Nanozyme based colorimetric detection of biogenic gaseous H2S using Ag@Au core/shell nanoplates with peroxidase-like activity.

A highly sensitive and facile colorimetric assay is introduced for detecting biogenic gaseous H2S using peroxidase (POD)-like catalytic activity of silver core/gold shell nanoplates (Ag@Au NPls). H2S can react with Ag@Au NPls to form Ag2S or Au2S on their surface, which can reduce POD-like activity of Ag@Au NPls and consequently decrease the absorbance at 650 nm due to oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). For in situ and multiple detection of H2S, we utilized a microplate cover with 24 polydimethylsiloxane inner wells where Ag@Au NPls reacted with H2S gas followed by treatment with TMB/H2O2. As a result, the change in absorbance at 650 nm showed a linear relationship with the H2S concentration in the range 0.33 to 2.96 µM (0.36 absorbance/µM H2S in PBS, R2 = 0.994) with a limit of detection of 263 nM and a relative standard deviation of 4.4%. Finally, this assay could detect H2S released from Eikenella corrodens, used as a model bacterium, in a short time (20 min) or at a low number of bacteria (1 × 104 colony forming units/mL). Therefore, this assay is expected to be applied for the study of H2S signaling in bacterial physiology, as well as measure H2S production released from other oral bacteria that cause halitosis and oral diseases, leading to the subsequent diagnosis.

Simple and Sensitive Detection of Bacterial Hydrogen Sulfide Production Using a Paper-Based Colorimetric Assay.

Hydrogen sulfide (H2S) is known to participate in bacteria-induced inflammatory response in periodontal diseases. Therefore, it is necessary to quantify H2S produced by oral bacteria for diagnosis and treatment of oral diseases including halitosis and periodontal disease. In this study, we introduce a paper-based colorimetric assay for detecting bacterial H2S utilizing silver/Nafion/polyvinylpyrrolidone membrane and a 96-well microplate. This H2S-sensing paper showed a good sensitivity (8.27 blue channel intensity/µM H2S, R2 = 0.9996), which was higher than that of lead acetate paper (6.05 blue channel intensity/µM H2S, R2 = 0.9959). We analyzed the difference in H2S concentration released from four kinds of oral bacteria (Eikenella corrodens, Streptococcus sobrinus, Streptococcus mutans, and Lactobacillus casei). Finally, the H2S level in Eikenella corrodens while varying the concentration of cysteine and treatment time was quantified. This paper-based colorimetric assay can be utilized as a simple and effective tool for in vitro screening of H2S-producing ability of many bacteria as well as salivary H2S analysis.

Rapid and simple colorimetric detection of hydrogen sulfide using an etching-resistant effect on silver nanoprisms.

A fast and sensitive colorimetric paper sensor has been developed using silver nanoprisms (Ag NPRs) with an edge length of ~50 nm for the detection of free H2S gas. We prepared two types of Ag NPRs-coated H2S sensing papers: a multi-zone patterned paper for passive (diffusion mode), and a single-zone patterned paper for pumped mode of H2S gas. The change in color intensity was quantitatively analyzed of Ag NPRs-coated paper after KCl treatment depending on the concentration of H2S gas, from yellow to purplish brown. As a result, Ag NPRs-coated H2S sensing paper showed good sensitivity with a linear range of 1.03 to 32.9 µM H2S, high selectivity, and good reproducibility and stability, together with a fast response time of 1 min. The developed H2S sensing paper was applied to detect the free H2S gas released from three types of garlic including crushed, peeled, and fresh garlic. Therefore, it can be utilized as a simple, fast, and reliable tool for on-site colorimetric detection of free H2S gas for quality control of dietary supplements and exhaled breath analysis.Graphical abstract.

Characterization of H2S releasing properties of various H2S donors utilizing microplate cover-based colorimetric assay.

In this study, we characterized the potential H2S-releasing properties of seven different H2S donors, including sodium sulfide (Na2S), sodium hydrosulfide (NaHS), diallyl disulfide (DADS), diallyl trisulfide (DATS), sodium thiosulfate (Na2S2O3), morpholin-4-ium 4-methoxyphenyl-morpholino-phosphinodithioate (GYY4137), and Lawesson's reagent, in three assay solutions, phosphate buffered saline (PBS, pH 7.4), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (HBS, pH 7.6), and cell growth media (GM), utilizing our microplate cover-based colorimetric assay. For quantitative analyses of H2S-releasing characteristics of the various donors, we evaluated four parameters, maximum concentration of H2S at the steady state (Cmax), the time required to reach half of Cmax (t1/2), maximum releasing rate of H2S (Rmax), and time at H2S (tr-max). The results showed that the H2S-releasing kinetics of each H2S donor were dependent on the type of assay solution. In particular, the addition of GSH to DATS in GM released the fastest and highest amounts of H2S among the four H2S donors in the following order: DATS > DADS > Na2S ~ NaHS. The H2S-releasing characteristics of the H2S donors were well-matched with cell viability results of human prostate cancer PC-3 cells. Therefore, the microplate cover-based colorimetric assay will be a useful tool for accurate and efficient measurements of H2S-releasing dynamics.

