ThiolâDisulfide Exchange Reaction for Cellular Glutathione Detection

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A Thiol-Disulfide Exchange Reaction for Cellular Glutathione Detection with Surface Enhanced Raman Scattering Chenghua Wei, Xiao Liu, Yun Gao, Yiping Wu, Xiaoyu Guo, Ye Ying, Ying Wen, and Hai-Feng Yang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 29 Aug 2018 Downloaded from http://pubs.acs.org on August 29, 2018

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Analytical Chemistry

A

Thiol-Disulfide

Exchange

Reaction

for

Cellular

Glutathione Detection with Surface Enhanced Raman Scattering

Chenghua Wei, Xiao Liu, Yun Gao, Yiping Wu*, Xiaoyu Guo, Ye Ying, Ying Wen, Haifeng Yang*

The Education Ministry Key Lab of Resource Chemistry, Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Municipal Education Committee Key Laboratory of Molecular Imaging Probes and Sensors and Department of Chemistry, Shanghai Normal University, Shanghai 200234, China

*Corresponding Authors Telephone: +86-21-64321701. E-mail: [email protected] [email protected] (Yiping Wu).

ACS Paragon Plus Environment

(Haifeng

Yang);

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Abnormal glutathione (GSH) levels are related to several diseases including cancer. In this paper, surface-enhanced Raman spectroscopy (SERS) based on a 2-(pyridyldithio) ethylamine (PDEA) modified AgNPs@Si wafer is proposed for intracellular glutathione (GSH) detection. PDEA plays multifunctional roles in the method, including the participation in the thiol-disulfide exchange reaction and the contribution with a great SERS signal reporter. With the addition of GSH, the disulfide bond of PDEA will be broken, releasing the pyridine ring in PDEA and resulting in a signal-off SERS response. The developed specific reaction-based SERS assay can detect GSH as low as 2.5×10-7 M. Where after, we employed this method to evaluate the cellular GSH levels, finding the levels in cancer cells are higher than that in normal cells. The PDEA modified AgNPs@Si-based SERS protocol demonstrates good selectivity and high sensitivity as well as robustness, which is suitable to evaluate cellular GSH levels.

Keywords: Glutathione, Surface-Enhanced Raman Scattering, Thiol-disulfide exchange reaction


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Introduction Glutathione is one of the most abundant antioxidant low-molecular-mass thiols in vivo, which occurs predominantly intracellular with the concentrations of 0.5 mM to about 10 mM. The intracellular glutathione exists in both reduced (GSH) and oxidized form (GSSG), while the majority (＞95%) of which is the reduced form1,2. Cellular GSH levels are related to various diseases including liver damage3, cystic fibrosis, Parkinson's disease4, acquired immune deficiency syndrome (AIDS), human immunodeficiency virus5,

6

(HIV) and cancer7-9. In tumor tissues, the GSH

concentrations are often elevated by as much as 2-fold to that in normal tissues, which is exploited to be a modulator for antitumor drug release10. Therefore, determination of cellular GSH with a simple and efficient method is important for drug delivery, cancer diagnosis and other biomedical applications. The classical method for thiol detection is the Ellman’s test, which is based on the thiol-disulfide exchange reaction occurring between the thiol and Ellman’s reagent (5, 5-dithiobis-(2-nitrobenzoic acid), DTNB). Thiols react with DTNB to form 2-nitro-5-thiobenzoate (TNB-) which will ionize to the TNB2- dianion in water at neutral or alkaline pH. TNB2− can be quantified in a spectrophotometer by measuring the absorbance at 412 nm. In turn, the quantitative determination of sulfhydryl group is achieved. In recent years, a variety of instrumental techniques have been developed for GSH detection, including fluorescentspectrometry11-13, colorimetric assay14-17, high performance liquid chromatography (HPLC)18,

