Controllable and Low-Loss Electrochemiluminescence Waveguide Supported by a Micropipette Electrode.

One-dimensional molecular crystal waveguide (MCW) can transmit self-generated electrochemiluminescence (ECL), but heavy optical loss occurs because of the small difference in the refractive index between the crystal and its surroundings. Herein, we report a micropipette electrode-supported MCW (MPE/MCW) for precisely controlling the far-field transmission of ECL in air with a low optical loss. ECL is generated from one terminal of the MCW positioned inside the MPE, which is transmitted along the MCW to the other terminal in air. In comparison with conventional waveguides on solid substrates or in solutions, the MPE/MCW is propitious to the total internal reflection of light at the MCW/air interface, thus confining the ECL efficiently in MCW and improving the waveguide performance with an extremely low-loss coefficient of 4.49 × 10-3 dB µm-1. Moreover, by regulation of the gas atmosphere, active and passive waveguides can be resolved simultaneously inside MPE and in air. This MPE/MCW offers a unique advantage of spatially controlling and separating ECL signal readout from its generation, thus holding great promise in biosensing without or with less electrical/chemical disturbance.

Intestinal microbiota and gene expression alterations in leopard coral grouper (Plectropomus leopardus) under enteritis.

Enteritis poses a significant threat to fish farming, characterized by symptoms of intestinal and hepatic inflammation, physiological dysfunction, and dysbiosis. Focused on the leopard coral grouper (Plectropomus leopardus) with an enteritis outbreak on a South China Sea farm, our prior scrutiny did not find any abnormalities in feeding or conventional water quality factors, nor were any specific pathogen infections related to enteritis identified. This study further elucidates their intestinal flora alterations, host responses, and their interactions to uncover the underlying pathogenetic mechanisms and facilitate effective prevention and management strategies. Enteritis-affected fish exhibited substantial differences in intestinal flora compared to control fish (P = 0.001). Notably, norank_f_Alcaligenaceae, which has a negative impact on fish health, predominated in enteritis-affected fish (91.76 %), while the probiotic genus Lactococcus dominated in controls (93.90 %). Additionally, certain genera with pathogenesis potentials like Achromobacter, Sphingomonas, and Streptococcus were more abundant in diseased fish, whereas Enterococcus and Clostridium_sensu_stricto with probiotic potentials were enriched in control fish. At the transcriptomic level, strong inflammatory responses, accompanied by impaired metabolic functions, tissue damage, and iron death signaling activation were observed in the intestines and liver during enteritis. Furthermore, correlation analysis highlighted that potential pathogen groups were positively associated with inflammation and tissue damage genes while presenting negatively correlated with metabolic function-related genes. In conclusion, dysbiosis in the intestinal microbiome, particularly an aberrantly high abundance of Alcaligenaceae with pathogenic potential may be the main trigger for this enteritis outbreak. Alcaligenaceae alongside Achromobacter, Sphingomonas, and Streptococcus emerged as biomarkers for enteritis, whereas some species of Lactococcus, Clostridium_sensu_stricto, and Enterococcus showed promise as probiotics to alleviate enteritis symptoms. These findings enhance our understanding of enteritis pathogenesis, highlight intestinal microbiota shifts in leopard coral grouper, and propose biomarkers for monitoring, probiotic selection, and enteritis management.

Gold Microbeads Enabled Proximity Electrochemiluminescence for Highly Sensitive and Size-Encoded Multiplex Immunoassays.

Developing highly sensitive multiplex immunoassays is urgently needed to guide medical research and improve clinical diagnosis. Here, we report the proximity electrochemiluminescence (ECL) generation enabled by gold microbeads (GMBs) for improving the detection sensitivity and multiplexing capacity of ECL immunoassays (ECLIAs). As demonstrated by microscopy and finite element simulation, GMBs can function as spherical ultramicroelectrodes for triggering ECL reactions in solutions. Employing GMBs as solid carriers in the bead-based ECLIA, the electrochemical oxidation of a coreactant can occur at both the GMB surface and the substrate electrode, allowing the coreactant radicals to diffuse only a short distance of ∼100 nm to react with ECL luminophores that are labeled on the GMB surface. The ECL generation via this proximity low oxidation potential (LOP) route results in a 21.7-fold increase in the turnover frequency of ECL generation compared with the non-conductive microbeads that rely exclusively on the conventional LOP route. Moreover, the proximity ECL generation is not restricted by the diffusion distance of short-lived coreactant radicals, which enables the simultaneous determination of multiple acute myocardial infarction biomarkers using size-encoded GMB-based multiplex ECLIAs. This work brings new insight into the understanding of ECL mechanisms and may advance the practical use of multiplex ECLIAs.