Cytotoxicity evaluation of sodium lauryl sulfate in a paper-based 3D cell culture system.

Because three-dimensional (3D) cell culture is more similar to in vivo cell microenvironments than two-dimensional (2D) cell culture, various 3D cell culture systems have been developed. Recently, paper has been used as a promising material for 3D cell culture and tissue models due to its flexibility, ease of manufacture, low cost, and widespread accessibility. In this study, we fabricated a paper-based 3D cell culture platform consisting of a hydrophilic region for cell attachment and a hydrophobic region printed with wax. Using this paper platform for 3D culture of L929 cells, we evaluated the cytotoxicity of a model substance, sodium lauryl sulfate (SLS), using water-soluble tetrazolium salt, Live/Dead, and luminescence assays. Then we compared those cytotoxicity results with results from a conventional 3D cell culture kit and 2D cell culture. We found that 3D cultured cells on paper responded more sensitively to SLS than 2D cultured cells, and the cytotoxicity of SLS to cells grown on the paper-based 3D cell culture platform was similar to that of cells grown using a commercially available 3D cell culture kit. Therefore, we expect that our paper-based 3D cell culture platform can be applied as a simple and facile tool for cell viability evaluation.

Targeting hydrogen sulphide signaling in breast cancer.

INTRODUCTION: Hydrogen sulphide (H2S) has been established as a key member of the gasotransmitters family that recently showed a pivotal role in various pathological conditions including cancer. OBJECTIVES: This study investigated the role of H2S in breast cancer (BC) pathogenesis, on BC immune recognition capacity and the consequence of targeting H2S using non-coding RNAs. METHODS: Eighty BC patients have been recruited for the study. BC cell lines were cultured and transfected using validated oligonucleotide delivery system. Gene and protein expression analysis was performed using qRT-PCR, western blot and flow-cytometry. In-vitro analysis for BC hallmarks was performed using MTT, BrdU, Modified Boyden chamber, migration and colony forming assays. H2S and nitric oxide (NO) levels were measured spectrophotometrically. Primary natural killer cells (NK cells) and T cell isolation and chimeric antigen receptor transduction (CAR T cells) were performed using appropriate kits. NK and T cells cytotoxicity was measured. Finally, computational target prediction analysis and binding confirmation analyses were performed using different software and dual luciferase assay kit, respectively. RESULTS: The H2S synthesizing enzymes, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), exhibited elevated levels in the clinical samples that correlated with tumor proliferation index. Knock-down of CBS and CSE in the HER2+ BC and triple negative BC (TNBC) cells resulted in significant attenuation of BC malignancy. In addition to increased susceptibility of HER2+ BC and TNBC to the cytotoxic activity of HER2 targeting CAR T cells and NK cells, respectively. Transcriptomic and phosphoprotein analysis revealed that H2S signaling is mediated through Akt in MCF7, STAT3 in MDA-MB-231 and miR-155/ NOS2/NO signaling in both cell lines. Lastly, miR-4317 was found to function as an upstream regulator of CBS and CSE synergistically abrogates the malignancy of BC cells. CONCLUSION: These findings demonstrate the potential role of H2S signaling in BC pathogenesis and the potential of its targeting for disease mitigation.

Hydrogen sulfide donor, NaHS, stimulates ANP secretion via the KATP channel and the NOS/sGC pathway in rat atria.

Hydrogen sulfide (H2S) is normally produced from l-cysteine in mammalian tissues and related to the pathogenesis of cardiovascular diseases. The aim of this study is to investigate the effects of H2S donor on atrial natriuretic peptide (ANP) secretion and define its mechanism using normal and isoproterenol (ISP)-treated rats. Several H2S donors were perfused into isolated beating rat atria, and atrial pressure (AP) and ANP secretion were measured. NaHS augmented high stretch-induced ANP secretion and decreased AP in a dose-dependent manner. The high stretch-induced ANP secretion was stimulated by Na2S but was not changed by GYY4137 and sodium thiosulfate. NaHS and Na2S produced very high amount of H2S rapidly whereas GYY4137 produced very low amount of H2S slowly. NaHS-stimulated ANP secretion was blocked by the pretreatment with inhibitor for KATP channel, nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC), phosphoinositol 3 kinase (PI3K) or protein kinase B. H2S synthesis enzyme inhibitor (DL-propargylglycine) did not show any significant changes in atrial parameters. However, the response of ANP secretion to NaHS markedly attenuated and DL-propargylglycine suppressed ANP secretion in ISP-treated rat atria. The expression of eNOS protein was decreased but the expression of cardiomyocyte-specific H2S producing enzyme, cystathione γ-lyase, was not changed in ISP-treated rat atria. The attenuation of NaHS-induced ANP secretion in ISP-treated rat atria may be due to the low expression of eNOS protein. These findings clarify that NaHS stimulates ANP secretion via the KATP channel and the PI3K/Akt/NOS/sGC pathway in rat atria.

