A Facile Raman Spectroscopy Method for Online Monitoring of Crystal Plane Orientation of Favipiravir.

The efficiency of pharmacotherapy is significantly influenced by the crystal habit and polymorphic form of the drugs. Especially due to the anisotropy of different facets in crystalline material, crystal habit impacts the physicochemical properties and behaviors of a drug, which has been rarely reported. This paper describes a facile method for online monitoring of crystal plane orientation of favipiravir (T-705) by Raman spectroscopy. Firstly, we investigated the synergy of multiple physicochemical fields (solvation, agitated flow fields, etc.), and then prepared favipiravir crystals with different orientations in a controllable manner. Secondly, to establish the connection between crystal planes and Raman spectra, the favipiravir crystals were theoretically analyzed at the molecular and structural levels using density functional theory (DFT) and three dimensional (3D) visualization tools. Finally, we based on standard samples and applied it to 12 actual samples to evaluate the crystal habit of favipiravir. The results are similar to the classical X-ray diffraction (XRD) method. Additionally, the XRD method is difficult to be monitored online, while the Raman method is non-contact, fast, and requires no sample preparation, showing a great application prospect in the pharmaceutical process.

Thermal Degradation Characteristics of Styrene-Butadiene-Styrene Copolymer Asphalt Binder Filled with an Inorganic Flame-Retarding Agent.

Asphalt binder is a complex mixture of dark brown polymers composed of hydrocarbons with generally poor fire resistance. To improve its flame retardancy when used in tunnel asphalt pavements, a new inorganic flame-retardant filler (FR) containing magnesium hydroxide, aluminum hydroxide, inorganic phosphate, and melamine salt was explored. Thereafter, limiting oxygen index (LOI) and smoke suppression tests for the flame-retarded asphalt binder (FRA) mastics mixed with FR and styrene-butadiene-styrene (SBS) copolymer asphalt binder were conducted. Thermogravimetric (TG) and differential scanning calorimetry (DSC) curves for the FRA were correspondingly generated. Based on the TG data, the reaction function g(α), apparent activation energy Ea, and pre-exponential factor A were quantitatively evaluated using kinetic analysis. In addition, a Fourier transform infrared spectrometry (FTIR) test was utilized to assess the effects of the presence of FR on the chemical composition of the asphalt binder. Dynamic shear rheometer (DSR) tests were also performed to evaluate the rheological behavior of FRA. Results show that the presence of the FR significantly reduced the LOI and improved the smoke suppression during combustion of the asphalt binder mastics. The presence of FR was found to increase the Ea and the complexity of the combustion reaction, thereby improving the flame retardancy of the asphalt binder. FTIR analysis indicated that the presence of FR did not induce any strong chemical reactions to significantly impact or alter the functional groups of the asphalt binder. Furthermore, it was also observed that the rutting parameter and critical failure temperature of FRA increased with the addition of FR due to the stiffening effect of the solid FR particles.

LDADN: a local discriminant auxiliary disentangled network for key-region-guided chest X-ray image synthesis augmented in pneumoconiosis detection.

Pneumoconiosis is deemed one of China's most common and serious occupational diseases. Its high prevalence and treatment cost create enormous pressure on socio-economic development. However, due to the scarcity of labeled data and class-imbalanced training sets, the computer-aided diagnostic based on chest X-ray (CXR) images of pneumoconiosis remains a challenging task. Current CXR data augmentation solutions cannot sufficiently extract small-scaled features in lesion areas and synthesize high-quality images. Thus, it may cause error detection in the diagnosis phase. In this paper, we propose a local discriminant auxiliary disentangled network (LDADN) to synthesize CXR images and augment in pneumoconiosis detection. This model enables the high-frequency transfer of details by leveraging batches of mutually independent local discriminators. Cooperating with local adversarial learning and the Laplacian filter, the feature in the lesion area can be disentangled by a single network. The results show that LDADN is superior to other compared models in the quantitative assessment metrics. When used for data augmentation, the model synthesized image significantly boosts the performance of the detection accuracy to 99.31%. Furthermore, this study offers beneficial references for insufficient label or class imbalanced medical image data analysis.

A Novel Integrated APCI and MPT Ionization Technique as Online Sensor for Trace Pesticides Detection.