19

and electrochemical

analysis20-22. Though these strategies are reliable in detecting GSH, they still suffer from the short comings of poor anti-interference ability, complicated sample preparation and expensive instruments. Over the past decade, surface-enhanced Raman scattering (SERS) spectroscopy has drawn considerable attentions in biological and medical detection applications owing to the specificity of molecular Raman spectrum combined with high sensitivity from plasmonic nanostructures23. As well known, Raman scattering signals are sharply amplified by putting the target molecules at or very close to the metallic nanostructure



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(normally, Au and Ag). The general accepted mechanisms for the enhancement is that the laser-excited surface-plasmon resonance of the metallic nanostructure amplifies the local electromagnetic (EM) fields, which improves the Raman signal of the surrounding probe molecules24-26. SERS should have a prospect for GSH detection in complex

bio-system.

Larsson

and

Lindgren

ever

used

gold-containing

chromatographic beads to record the spectra of tripeptide glutathione27. Since the low polarization of GSH, the direct SERS detection of GSH was not sensitive. After that, Ozaki et. al proposed a “heat-induced SERS sensing method” for rapid determination of glutathione in aqueous solutions28. Though a low detection limit was achieved, the interference is still inevitable. Moving on, competitive surface-enhanced Raman spectroscopy was developed for GSH measurement. In these methods, the added GSH would replace the Raman-probe, crystal violet (CV) or 4, 4’-dipyridyl (Dpy), on the surface of the substrate leading to a signal decrease25, 29. For GSH, the Raman-probe mediate competitive method is helpful, but the uses of sol-gel-based SERS substrates in the works suffer from a fatal deficiency of easy-aggregation. Inspired by the aforementioned thiol-disulfide exchange reaction, in present work, we developed a PDEA modified AgNPs@Si substrate for sensitive SERS detection of trace GSH. PDEA is a molecule with multi-functional groups including amino-group, disulfide bond and pyridine ring. Among them, amino-group helps PDEA easily assemble on the surface of AgNPs@Si though the Ag-N bond, the pyridine ring of PDEA contributes to a high SERS response and the disulfide bond participates in the thiol-disulfide exchange reaction with GSH. The broken of disulfide bond by GSH will release pyridine ring from the surface of AgNPs@Si, resulting in a decrease of the SERS signal. Consequently, the SERS signal is closely related to the content of GSH. By this specific surface reaction strategy, the rapid and sensitive determination of GSH is achieved in a robust way.

Experimental Section Chemicals and materials

Silicon (100) wafer (phosphate-doped, p-type,

0.01-0.02 Ω sensitivity) was bought from Hefei Kejing Materials Technology Co., Ltd. ACS Paragon Plus Environment

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(Hefei, P. R. China). Hydrofluoric acid (HF) was bought from Greagent (Shanghai, P. R. China). Silver nitrate (AgNO3) was purchased from Shanghai Chemical Reagent Co., Ltd (Shanghai, China). 2-(Pyridyldithio) ethylamine hydrochloride (PDEA) was obtained from MedChemExpress Co., Ltd (Shanghai, P. R. China). Rhodamine 6G (R6G), human srum albumin (HAS) and gutathione (GSH) were from Sigma-Aldrich (St. Louis, MO, USA). Methionine (Met), lysine (Lys), histidine (His), glucose, glycine (Gly), glutamic acid (Glu) and cysteine (Cys) and homocysteine (Hcy) were bought from Shanghai Macklin Biochemical Technology Co., Ltd (Shanghai, P. R. China). The N-ethylmaleimide (NEM) was purchased from Adamas Reagent, Ltd (Shanghai, China) for thiol-blocking30. Sulfosalicylic acid (SSA) was bought from Sangon Biotech Co., Ltd (Shanghai, China). Glutathione quantification kit was purchased from Dojindo China Co., Ltd (Shanghai, China). Three human cell lines, including one normal cell (293T, 5×106 cells) and two cancer cells (A549, 5×106 cells and HeLa, 1×106 cells), were supplied by Professor feizhen Wu (Institutes of Biomedical Sciences Fudan University). All chemicals were used without further purification. All aqueous solutions were prepared and diluted with ultrapure water (18.2 MΩ.cm) from the Shanghai senkang Milli-Q system. Preparation of PDEA modified AgNPs@Si Chip