Multicolor Iridium(III) Complexes with Host-Guest Recognition Motifs for Enhanced Electrochemiluminescence and Modular Labeling.

Cyclometalated Ir(III) complexes with high electrochemiluminescence (ECL) efficiency and appropriate bioconjugation sites are urgently needed in ECL immunoassays (ECLIA). Herein, we report the synthesis, photophysics, electrochemistry, and ECL of six new Ir(III) complexes bearing naphthyl (nap) or adamantane phenyl (adap) substitutions, four of which emit cyan, green, or red light and display 1.7- to 7.5-fold increases in ECL intensity. In combination with DFT/TDDFT calculations, this enhancement is rationalized to the augmented radiative rate that arises from both the strengthened spin-orbit coupling (SOC) and the increased transition dipole moment. In addition, the adap-based Ir(III) complex shows high binding affinity with ß-cyclodextrin (ß-CD) due to the strong hydrophobic interaction, which enables us to develop a modular strategy for the labeling of Ir(III) complexes with biomolecules and to use hydrophobic luminophores in the aqueous-phase detection. As demonstrated, a novel ECLIA is built up and exhibits a wide linear range from 1 ng/mL to 10 µg/mL and a detection limit of 72 pg/mL for the determination of C-reactive protein (CRP). These findings provide new insights into the design, synthesis, and bio-labeling of highly emissive Ir(III) complexes and pave the way for the development of novel ECLIA based on host-guest recognition motifs.

Development and application of a sensitive droplet digital PCR-based method to detect tilapia parvovirus.

Tilapia parvovirus (TiPV) causes severe mortality rates in cultured tilapia, resulting in substantial losses to the fish industry. Droplet digital PCR (ddPCR) is a sensitive, accurate, and absolute quantitation method, plus it does not require a standard curve. Herein we report the development and application of a sensitive ddPCR-based method to rapidly detect and quantify TiPV. Optimal annealing temperature was determined to be 59.3°C, and optimal primer and probe concentrations were 900 nmol/L and 250 nmol/L, respectively. Our ddPCR method was highly specific to TiPV and showed no cross-reactivity with other viruses. Further, the detection limit of ddPCR was 0.07 copies/µl, being lower than that of real-time PCR (qPCR, 4.63 copies/µl). We also investigated the ability of ddPCR to detect TiPV in 50 samples and compared the outcome with qPCR data in terms of sensitivity and accuracy. The results showed that the positive detection rate of ddPCR (32%) was higher than that of qPCR (18%). To conclude, our ddPCR method was effective at detecting TiPV in samples with low viral loads. We believe that its application can facilitate the surveillance of sources and transmission routes of TiPV.

Establishment of a new fish cell line from the brain of humpback grouper (Cromileptes altivelis) and its application in toxicology and bacterial susceptibility.

Cromileptes altivelis, humpback grouper, belongs to the family Epinephelidae and is one popular farmed fish species because of its high economic value and ornamental value. However, more and more diseases outbreaks have been reported with C. altivelis aquaculture. Today, a new brain cell line of C. altivelis (named CAB) was established and characterized. Our results showed that CAB cells were suitable for growth at 26 °C in L-15 medium supplemented with 15% fetal bovine serum (FBS). The results of 18S rRNA gene sequencing confirmed that CAB cell line was derived from C. altivelis. Moreover, chromosomal aneuploidy was observed in CAB cells, and the modal chromosome number of CAB cells was 48 by chromosome analysis. In addition, CAB cells could transfect pEGFP-N3 plasmid with high transfection efficiency, indicating that CAB cell line has the potential to investigate the function of exogenous genes in vitro. Furthermore, the bacterial susceptibility results suggested that CAB cells were susceptive to Vibrio harveyi and Edwardsiella tarda. And, heavy metals (Hg, Cd, and Cu) were toxic to the CAB cells, and the toxic effect was dose-dependent. In summary, the CAB cell line could be a powerful tool in vitro to study functional genes and has the potential application in bacterial susceptibility and toxicology.