The misuse of pesticides poses a tremendous threat to human health. Excessive pesticide residues have been shown to cause many diseases. Many sensor detection methods have been developed, but most of them suffer from problems such as slow detection speed or narrow detection range. So, the development of rapid, direct and sensitive means of detecting trace amounts of pesticide residues is always necessary. A novel online sensor technique was developed for direct analysis of pesticides in complex matrices with no sample pretreatment. The portable sensor ion source consists of an MPT (microwave plasma torch) with desolventizing capability and an APCI (atmosphere pressure chemical ionization), which provides abundant precursor ions and a strong electric field. The performance which improves the ionization efficiency and suppresses the background signal was verified by using pesticide standard solution and pesticide pear juice solution measurements with an Orbitrap mass spectrometer. The limit of detection (LOD) and the limit of quantization (LOQ) of the method were measured by pear juice solutions that were obtained in the ranges of 0.034-0.79 µg/L and 0.14-1 µg/L. Quantitative curves were obtained ranging from 0.5 to 100 µg/L that showed excellent semi-quantitative ability with correlation coefficients of 0.985-0.997. The recoveries (%) of atrazine, imidacloprid, dimethoate, profenofos, chlorpyrifos, and dichlorvos were 96.6%, 112.7%, 88.1%, 85.5%, 89.2%, and 101.9% with the RSDs ranging from 5.89-14.87%, respectively. The results show that the method has excellent sensitivity and quantification capability for rapid and direct detection of trace pesticide.

Dual ambient plasma source ionization mass spectrometry for the rapid detection of trace sterols in urban water: Rapid detection of trace sterols in urban water.

A direct analytical method based on dual ambient plasma ion source tandem mass spectrometry was used for the simultaneous determination of four sterols in the environment. This technology has very high sensitivity and the method detects the four sterols in methanol-water (1:3) solutions with limits of detection (LOD) and limits of quantification (LOQ) ranging from 1.2 ng/L to 6.9 ng/L and 7.6 ng/L to 10.0 ng/L, respectively. The method was also used to test water quality at three locations within the city and successfully detected all four sterols at very low concentrations. The dual plasma source tandem mass spectrometry technique is extremely simple, rapid, sensitive and highly efficient compared to other traditional methods, providing a useful screening tool for sterols in water.

An Adversarial Learning Approach for Super-Resolution Enhancement Based on AgCl@Ag Nanoparticles in Scanning Electron Microscopy Images.

Scanning electron microscopy (SEM) plays a crucial role in the characterization of nanoparticles. Unfortunately, due to the limited resolution, existing imaging techniques are insufficient to display all detailed characteristics at the nanoscale. Hardware-oriented techniques are troubled with costs and material properties. Computational approaches often prefer blurry results or produce a less meaningful high-frequency noise. Therefore, we present a staged loss-driven neural networks model architecture to transform low-resolution SEM images into super-resolved ones. Our approach consists of two stages: first, residual channel attention network (RCAN) with mean absolute error (MAE) loss was used to get a better peak signal-to-noise ratio (PSNR). Then, discriminators with adversarial losses were activated to reconstruct high-frequency texture features. The quantitative and qualitative evaluation results indicate that compared with other advanced approaches, our model achieves satisfactory results. The experiment in AgCl@Ag for photocatalytic degradation confirms that our proposed method can bring realistic high-frequency structural detailed information rather than meaningless noise. With this approach, high-resolution SEM images can be acquired immediately without sample damage. Moreover, it provides an enhanced characterization method for further directing the preparation of nanoparticles.

60-nt DNA Direct Detection without Pretreatment by Surface-Enhanced Raman Scattering with Polycationic Modified Ag Microcrystal Derived from AgCl Cube.

Direct detection of long-strand DNA by surface-enhanced Raman scattering (SERS) is a valuable method for diagnosis of hereditary diseases, but it is currently limited to less than 25-nt DNA strand in pure water, which makes this approach unsuitable for many real-life applications. Here, we report a 60-nt DNA label-free detection strategy without pretreatment by SERS with polyquaternium-modified Ag microcrystals derived from an AgCl cube. Through the reduction-induced decomposition, the size of the about 3 × 3 × 3 µm3 AgCl cube is reduced to Ag, and the surface is distributed with the uniform size of 63 nm silver nanoparticles, providing a large area of a robust and highly electromagnetic enhancement region. The modified polycationic molecule enhances the non-specific electrostatic interaction with the phosphate group, thereby anchoring DNA strands firmly to the SERS enhanced region intactly. As a result, the single-base recognition ability of this strategy reaches 60-nt and is successfully applied to detect thalassemia-related mutation genes.