The AgNPs@Si chip was

prepared facilely via in situ growth of AgNPs on the silicon wafer by an established HF-etching method. To remove organics, the Si wafer was first cleaned according to the reported method31. Then, the cleaned Si wafer (0.5 cm × 0.5 cm) was immersed into HF solution (5%) for 30 min to obtain a Si-H bonds covered surface32. After that, the modified chip was placed into AgNO3 solution with 10% HF immediately for 90 s. The resultant AgNPs@Si chip was thoroughly washed with high-purity water and blown dry with nitrogen for further use. The morphology of AgNPs@Si chip was characterized by scanning electron microscopy (SEM). SEM was carried out using a Hitachi S-4800 scanning electron microscope. AgNPs@Si chip was modified with PDEA by N-Ag-bond-based self-assembly method. In brief, 20 µL 10-4 M PDEA was dropped on the surface of AgNPs@Si



chip and incubated for 2 h. Then, it was rinsed with pure water to remove the nonspecific adsorption PDEA and dried with nitrogen. SERS measurement of GSH

The PDEA modified AgNPs@Si chip was incubated

with different concentrations of GSH for 2 h. Then, the chip was washed with pure water and blown dry with nitrogen for SERS measurement. The selectivity of the chip assay was also investigated with a variety of the interfering species including Met, Lys, His, glucose, Gly, Glu, Cys, Hcy, HAS and their mixture. The Raman detection was carried out on a Dilor confocal laser Raman system (Super LabRam II) with a 5 mW He-Ne laser at 632.8 nm. The acquisition time was 8 s with 3 accumulations. Evaluation of cellular GSH levels

To compare the GSH levels in different cells,

three human cells lines were selected for the cellular GSH level evaluation. The cells were first detached from the dish using trypsin and washed with phosphate buffer solution (PBS), followed by a centrifugation (4 0C, 200 g, and 10 min) for collection. To obtain the cellular content, 80 µL 10 mM HCl was added for cell broken followed by repeated freezing and thawing twice. After that, 20 µL 5% SSA were added into the solution, followed by a centrifugation (4 0C, 8000 g, and 10 min) to remove the cell debris. About 100 µL supernatant was collected and transferred to a new tube for the following GSH assay. A commercial GSH kit and the PDEA modified AgNPs@Si chip were employed to evaluate the cellular GSH levels. The detection principle of the kit was described in Introduction part. The cell lysate was diluted 400 fold for GSH kit and diluted 800 fold for SERS measurement.

Results and discussion Characterization of AgNPs@Si Chip

The AgNPs@Si is prepared via in situ

growth of AgNPs on the silicon wafer by reducing Ag ions with the Si-H bonds. Scanning electronic microscopy (SEM) images of the AgNPs@Si prepared in different concentrations of AgNO3 are presented in Figure 1. Obviously, the AgNPs prepared under different concentrations of AgNO3 are different in morphology and


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sizes. The AgNPs prepared under 3 mM AgNO3 (see Figure 1A-c) is like broad bean distributing uniformly on the surface of silicon wafer, denoted as AgNPs-3@Si. Next, taking Rhodamine 6G (R6G) as the probe, SERS measurement of the different AgNPs@Si chips were conducted. As shown in Figure 1B, the Raman intensity of 10-6 M R6G on the surface of AgNPs-3@Si chip is the greatest. In addition, the Raman spectra of R6G with AgNPs sol and on the pure silicon wafer were also recorded (Figure S1). The result reveals that to the Raman probe the prepared AgNPs-3@Si has the best Raman enhancement effect. Figure 2A is the concentration dependent SERS spectra of R6G on the optimal AgNPs-3@Si surface and the lowest detectable concentration reaches down to 10-10 M. According to the previous reports33, 34

, the great enhancement mechanism of AgNPs@Si may be from the boiling

electromagnetic field in hot spots which were formed by the strongly coupled and interconnected AgNPs distributing on the semiconducting silicon wafer.