Spatially Selective Imaging of Cell-Matrix and Cell-Cell Junctions by Electrochemiluminescence.

Cell junctions are protein structures located at specific cell membrane domains that determine key processes in multicellular development. Here we report spatially selective imaging of cell junctions by electrochemiluminescence (ECL) microscopy. By regulating the concentrations of luminophore and/or co-reactant, the thickness of ECL layer can be controlled to match with the spatial location of different cell junctions. At a low concentration of luminophore, ECL generation is confined to the electrode surface, thus revealing only cell-matrix adhesions at the bottom of cells. While at a high concentration of luminophore, the ECL layer can be remarkably extended by decreasing the co-reactant concentration, thus allowing the sequential imaging of cell-matrix and cell-cell junctions at the bottom and near the apical surface of cells, respectively. This strategy not only provides new insights into the ECL mechanisms but also promises wide applications of ECL microscopy in bioimaging.

Microtube Electrodes for Imaging the Electrochemiluminescence Layer and Deciphering the Reaction Mechanism.

Electrochemiluminescence (ECL) is a powerful transduction technique in biosensing and diagnostics, while mechanistic studies are still scarce. Herein we report the combined use of microtube electrode (MTE) and microscopy to measure the thickness of ECL layer (TEL) to decipher reaction mechanisms. For the classical system involving tris(2,2'-bipyridyl)ruthenium and tri-n-propylamine, the ECL pattern generated at the MTE tends to change from ring to spot upon increasing the luminophore concentration, with the TEL varying from ca. 3.1 µm to >4.5 µm. This variation is rationalized to arise from the contribution of the so-called catalytic route. While using 2-(dibutylamino)ethanol as the co-reactant, the ECL pattern remains ring-shaped and independent on the luminophore concentration. The TEL in this case is ca. 2.1 µm, implying that ECL generation is always surface-confined. MTEs can thus act as optical rulers for measuring the TEL and providing insightful mechanistic information.

Electrochemiluminescence Self-Interference Spectroscopy with Vertical Nanoscale Resolution.

Here we report an electrochemiluminescence (ECL) self-interference spectroscopy technique (designated as ECLIS) with spatial resolution in the normal direction of the electrode surface. Self-interference principally originates from the superposition of ECL emitted directly by luminophores and that reflected from electrode surfaces, resulting in a spectrum consisting of orderly distributed peaks. On the basis of this spectrum and theoretical analysis by the matrix propagation model, the distance between luminophores and the electrode surface can be probed with a vertical resolution on the nanometer scale. We demonstrated first in this work that the height of ECL luminophores assembled on the electrode surface using different molecular linkers, such as double-stranded DNA, could be determined, as well as the possible conformation of linker molecules at the surface. Moreover, the thickness of the ECL emitting layer adjacent to the electrode surface was estimated for the classical coreactant ECL systems involving freely diffusing Ru(bpy)32+ and tri-n-propylamine in solutions. The thickness was found to vary from ∼350 nm to nearly 1 µm depending on the concentration of Ru(bpy)32+. We believe that ECLIS with a high vertical resolution will provide an easy way to collect molecular conformation information and study ECL reaction mechanisms at electrode interfaces.

Construction and analysis of the immune effect of Vibrio harveyi subunit vaccine and DNA vaccine encoding TssJ antigen.