A new self-passivating template with the phosphorothioate strategy to effectively improve the detection limit and applicability of exponential amplification reaction.

Exponential amplification reaction (EXPAR) has attracted much attention due to its simple primers and high amplification efficiency, but its applications are hindered by severe non-specificity amplification. Convenient exogenous chemical modification methods modified the entire template while inhibiting both non-specific and specific amplification. In this paper, we proposed a new self-passivating template with the phosphorothioate strategy to effectively improve the detection limit and applicability of EXPAR. We phosphorothioated several bases where the sequence was prone to form transient intermolecular 3'-end hybridization, thereby inhibiting the non-specific interactions and preventing the extension of templates by DNA polymerase. The melting temperature (Tm) curve and density functional theory (DFT) proved that the stability of hydrogen bonds between phosphorothioated bases did decrease. Benefitting from this strategy, the detection limit had been improved by 3 orders of magnitude. Moreover, due to the antioxidation property of phosphorothioate, this strategy showed good stability in serum, reflecting its excellent prospects in clinical sampling and detection.

Surface-enhanced Raman spectroscopy integrated with aligner mediated cleavage strategy for ultrasensitive and selective detection of methamphetamine.

New drugs and illicit synthesized mixtures detection at crime scenes is a great challenge for detection method, which requires anti-interference and ultrasensitive methods to detect methamphetamine (METH) in seized street samples and biological fluids. Herein, we constructed a surface-enhanced Raman sensing method based on aligner mediated cleavage (AMC) of nucleic acid for quantitative detection of METH for the first time. This method we proposed relied on AMC to achieve programmable sequence-specific cleavage of METH aptamer linked by gold nanoparticles (METH aptamer-Au NPs), the cleavage product-Au NPs conjugates (cleavage aptamer-Au NPs) would hybridize with complementary DNA (cDNA)-Au NPs, resulting in the aggregation of the Au NPs and concomitant plasmonic coupling effect. Besides, due to the base number of METH aptamer-Au NPs was decreased, the interparticle distance of the Au NPs was shortened, which increased the electric field enhancement factor. Thus, under the irradiation of the laser, rhodamine 6G (R6G) adsorbed on Au NPs generated a strong Raman signal. The detection limit reached 7 pM, the linear range was from 10 pM to 10 nM, and this detection method also showed good anti-interference ability and reproducibility in serum.

Feedback activation of STAT3 limits the response to PI3K/AKT/mTOR inhibitors in PTEN-deficient cancer cells.

The PI3K/AKT/mTOR signaling pathway is constitutively active in PTEN-deficient cancer cells, and its targeted inhibition has significant anti-tumor effects. However, the efficacy of targeted therapies is often limited due to drug resistance. The relevant signaling pathways in PTEN-deficient cancer cells treated with the PI3K/mTOR inhibitor BEZ235 were screened using a phosphokinase array, and further validated following treatment with multiple PI3K/AKT/mTOR inhibitors or AKT knockdown. The correlation between PTEN expression levels and STAT3 kinase phosphorylation in the tissue microarrays of gastric cancer patients was analyzed by immunohistochemistry. Cell proliferation and clonogenic assays were performed on the suitably treated PTEN-deficient cancer cells. Cytokine arrays, small molecule inhibition and knockdown assays were performed to identify related factors. PTEN-deficient tumor xenografts were established in nude mice that were treated with PI3K/AKT/mTOR and/or STAT3 inhibitors. PTEN deficiency was positively correlated with low STAT3 activity. PI3K/mTOR inhibitors increased the expression and secretion of macrophage migration inhibitory factor (MIF) and activated the JAK1/STAT3 signaling pathway. Both cancer cells and in vivo tumor xenografts showed that the combined inhibition of PI3K/AKT/mTOR and STAT3 activity enhanced the inhibitory effect of BEZ235 on the proliferation of PTEN-deficient cancer cells. Our findings provide a scientific basis for a novel treatment strategy in cancer patients with PTEN deficiency.