Figure 1. (A) SEM images of AgNPs@Si chips prepared by different concentrations of AgNO3, a) 1 mM, b) 2 mM, c) 3 mM, and d) 4 mM. (B) SERS spectra of 10-6 M R6G dispersed on the surface of different AgNPs@Si chips. To evaluate the reproducibility of the AgNPs-3@Si chip, Raman spectra of R6G was recorded from 10 random spots on the substrate with an area of 0.5 cm × 0.5 cm. The marked peaks in Figure 2B are all corresponding to the Raman bands of R6G. Through statistic on the most prominent band of R6G at 1361 cm-1, the relative standard deviation (RSD) is 4.44%, suggesting the excellent uniformity and reproducibility of Raman signals on the prepared AgNPs-3@Si. The improved reproducibility of the substrate is due to the in situ growth of AgNPs on the Si wafer,



which can tightly fix the nanoparticles on the surface and effectively prevent the random aggregation.

Figure 2. (A) AgNPs-3@Si-based SERS spectra of different concentrations of R6G, from a to f: 10-6 M, 10-7 M, 10-8 M, 10-9 M, 10-10 M, and 0 M. (B) AgNPs-3@Si-based SERS spectra of 10−6 M R6G collected from 10 random spots on the surface of the chip. PDEA modified substrate

Although the AgNPs-3@Si-based SERS method

demonstrated a high enhancement of R6G Raman scattering, the direct detection of trace GSH with SERS technique is still difficult since GSH has tiny Raman scattering section. As mentioned above, the visible spectrophotometric measurement of protein sulfhydryls with Ellman’s reagent is based on a thiol-disulfide exchange reaction. By extending this exchange reaction into SERS assay, we proposed a signal-off strategy to achieve the detection of low amount of GSH, as cartooned in Scheme 1. PDEA is a novel disulfide intercalating cross-linking reagent used in the preparation of a drug-octreotide conjugate. With the special molecular structure, it is a perfect candidate for the construction of the highly selective and sensitive GSH Raman sensor as well. The amino group in PDEA is beneficial for self-assembly on the metal surface and the pyridine ring in the molecule has a high SERS response. As shown in Figure 3A, the dominated vibrations at 1000, 1050, 1082, and 1116 cm-1 are all from the pyridine ring. With the addition of GSH, the disulfide bond of PDEA will be broken efficiently via the thiol-disulfide exchange reaction. The released pyridine ring from the surface of AgNPs-3@Si results in the decrease of the SERS signal. By this, a signal-off type Raman sensor for trace GSH analysis is developed.


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Scheme 1. Schematic illustration of the GSH detection

To optimize the amount of PDEA modified on the surface of the AgNPs-3@Si chip, the PDEA-dependent SERS responses were acquired on the AgNPs-3@Si chip. As illustrated in Figure 3B, the SERS signal first increased with PDEA from 10-6 M to 10-4 M. Then, it reaches a platform when the concentration is higher than 10-4 M, suggesting a full cover of PDEA on the surface of AgNPs-3@Si. Therefore, 10-4 M PDEA is used for the construction of the GSH Raman sensor in the following experiment.

Figure 3. (A) The Raman spectra of (a) AgNPs-3@Si, (b) powder PDEA, and (c) SERS spectrum of 10-4 M PDEA on the surface of AgNPs-3@Si. (B) The SERS spectra of different concentrations of PDEA modified AgNPs-3@Si, from a to e: 10-2 M, 10-3 M, 10-4 M, 10-5 M, and 10-6 M. SERS detection of GSH

Glutathione is a small molecule peptide, containing

three amino acid residues glutamate, cysteine and glycine. Due to the small Raman scattering section areas of amino acid residues, GSH molecule has weak SERS response. Undoubtedly, the SERS response of 10-3 M GSH recorded on the surface of



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AgNPs-3@Si is basically the same as the back ground (Figure 4), revealing the difficult of direct GSH determination with AgNPs-3@Si. The SERS signal of the substrate may be from the nonspecific adsorption of nitrate since the AgNPs@Si chip was prepared facilely via in situ growth of AgNPs on the clean silicon wafer by an established HF-etching method and no additional reagents were added except for silver nitrate as the silver source. The added GSH with the low polarization itself do not have SERS response but it can replace the nonspecific adsorbed nitrate on the surface of the substrate leading to a weaker SERS response.