Paper-Based Surface-Enhanced Raman Spectroscopy for Diagnosing Prenatal Diseases in Women.

We report the development of a surface-enhanced Raman spectroscopy sensor chip by decorating gold nanoparticles (AuNPs) on ZnO nanorod (ZnO NR) arrays vertically grown on cellulose paper (C). We show that these chips can enhance the Raman signal by 1.25 × 107 with an excellent reproducibility of <6%. We show that we can measure trace amounts of human amniotic fluids of patients with subclinical intra-amniotic infection (IAI) and preterm delivery (PTD) using the chip in combination with a multivariate statistics-derived machine-learning-trained bioclassification method. We can detect the presence of prenatal diseases and identify the types of diseases from amniotic fluids with >92% clinical sensitivity and specificity. Our technology has the potential to be used for the early detection of prenatal diseases and can be adapted for point-of-care applications.

A label-free cellulose SERS biosensor chip with improvement of nanoparticle-enhanced LSPR effects for early diagnosis of subarachnoid hemorrhage-induced complications.

It is very difficult to predict some complications after subarachnoid hemorrhage (SAH), despite rapid advances in medical science. Herein, we introduce a label-free cellulose surface-enhanced Raman spectroscopy (SERS) biosensor chip with pH-functionalized, gold nanoparticle (AuNP)-enhanced localized surface plasmon resonance (LSPR) effects for identification of SAH-induced cerebral vasospasm and hydrocephalus caused by cerebrospinal fluid (CSF). The SERS biosensor chip was implemented by the synthesis reaction of the AuNPs, which were charged positively through pH level adjustment, onto a negatively-charged cellulose substrate with ξ = -30.7 mV. The zeta potential, nanostructural properties, nanocrystallinity, and computational calculation-based electric field distributions of the cellulose-originated AuNPs were optimized to maximize LSPR phenomena and then characterized. Additionally, the performance of the SERS biosensor was compared under two representative excitation laser sources in the visible region (532 nm) and near-infrared region (785 nm). The Raman activities of our SERS biosensor chip were evaluated by trace small molecules (crystal violet, 2 µL), and the biosensor achieved an enhancement factor of 3.29 × 109 for the analytic concept with an excellent reproducibility of 8.5% relative standard deviation and a detection limit of 0.74 pM. Furthermore, the experimental results revealed that the five proposed SERS-based biomarkers could provide important information for identifying and predicting SAH-induced cerebral vasospasm and hydrocephalus complications (91.1% reliability and 19.3% reproducibility). Therefore, this facile and effective principle of our SERS biosensor chip may inspire the basis and strategies for the development of sensing platforms to predict critical complications in various neurosurgical diagnoses.

Colorimetric detection of endogenous hydrogen sulfide production in living cells.

Hydrogen sulfide (H2S) has received great attention as a third gaseous signal transmitter, following nitric oxide and carbon monoxide. In particular, H2S plays an important role in the regulation of cancer cell biology. Therefore, the detection of endogenous H2S concentrations within biological systems can be helpful to understand the role of gasotransmitters in pathophysiology. Although a simple and inexpensive method for the detection of H2S has been developed, its direct and precise measurement in living cells remains a challenge. In this study, we introduced a simple, facile, and inexpensive colorimetric system for selective H2S detection in living cells using a silver-embedded Nafion/polyvinylpyrrolidone (PVP) membrane. This membrane could be easily applied onto a polystyrene microplate cover. First, we optimized the composition of the coating membrane, such as the PVP/Nafion mixing ratio and AgNO3 concentration, as well as the pH of the Na2S (H2S donor) solution and the reaction time. Next, the in vitro performance of a colorimetric detection assay utilizing the silver/Nafion/PVP membrane was evaluated utilizing a known concentration of Na2S standard solution both at room temperature and at 37°C in a 5% CO2 incubator. As a result, the sensitivity of the colorimetric assay for H2S at 37°C in the incubator (0.0056Abs./µM Na2S, R2=0.9948) was similar to that at room temperature (0.0055Abs./µM Na2S, R2=0.9967). Moreover, these assays were less sensitive to interference from compounds such as glutathione, l-cysteine (Cys), and dithiothreitol than to the H2S from Na2S. This assay based on the silver/Nafion/PVP membrane also showed excellent reproducibility (2.8% RSD). Finally, we successfully measured the endogenous H2S concentrations in live C6 glioma cells by s-(5'-adenosyl)-l-methionine stimulation with and without Cys and l-homocysteine, utilizing the silver/Nafion/PVP membrane. In summary, colorimetric assays using silver/Nafion/PVP-coated membranes can be simple, robust, and reliable tools for the detection of H2S that can avoid the complicated and labor-intensive analytical approach used in conventional biology. In addition, we expect that this assay will demonstrate a powerful ability to study pathophysiological pathways that involve H2S.