Determination of polychlorinated biphenyls and organochlorine pesticides in Chinese mitten crabs (Eriocheir sinensis) using modified QuEChERS followed by GC-MS.

Chinese mitten crabs (Eriocheir sinensis) are one of the most popular food items with a high nutritional value. The use of pesticides and environmental pollutants directly influenced improvements in productivity of mitten crabs. However, there is an increasing number of food safety issues raising consumer concerns. It is necessary to determine whether the pollutants that crabs contain are below the maximum residue limits to guarantee food safety. Moreover, there has been very little research on detecting pesticide residues and other pollutants in Chinese mitten crabs due to their complicated matrix effects. In the current study, a Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method has been modified and validated for the simultaneous analysis of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) in mitten crabs. By using acetonitrile extraction, low temperature (freezing) precipitation and clean-up by mixed sorbents of C18, PSA and Florisil (magnesium silicate), the method was proved to be valid with linearity, precision, recovery, limit of quantification (LOQ), and limit of detection (LOD) with recoveries ranging from 85.9-119.8% and LOQ within 0.1-3.6 µg kg-1. The method was also applied to detect PCBs and OCPs in 48 crab samples from Jiangxi Province, China. The results showed that most of the crab samples contained PCB 118, beta-HCH and p,p'-DDT, and the content of p,p'-DDD in mitten crabs of Jiangxi Province showed sex-specific differences, which might be connected with sex-differential regulation.

Bioinspired Brochosomes as Broadband and Omnidirectional Surface-Enhanced Raman Scattering Substrates.

Surface-enhanced Raman scattering (SERS) substrates capable of working under laser excitation in a broad wavelength range are highly desirable in diverse application fields. Here, we demonstrate that the bioinspired Ag brochosomes, hollow microscale particles with submicroscale pits, have broadband and omnidirectional SERS performance. The SERS performance of the Ag brochosomes under near-infrared laser excitation makes them promising for applications in biosensing fields, such as the sensitive detection of Staphylococcus aureus bacteria and bovine hemoglobin protein. Additionally, the SERS intensity was insensitive to the incident angle of the laser beam, resulting from the spherical structure of the Ag brochosomes. The omnidirectional SERS performance makes the Ag brochosomes have application potential for in-the-field analysis using a hand-held Raman spectrometer for which it is difficult to accurately control the laser beam normal to the SERS substrates. Overall, the broadband and omnidirectional brochosome SERS substrates will find applications in diverse fields, particularly in biomedicine and in-the-field analysis.

Functional conservation and divergence of duplicated the suppressor of cytokine signaling 1 in blunt snout bream (Megalobrama amblycephala).

The suppressor of cytokine signaling 1 (SOCS1) is an essential feedback regulator extensively involved in many different cytokine signaling pathways, such as regulation of the immune system and growth of organism. However, the molecular and functional information on socs1 genes in freshwater fish is unclear. In the present paper, we identified and characterized the full-length closely related but distinct socs1 genes (socs 1a and -1b) in blunt snout bream (Megalobrama amblycephala). The bioinformatic analysis results showed that duplicated socs1s shared majority conserved motifs with other vertebrates. Both socs1a and -1b mRNAs were detected throughout embryogenesis, and gradually increase and then constantly expressed after 16 hpf. Whole-mount in situ hybridization demonstrated that socs1a and socs1b mRNAs were detected in the brain at 12hpf and 24hpf, and in the notochord and brain at 36hpf. In adult fish, the socs1a mRNA were strongly expressed in the heart, eye, kidney, spleen and gonad, but were found to be relatively low in the intestine and liver. On the other hand, the expression of socs1b mRNA was significantly high in the muscle, eye and spleen, and relatively low in the intestine, liver, skin and heart. The results of hGH treatment experiment showed that socs1a and 1b mRNAs were upregulated markedly in the kidney, muscle and liver. Overexpression of socs1s significantly inhibit the GH and JAK/STAT factor stat3 and the inhibitory effect of SOCS1s on GH may be involved in JAK-STAT signaling pathway. These results indicate that SOCS1 plays an important role in regulating growth and development.

Feedback Activation of SGK3 and AKT Contributes to Rapamycin Resistance by Reactivating mTORC1/4EBP1 Axis via TSC2 in Breast Cancer.