Vibrio harveyi, a severe pathogen infects different kinds of sea animals, causes huge economic loss in aquaculture industry. In order to control the Vibriosis disease caused mainly by V. harveyi and other Vibrio spp., the best solution lies in developing corresponding efficient vaccines. In this study, we have cloned and analysed a putative antigen TssJ from the T6SS of V. harveyi, which has the potential as a vaccine against infection. The sequence analysis and western blotting experiments indicated that TssJ anchored in outer membrane and there were several antigenic determinants existed on its extracellular region. Two forms of universal vaccines, subunit vaccine and DNA vaccine, were developed based on TssJ and applied in Trachinotus ovatus. The results showed that both of the two vaccines could generate a moderate protection in fish against V. harveyi. The relative percentage survival (RPS) of subunit vaccine and DNA vaccine were 52.39% and 69.11%, respectively. Immunological analysis showed both subunit vaccine and DNA vaccine enhanced acid phosphatase, alkaline phosphatase, superoxide dismutase, and lysozyme activities. Specific serum antibodies against TssJ in the fish vaccinated with subunit vaccine was much higher than that in the DNA vaccine group. Several immune-related genes, i.e., IL10, C3, MHC Iα, MHC IIα, and IgM, were induced both by the two forms of vaccines. TNFα and Mx were only upregulated in the DNA vaccine group. However, the induction levels of these genes induced by DNA vaccine were higher than subunit vaccine. All these findings suggested that TssJ from V. harveyi had a potential application value in vaccine industry.

Establishment and characterization of a new cell line from the muscle of humpback grouper (Cromileptes altivelis).

The humpback grouper (Cromileptes altivelis) is a commercially important species of the family Epinephelidae. With the development in aquaculture industry, C. altivelis breeding has gradually increased in volumetric production, leading to the occurrence of various diseases. In this study, we established a new cell line (CAM) derived from the muscle tissue of C. altivelis. Our results showed that the optimal growth temperature and working concentration of fetal bovine serum (FBS) of CAM cells were 28 °C and 15%, respectively. DNA sequencing and comparative analysis of 18S rRNA gene sequence showed that CAM cell line was originated from C. altivelis. Chromosome analysis showed that the modal chromosome number of CAM cells was 48. After transfection using pEGFP-N3 plasmid, CAM cells exhibited high transfection efficiency, indicating that CAM cells could be used in foreign gene expression studies. Further, cytotoxicity analysis revealed that CAM cells were sensitive to Vibrio harveyi and Edwardsiella tarda. Moreover, the cytotoxicity of heavy metals (Hg, Cd, and Cu) to CAM cells was dose-dependent. This CAM cell line might be used as an ideal tool in vitro for analyzing and understanding the mechanisms of pathogenesis, host-pathogen interactions, and toxicity assay of heavy metals.

Imaging Cell-Matrix Adhesions and Collective Migration of Living Cells by Electrochemiluminescence Microscopy.

Cell-matrix adhesions play essential roles in a variety of biological processes. Herein, we report a label-free method to map cell-matrix adhesions of single living cells on an electrode surface by electrochemiluminescence (ECL). An indium tin oxide electrode modified with a silica nanochannel membrane was used as the substrate electrode, at which the ECL generation from freely diffusing luminophores provided a distinct visual contrast between adhesion sites and noncontacted domains, thus selectively revealing the former in a label-free manner. With this methodology, we studied the spatial distribution, as well as dynamic variation, of cell-matrix adhesions and the adhesion strength at the subcellular level. Cell-matrix adhesions of an advancing cell sheet were finally imaged to study the movement of cells in collective migration. A statistical analysis suggests that cells on the far side of leading edge also have the propensity to migrate and do not act as just passive followers.

Electrochemiluminescence Waveguide in Single Crystalline Molecular Wires.

Here we report the first observation of active waveguide of electrochemiluminescence (ECL) in single crystalline molecular wires self-assembled from cyclometalated iridium(III) complexes, namely tris(1-phenylisoquinoline-C2 , N) (Ir(piq)3 ). Under dark conditions, the molecular wires deposited on the electrode surface can act as both ECL emitters and active waveguides. As revealed by ECL microscopy, they exhibit the typical characteristics of optical waveguides, transmitting ECL and generating much brighter ECL emission at their terminals. Moreover, self-generated ECL can be confined inside the molecular wire and propagates along the longitudinal direction as far as ≈100 µm to the terminal out of touch with the electrode. Therefore, this one-dimensional crystalline molecular wire-based waveguide offers the opportunity to switch the electrochemically generated ECL to remote light emission in non-conductive regions and is promising for contactless electrochemical analysis and study of (bio)chemical systems.