Figure 4. The SERS spectra of AgNPs-3@Si chip (black) and 10-3 M GSH on its surface (red) Figure 5A is the SERS spectra of PDEA modified AgNPs-3@Si recorded in the presence of various concentrations of GSH. Obviously, the SERS signal of PDEA decreased rapidly with the increase of GSH amount. When the concentration of GSH reached 1.0 µM, the SERS intensity of PDEA faded off almost completely, indicating the finish of the thiol-disulfide exchange reaction at the surface of AgNPs-3@Si. The highest SERS peak of PDEA at 1000 cm-1 is used as the marker band for GSH determination since the peak intensity is closely related to the varying GSH concentrations. The inset in Figure 5B shows a linear relationship between the

peak

intensity (1000 cm-1) and GSH concentration in the range from 0 to 1.0 µM with R2=0.9638. The error bars were estimated from 3 times repeating measurements. The detection of GSH is as low as 2.5×10-7 M. Since the proposed method is simple with


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a good sensitivity, this assay shows a good prospect in practical application.

Figure 5. (A) SERS spectra of PDEA modified AgNPs-3@Si with different concentrations of GSH, from a to i: 0, 0.250, 0.500, 0.750, 1.00, 1.25, 1.50, 1.75 and 2.00 µM, respectively. (B) The plot of intensities of SERS peak at 1000 cm-1 versus GSH concentrations. Inset: the linear calibration plotted in the concentration range from 0 to 1.0 µM. Considering organic molecules, especially the amino acids or proteins containing the sulfhydryl group, might disturb the SERS detection of GSH, the selectivity of the proposed sensor was investigated with Cys, Hcy, Met, Lys, His, Gly, Glu, glucose and HAS. As shown in Figure 6, most of the interferences from the investigated molecules could be negligible except that of Cys and Hcy. The free-SH group in small molecules Cys and Hcy may be involved in the thiol-disulfide exchange reaction resulting in a slight signal decline. As for HAS, it would not interfere with GSH detection as well. Though there may be free thiols in proteins, the large sizes of proteins hinder the thiol-disulfide exchange reaction between PDEA and thiols. Considering Cys is the most abundant plasma aminothiol (total concentration, ～250 µM), which is much higher than plasma glutathione (total concentration, ～ 6 µM)35,

36

, the PDEA

modified AgNPs-3@Si chip is suitable for the detection of the total amount of small thiol molecules in plasma.



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Figure 6. (A) Selectivity of the PDEA modified AgNPs-3@Si based SERS sensor. The concentration of GSH, Cys and Hcy are 1.5 × 10 −6 M. The concentration of HAS is 1 mg/mL. The others are 10-4 M. (B) The corresponding histogram.

Evaluation of cellular GSH levels

To apply the PDEA modified AgNPs-3@Si

chip for a biomedical detection problem, we employ the method to evaluate the cellular glutathione levels. The intracellular condition is different from that of plasma. Glutathione in cytoplasm is the most abundant biothiol with a level of 0.5-10 mM, which is much higher than that of Cys/Hcy (µM)