The mTORC1 inhibitors, such as rapamycin and its analogs, show limited antitumor activity in clinic, reasons for which have not been clearly elucidated. Here, we undertook an effort to uncover the mechanisms underlying the limited efficacy of rapamycin, and found that the transit suppression of 4EBP1 phosphorylation led to cap-dependent translation and cell proliferation in breast cancer cells. AKT only partially contributed to 4EBP1 re-phosphorylation. By taking advantage of mass spectrometry-based phosphoproteomic analysis, we identified SGK3 as a potent kinase involved in 4EBP1 re-phosphorylation. SGK3 deletion inhibited 4EBP1 phosphorylation and cap-dependent translation. Importantly, 4EBP1 phosphorylation was positively correlated with SGK3 activity in 67 clinical breast cancer specimens. Moreover, SGK3 deletion in combination with AKT inhibition almost blocked the 4EBP1 re-phosphorylation that was induced by rapamycin and profoundly enhanced rapamycin-induced growth inhibition in vitro and in an MCF7 breast cancer mouse xenograft model in vivo. Mechanistically, the feedback activation of SGK3 by rapamycin was dependent on hVps34 and mTORC2, and reactivated mTORC1/4EBP1 axis by phosphorylating TSC2. Collectively, our study reveals a critical role of SGK3 in mediating rapamycin resistance, and provides a rationale for targeting SGK3 to improve mTOR-targeted therapies.

Aligner mediated cleavage of nucleic acids for site-specific detection of single base mismatch.

Single base mismatch can always connect with various gene-related diseases, whose determination has aroused widespread interest. So far, various methods have been developed to determine the common base mismatch. However most of them are complex, time-consuming. Herein, we report a novel method, which only need one conventional endonuclease (NEase) and achieve site-specific cleavage in a programmable way, to detect single base mismatch, termed aligner-mediated cleavage-based single base mismatch discrimination (AMCMD). The DNA aligner (DA) is in a stem-loop structure, consistent with an incomplete recognition site of NEase on its stem and a 5'-side arm complementary to the target sequence (TS). Once TS contains matched base and hybridizes with DA, the complete recognition site of NEase is formed, and the TS will be cleavaged with fast speed, while converse is not. Based on it, the method can clearly distinguish mismatched and complementary bases. Without sample pre-processing, we were able to obtain and verify all the test result in about 30 min through the polyacrylamide gel electrophoresis analysis. This endows the proposed method with a simpler advantage. Then we combined AMCMD and EXPAR to create a new method for single base mismatch discrimination, the short sequence obtained by AMCMD as a target to trigger EXPAR, with a detection limit at 1pM level. Another process with human serum underlines that AMCMD is compatible with the complex biological sample, thus it has the potentials for practical applications.

Bridging the gap between transition metal- and bio-catalysis via aqueous micellar catalysis.

Previous studies have shown that aqueous solutions of designer surfactants enable a wide variety of valuable transformations in synthetic organic chemistry. Since reactions take place within the inner hydrophobic cores of these tailor-made nanoreactors, and products made therein are in dynamic exchange between micelles through the water, opportunities exist to use enzymes to effect secondary processes. Herein we report that ketone-containing products, formed via initial transition metal-catalyzed reactions based on Pd, Cu, Rh, Fe and Au, can be followed in the same pot by enzymatic reductions mediated by alcohol dehydrogenases. Most noteworthy is the finding that nanomicelles present in the water appear to function not only as a medium for both chemo- and bio-catalysis, but as a reservoir for substrates, products, and catalysts, decreasing noncompetitive enzyme inhibition.

Simple and efficient genome recombineering using kil counter-selection in Escherichia coli.

Seamless modification of the Escherichia coli genome using positive selection / negative selection is widely used in metabolic engineering and functional genome analysis. Some excellent negative selection systems have been reported, of which tetA-sacB and inducible toxins system are prominent. To expand the existing negative selection toolkit, we constructed a new negative selection marker system based on kil gene of lambda prophage. The selection stringency of kil was measured and compared with the most widely used counter-selection gene, sacB, at the lacI, ack, and dbpa loci using different E. coli strains. At all these loci of tested strains, the selection stringency of kil significantly exceeds that of sacB by 2- to 28-fold. When dsDNA fragments were employed for recombination, the efficiency for isolating the correct recombinant of kil was significantly higher than that of sacB. This new negative selection system does not require special media or extended incubation time. However, our system cannot be used in host strains containing temperature-sensitive kil gene. A Red system providing plasmid without kil gene is recommended for use together with our system. Our counter-selection system is expected to be an addition to the engineering arsenal of E. coli.