Evaluation of Lactococcus lactis HNL12 combined with Schizochytrium limacinum algal meal in diets for humpback grouper (Cromileptes altivelis).

The humpback grouper (Cromileptes altivelis) is a commercially valuable species of the family Epinephelidae; however, its marketization suffers from slow growth speed, low survival rate, and various pathogenic diseases. Lactococcus lactis and Schizochytrium limacinum are commonly used as immunostimulants due to their health benefits for the aquatic organisms. In the present study, we assessed the effects of dietary supplementation with L. lactis HNL12 combined with S. limacinum algal meal on the growth performances, innate immune response, and disease resistance of C. altivelis against Vibrio harveyi. The results showed that fish fed with a combination diet of L. lactis and S. limacinum exhibited significantly higher final weight, percent weight gain, and specific growth rate compared with groups fed with them alone. A bacterial challenge experiment indicated that the group fed with the L. lactis combined with S. limacinum diet achieved the highest relative percent of survival value (68.63%), suggesting that L. lactis and S. limacinum significantly improved the disease resistance against V. harveyi after a 4-week feeding trial. Moreover, the respiratory burst activity of macrophages of fish fed with a L. lactis combined with S. limacinum diet was significantly higher than that of fish fed the control diet after 1, 2, and 3 weeks of feeding. The serum superoxide dismutase of fish fed with a L. lactis combined with S. limacinum diet significantly increased compared to those fed the control diet after 1 and 2 weeks of feeding, while the serum alkaline phosphatase of fish fed with a L. lactis combined with S. limacinum diet after 2 and 4 weeks was significantly increased, compared to the control group. The serum lysozyme activities of fish fed with a L. lactis combined with S. limacinum diet significantly increased compared to the control group after 2 weeks of feeding. Furthermore, transcriptome sequencing of the C. altivelis head kidney was conducted to explore the immune-regulating effects of the L. lactis combined with S. limacinum diet on C. altivelis. A total of 86,919 unigenes, annotated by at least one of the reference databases (Nr, Swiss-Prot, GO, COG, and KEGG), were assembly yielded by de novo transcriptome. In addition, 157 putative differentially expressed genes (DEGs) were identified between the L. lactis combined with S. limacinum group and the control group. For pathway enrichment, the DEGs were categorized into nine KEGG pathways, which were mainly related to infective diseases, antigen processing and presentation, digestive system, and other immune system responses. The findings of this study suggest that the L. lactis combined with S. limacinum diet can induce positive effects on the growth, immunity, and disease resistance of C. altivelis against V. harveyi. This study expands our understanding of the synergistic combinations of probiotics and prebiotics in aquaculture.

Comparative analysis of the expression patterns of IL-1ß, IL-11, and IL-34 in golden pompano (Trachinotus ovatus) following different pathogens challenge.

Interleukins (ILs) are a subgroup of cytokines, which are molecules involved in the intercellular regulation of the immune system. These cytokines have been extensively studied in mammalian models, but systematic analyses of fish are limited. In the current study, 3 IL genes from golden pompano (Trachinotus ovatus) were characterized. The IL-1ß protein contains IL-1 family signature motif, and four long helices (αA - αD) in IL-11 and IL-34, which were well conserved. All 3 ILs clustered phylogenetically with their respective IL relatives in mammalian and other teleost species. Under normal physiological conditions, the expression of IL-1ß, IL-11, and IL-34 were detected at varied levels in the 11 tissues examined. Most of the 3 ILs examined were highly expressed in liver, spleen, kidney, gill, or skin. Following pathogenic bacterial, viral, or parasitic challenge, IL-1ß, IL-11, and IL-34 exhibited distinctly different expression profiles in a time-, tissue-, and pathogen-dependent manner. In general, IL-1ß was expressed at higher levels following challenge with all pathogens examined than was observed for IL-11 and IL-34. Furthermore, Streptococcus agalactiae and Cryptocaryon irritans caused higher levels of IL-1ß and IL-11 expression than Vibrio harveyi and viral nervous necrosis virus (VNNV). The increased expression of IL-34 caused by VNNV and C. irritans were higher than that caused by V. harveyi and S. agalactiae. These results suggest that these 3 ILs in T. ovatus may play different effect pathogen type specific responses.