2, 37, 38

. Therefore, with proper

sample dilution, the interference from Cys/Hcy and other biothiols is completely negligible in the case of intracellular glutathione evaluation. In this study, the developed method was employed to evaluate three human cell lines, including one normal cell line (293T) and two cancer cell lines (A549 and HeLa). For comparison, a commercial GSH kit was employed to evaluate the cellular GSH levels as well. Both the methods were used for evaluation the GSH levels in cell lysate. As shown in Figure 7, the results obtained by the two approaches have good correlation. GSH level in A549 cell is the highest (0.60 mM), followed by 293T (0.29 mM), and HeLa (0.21 mM). The GSH level in HeLa cells is the lowest, which is inconsistent with those previously reported10,

38

. This may be due to the difference in cell density. As

indicated in the experiment section, the cell density of 293T and 549A is five-fold more than that of HeLa. Compared with the GSH kit, our approach has advantages in terms of operation. The GSH kit is time-consuming and may be compromised due to the enzyme inactivity, while our chip is simple preparation and convenient storage. Taking together, the above results demonstrate that the developed PDEA modified


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AgNPs-3@Si-based SERS method is a reliable, rapid and sensitive technique for cellular GSH evaluation.

Figure 7. (A) The SERS spectra of the real samples. The cell lysate were diluted 800 fold for SERS measurement. Blank is the cell lysate previously reacted with equivoluminal 10-4 M NEM for thiol-blocking. (B) Schematic illustration of the developed approach and the commercial GSH kit for the qualitative evaluation of cellular GSH levels. The number marked on the histogram is the cell density. The error bars represent standard deviation of three measurements.

Conclusion We have proposed a rapid, effective, and sensitive SERS method for intracellular GSH determination. The SERS detection strategy was designed based on a thiol-disulfide exchange reaction via constructing a PDEA modified AgNPs-3@Si substrate. Emphatically, the AgNPs-3@Si prepared by a simple HF-etching method exhibited pronounced SERS activity. The PDEA modified AgNPs-3@Si induces the GSH approaching into the vicinity of hot spots of AgNPs and participating in the thiol-disulfide exchange reaction. The assay realized the determination of GSH in cell. As a result, such specifically reactive modification SERS assay has great potential for the GSH detection in biomedical diagnosis and can provide guidance for biomedical applications that highly depend on intracellular GSH levels.



Supporting Information The Supporting Information is available free of charge. It includes Raman spectra of 10-7 M R6G on the different substrates; The UV-visible adsorption of NEM after reaction with Cys/GSH; The SERS spectrum of the sample after thiol-blocking; the data of GSH kit.

Acknowledgements This work is supported by the National Natural Science Foundation of China (21475088, 21507087), PCSIRT (IRT1269), Chenguang Program of Shanghai Municipal Education Commission, International Joint Laboratory on Resource Chemistry (IJLRC) and Shanghai Key Laboratory of Rare Earth Functional Materials.

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and

Ultraviolet

Detection

after

Pre-column

Derivatization

with

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For TOC Only



Figure 1. (A) SEM images of AgNPs@Si chips prepared by different concentrations of AgNO3, a) 1 mM, b) 2 mM, c) 3 mM, and d) 4 mM. (B) SERS spectra of 10-6 M R6G dispersed on the surface of different AgNPs@Si chips. 501x178mm (150 x 150 DPI)


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Figure 6. (A) Selectivity of the PDEA modified AgNPs-3@Si based SERS sensor. The concentration of GSH, Cys and Hcy are 1.5 × 10 −6 M. The concentration of HAS is 1 mg/mL. The others are 10-4 M. (B) The corresponding histogram. 58x20mm (600 x 600 DPI)



Figure 7. (A) The SERS spectra of the real samples. The cell lysate were diluted 800 times for SERS measurement. Blank is the cell lysate previously reacted with equivoluminal 10-4 M NEM for thiol-blocking. (B) Schematic illustration of the developed approach and the commercial GSH kit for the qualitative evaluation of cellular GSH levels. The number marked on the histogram is the cell density. The error bars represent standard deviation of three measurements.

2155x843mm (96 x 96 DPI)


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ThiolâDisulfide Exchange Reaction for Cellular Glutathione Detection

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ThiolâDisulfide Exchange Reaction for Cellular Glutathione Detection