One-pot synthesis of hollow hydrangea Au nanoparticles as a dual catalyst with SERS activity for in situ monitoring of a reduction reaction.

The controlled synthesis of metallic nanomaterials has attracted the interest of many researchers due to their shape-dependent physical and chemical properties. However, most of the synthesized nanocrystals cannot be combined with spectroscopy to measure the reaction kinetics, thus limiting their use in monitoring the catalytic reaction process to elucidate its mechanism. As a powerful analytical tool, surface-enhanced Raman spectroscopy (SERS) can be used to achieve in situ monitoring of catalytic reactions by developing bifunctional metal nanocrystals with both SERS and catalytic activities. Herein, we have developed a simple one-pot synthesis method for the large-scale and size-controllable preparation of highly rough hydrangea Au hollow nanoparticles. The growth mechanism of flower-like Au hollow nanostructures was also discussed. The hollow nanostructure with a 3D hierarchical flower shell combines the advantages of hollow nanostructure and hierarchical nanostructure, which possess high SERS activity and good catalytic activity simultaneously. Furthermore, the hydrangea Au hollow crystals were used as a bifunctional nanocatalyst for in situ monitoring of the reduction reaction of 4-nitrothiophenol to the 4-aminothiophenol.

EphA3 contributes to tumor growth and angiogenesis in human gastric cancer cells.

Eph receptor tyrosine kinases and their ephrin ligands, mediate an important cell communication system both in normal and oncogenic development, and play central roles in a series of processes including angiogenesis, stem cell maintenance and cancer metastasis. Eph receptor A3 (EphA3), commonly overexpressed in a broad range of cancers, including gastric cancer (GC), is related to tumor progression. Our previous study revealed that EphA3 may play important roles in tumorigenesis and angiogenesis in GC. However, its exact role and the mechanisms underlying its function in GC remain unclear. In the present study, lentivirus­mediated RNA interference was employed to knock down the expression of EphA3 in GC HGC­27 cells. Functional analyses indicated that depletion of EphA3 expression inhibited the cell growth and tumorigenicity of HGC­27 cells in vitro and in vivo. Furthermore, knockdown of the expression of EphA3 in HGC­27 cells inhibited tube formation and migration of HUVEC endothelial cells. Tumor angiogenesis in vivo was also inhibited upon EphA3 knockdown in HGC­27 cells, with reduced microvessel density (MVD) in xenograft models. We further revealed that EphA3 depletion inhibited tumor angiogenesis and migration through the signal transducer and activator of transcription 3/vascular endothelial growth factor (STAT3/VEGF) signaling pathway. These results indicated that EphA3 may be an effective prognostic indicator and a potential target for GC therapy.

Circumventing silver oxidation induced performance degradation of silver surface-enhanced Raman scattering substrates.

Surface-enhanced Raman scattering (SERS) has been recognized as a promising sensing technique in biomedical/biosensing applications and analytical chemistry. Silver (Ag) nanostructures have the strongest SERS enhancement, but suffer from severe enhancement degradation induced by oxidation. Here, we introduce electrochemical reduction of silver oxide to produce Ag SERS substrates on request to partially circumvent the SERS enhancement degradation problem of Ag SERS substrates. Silver oxide nanostructures were first prepared in pure silver citrate aqueous solutions with controllable morphologies depending on the electrodeposition parameters. The transition process from silver oxide to Ag was investigated by density functional theory calculations. Based on the understanding of the transition mechanism, heating treatment, applying reducing agent, and electrochemical reduction were adopted to transform silver oxide to Ag. Notably, no organic agents were introduced neither in the electrodeposition of silver oxide nor electrochemical transformation of silver oxide to Ag. The electrochemical reduction strategy could produce Ag SERS substrates with a 'clean' surface with outstanding SERS performance in a simple as well as cost and time effective manner. Ag SERS substrates can be used in biomedical/biosensing fields. The approach through electrochemical reduction of silver oxide to generate Ag SERS substrate may push forward practical application process of Ag SERS substrates.