TroCCL4, a CC chemokine of Trachinotus ovatus, is involved in the antimicrobial immune response.

CC chemokines are a large subfamily of chemokines that play an important role in the innate immune system. To date, several CC chemokines have been identified in fish species; however, the activities and functions of these putative chemokines remain ambiguous in teleosts, especially in the golden pompano, Trachinotus ovatus. Here, we characterized CC chemokine ligand 4 from T. ovatus (TroCCL4) and studied its functions. TroCCL4 contains a 294 bp open reading frame that encodes a putative peptide comprising 97 amino acids. TroCCL4 shares a high amino acid sequence similarity of 31.11%-78.35% with other CC chemokines sequences in humans and teleosts and has four cysteine residues that are conserved among other CC chemokines. TroCCL4 is also related to the macrophage inflammatory protein (MIP) group of CC chemokines. TroCCL4 expression was most abundant in immune organs and significantly upregulated in a time-dependent manner following Edwardsiella tarda infection. Recombinant TroCCL4 (rTroCCL4) induced the migration of peripheral blood leukocytes and the cellular proliferation of head kidney lymphocytes. In addition, rTroCCL4 inhibited the growth of Escherichia coli and E. tarda, indicating an antimicrobial function. Furthermore, the results of in vivo analysis showed that TroCCL4 overexpression in T. ovatus significantly enhanced macrophage activation; upregulated the gene expression of interleukin 1-ß (IL-1ß), interleukin 15 (IL15), interferon-induced Mx protein (Mx), tumor necrosis factor α (TNFα), complement C3, and major histocompatibility complex (MHC) class Iα and class IIα; and protected against bacterial infection in fish tissues. In contrast, knockdown of TroCCL4 expression resulted in increased bacterial dissemination and colonization in fish tissues. Taken together, our results provide evidence indicating that TroCCL4 has the ability to stimulate leukocytes and macrophages and enhance host immunity to defend against bacterial infection.

Potential-Resolved Multicolor Electrochemiluminescence for Multiplex Immunoassay in a Single Sample.

Electrochemiluminescence (ECL) is a highly successful technique used in commercial immunoassays for clinical diagnosis. Developing an ECL-based multiplex immunoassay, with the potential to enable high-throughput detection of multiple biomarkers simultaneously, remains a current research interest yet is limited by a narrow choice of ECL luminophores. Herein we report the synthesis, photophysics, electrochemistry, and ECL of several new ruthenium(II) and iridium(III) complexes, three of which are eventually used as signal reporters for multiplex immunoassay. The ECL behaviors of individual luminophores and their mixtures were investigated in multiple modes, including light intensity, spectrum, and image measurements. The spectral peak separation between Ru(bpy)2(dvbpy)2+ (bpy = 2,2'-bipyridine, dvbpy = 4,4'-bis(4-vinylphenyl)-2,2'-bipyridine), and Ir(dFCF3ppy)2(dtbbpy)+ (dFCF3ppy = 3,5-difluoro-2-[5-(trifluoromethyl)-2-pyridinyl]phenyl, dtbbpy = 4,4'-bis( tert-butyl)-2,2'-bipyridine) was up to 145 nm, thus providing the spectrum-resolved possibility of identifying light signals. The potential-resolved ECL signals were achieved for the mixtures of Ir(ppy)3 (ppy = 2-phenylpyridine) with either Ru(bpy)2(dvbpy)2+ or Ir(dFCF3ppy)2(dtbbpy)+, due to the self-annihilation ECL of Ir(ppy)3 at higher potentials, as confirmed by electrochemistry-coupled mass spectrometry. A multiplex immunoassay free of spatial spotting antibodies on plates or substrates was ultimately devised by combining luminophore-loaded polymer beads with the homogeneous sandwich immunoreaction. Using potential and spectrum dual-resolved ECL as the readout signal, simultaneous recognition of three antigens, namely, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and beta-human chorionic gonadotropin (ß-HCG), was demonstrated in a single run for a sample volume of 300 µL. These results contribute to the understanding of ECL generation by multiple luminophores and devising spot-free multiplex immunoassays with less sample consumption.

