar concentration, 5 mM L-Cys (analyte 14) reduced the reflection intensity of silver needles most significantly, but the reduction degree was much lower than 1 μM Na2S. There was another concern that the presence of chloride would affect the reflection of silver needles by formation of AgCl (pKsp = 9.74). But the extremely low solubility of Ag2S (pKsp = 50.83) made the formation of Ag2S dominant in this process so that the reflection of silver needles

Figure 4. Reflection intensity changes (1 − I/I0) of silver needles after 3 min reactions with different analytes. Analytes 1−18 represent (1) 1 μM Na2S, (2) 10 μM Na2S, (3) 1 mM H2O2, (4) 100 μM NaNO3, (5) 100 μM NaNO2, (6) 100 μM Na2CO3, (7) 100 μM NaHCO3, (8) 100 μM Na2SO4, (9) 50 μM Na2SO3, (10) 5 mM PB (pH = 7.4) with 0.1 M NaCl in it, (11) 100 μM SNP, (12) 50 μM L-Cys, (13) 500 μM LCys, (14) 5 mM L-Cys, (15) 100 μM GSH, (16) 1 mM GSH, (17) 250 μM GSSG, and (18) 2.5 mM GSSG, respectively. Each error bar represents the standard deviation of 3 replicates.

barely changed in 5 mM PB (pH = 7.4) with 0.1 M NaCl in it. This was why we used this buffer for intracellular H2S detection. We also tested the interference of SNP which was used as a stimulator for increasing endogenous H2S in A549 cells. As a sulfide-free reagent, SNP barely changed the reflection intensity of silver needles (analyte 11). Rapid H2S Detection in Live Cells. To determine whether our silver needles could respond to intracellular H2S, we inserted live HepG2 cells cocultured with Na2S-containing medium. Considering that the o.d. of a silver needle tip was 1− 2 μm and the smallest step of the manipulator was 65 nm, we could obtain sampling resolution of 2 × 2 μm2 in live cells so that we could easily target extranuclear or intranuclear areas as the average diameter of the nucleus is approximately 6 μm in mammalian cells.33 As shown in Figure 5, the reflection intensity of silver needles reduced after insertion into HepG2 cells, indicating the increase of intracellular H2S concentration after Na2S coculture. Also, the reflection intensity reduced more significantly with the increase of coculture time due to the H2S enrichment in extranuclear areas (Figure 5C). Interestingly, the reflection reduction of silver needles inserted in intranuclear areas was much less than in extranuclear areas (Supporting Information Figure S6), implying that the H2S level of cell nucleus might not rise up as much as cytoplasm during Na2S coculture. By the way, H2S in cytoplasm would react with silver needles if we let the tip stay in cytoplasm for an extended period (e.g., 5 min) before entering the cell nucleus (Supporting Information Figure S7). However, it took about 15 s for a silver needle to enter the nucleus from cytoplasm normally, and the interference of H2S in cytoplasm during such a short time was negligible. Notably, most fluorescent probes for H2S could not go into the cell nucleus easily23,25 while our silver needles could insert cell nucleus for H2S evaluation due to the accurate-localization ability. The fast response time (3 min) also offered the potential for efficient real-world applications. We also tried to perform in situ H2S monitoring in live cells cocultured with Na2S. As shown in Supporting Information 7737

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Figure 5. Microscopic images of silver needles before and after sampling HepG2 cells and the sampling position images: (A) HepG2 cells without Na2S treatment, (B) HepG2 cells treated with 50 μM Na2S for 10 min, and (C) HepG2 cells treated with 50 μM Na2S for 30 min. The light source is 559 nm.

the nanomolar range of endogenous H2S level reported by Kevil’s group.36 A 10 min Na2S coculture raised the H2S level in cellular cytoplasm of HepG2 cells to around 1 μM while the intranuclear H2S level still remained in the nanomolar range. With the coculture time extended to 30 min, the extranuclear H2S level of HepG2 cells exceeded 20 μM, but the increase of intranuclear H2S was much smaller. For A549 cells pretreated with SNP, the endogenous H2S level of cytoplasm raised to around 1 μM while H2S of the nucleus increased slightly like Na2S cocultured HepG2 cells.

Figure S8, the reflection intensity of needle tips was much weaker due to the angle of about 30° between the silver needle and objective lens. However, as time went on, we still could detect the reflection intensity decrease of the silver tip in a Na2S cocultured HepG2 cell (Supporting Information Figure S8C). We plan to design a new optical path system for better focusing and in situ reflection recording in our ongoing research. On the other hand, on the basis of irreversible Ag2S formation, this approach cannot tell reduction of intracellular H2S level. Endogenous H2S Evaluation. To further examine the sensitivity of this approach toward endogenous H2S in live cells, we used SNP pretreated A549 cells as a study model. According to the previous report,23 SNP, as a commercial NO donor, could upregulate the activity of H2S-related enzymes like CSE (cystathionine γ-lyase) and CBS (cystathionine β-synthase),34 stimulating the generation of endogenous H2S in A549 cells. As shown in Supporting Information Figure S9, we inserted both cell nucleus and cytoplasm to evaluate endogenous H2S level in these areas. The reflection intensity of the silver needle inserted in extranuclear area of an SNP pretreated cell reduced obviously as expected (Supporting Information Figure S9C). Also, similar to Na2S cocultured HepG2 cells, H2S concentration of A549 cell nucleus barely increased due to the SNP treatment (Supporting Information Figure S9D). It was consistent with the reports of endogenous H2S mainly generated from mitochondria.18,35 Finally, we summed up these results in Figure 6 and evaluated H2S level in specific areas of live cells through comparing Figure 6 and Figure 3B. The endogenous H2S of HepG2 cells without Na2S coculture and A549 cells without SNP pretreatment was undetectable, which was consistent with

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CONCLUSIONS In summary, we have developed a rapid and effective H2S detection strategy with accurate localization in live cells inspired by the poison test approach of ancient China. The reflection decrease of silver needles, which can readily insert live cells with high spatial resolution, illustrates the H2S level just like silverware turning black after reaction with sulfide residue. The success of endogenous H2S detection in specific areas of live cells indicates not only the powerful ability of precise positioning in single-cell analysis, but also great application potential of reflected light in analytical chemistry. In our ongoing studies, we are exploiting both labeling strategies on silver needles and reflection recording approaches in tissue samples to perform DNA/RNA or protein biomarker detection in live cells and in vivo medical diagnosis.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (+86)10-62787678. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the 973 program (2013CB933800), the National Natural Science Foundation of China (No. 21390411 and 21125525), and the Ministry of Science and Technology of China (No. 2012IM030400).

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Figure 6. Reflection intensity changes (1 − I/I0) of silver needles after sampling in extranulcear or intranuclear areas of different cell lines. Each error bar represents the standard deviation of 5 replicates.

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