Electrochemical detection of Alzheimer's disease related substances in biofluids by silica nanochannel membrane modified glassy carbon electrodes.

Alzheimer's disease (AD) affects middle- and old-age populations, and causes loss of brain weight, degradation of brain functions and memory loss. So the fast and accurate detection of AD related markers is highly important in diagnosis. We report in this work the detection of Cu2+ and dopamine (DA), which are markers related to AD, by direct electrochemistry (DEC) and electrochemiluminescence (ECL) using a silica nanochannel membrane modified glassy carbon electrode (SNM/GCE). By DEC, the detection of both Cu2+ and DA in buffer solutions was achieved with a wide linear range and a low limit of detection (LOD). The determination of DA was also achieved in terms of its quenching effect on the ECL of the tris(2,2'-bipyridyl)ruthenium(ii)/tri-n-propylamine co-reactant system in both intensity and image modes, yielding a particularly high sensitivity in the former case. In comparison with a bare GCE, the analytical sensitivity and selectivity of the SNM/GCE were superior, most likely due to the analyte pre-concentration and permselective effects of the SNM. Moreover, given that the SNM consists of perpendicular channels with a negatively charged surface, high channel density (ca. 7.5 × 1012 cm-2) and uniform size (ca. 2.3 nm in diameter), it displays a high molecular permeability and meanwhile a high selectivity in terms of molecular size and charge. So the SNM/GCE exhibited an excellent anti-fouling ability in biofluids, such as human blood and artificial cerebrospinal fluid, suppressing effectively the interference of coexisting substances (such as cells, proteins, and other big and small molecules) and providing excellent signal stability.

Transcriptome analysis provides insights into the immune responsive pathways and genes in the head kidney of tiger grouper (Epinephelus fuscoguttatus) fed with Spatholobus suberectus, Phellodendron amurense, or Eclipta prostrata.

The tiger grouper, Epinephelus fuscoguttatus, is an economically important fish in Southeast Asia but has been plagued by several diseases. Spatholobus suberectus (S), Phellodendron amurense (P), and Eclipta prostrate (E) are three commonly used Chinese medicinal herbs. Although previous pharmacological and clinical studies indicated that S, P, and E possess a variety of beneficial functions in mammals, little is known about their functions in farmed fish and the underlying molecular mechanism of their actions. Challenge tests in this study showed that after 14 days of diet supplement, all these herbs could effectively enhance the disease resistance of E. fuscoguttatus against Vibrio harveyi. However, the non-specific immune parameters of the herb-supplemented groups were not significantly different from the control group. To further explore the molecular mechanism of herbal immune-regulating effects on E. fuscoguttatus, transcriptome sequencing and RNA-Seq technique were applied on E. fuscoguttatus kidney. De novo transcriptome assembly of E. fuscoguttatus kidney yield 80,014 unigenes, among which, 44,901 (56.12%) were annotated with at least one of the public databases (Nr, Nt, Swiss-Prot, KEGG, COG, GO). Among these, 22,738, 11,700 and 27,457 unigenes were assigned to 57, 25 and 258 categories of GO, COG and KEGG databases, respectively. Using Solexa/Illumina's DGE platform, a total of 231, 186 and 144 putative differentially expressed genes (DEGs) were detected in P, E and S group compared with the control group. GO analysis indicated that in P and E, down-regulated DEGs were dominant in almost every GO term; whereas in S, up-regulated DEGs were more dominant. KEGG pathway analysis revealed that putative DEGs in all three herb groups were obviously enriched in the pathways related to infective diseases and immune system. We also identified a number of immune relative genes and pathways (TLR5, IL8 and MAPK pathway, for instance) associated with P, E and S's regulatory effects on E. fuscoguttatus. This study will enrich the E. fuscoguttatus transcriptome database, contribute to a better understanding of the molecular mechanisms associated with the immunoregulatory activities of Chinese medicinal herbs on teleost and provide valuable information on the prevention of grouper Vibrio diseases using Chinese medicinal herbs.

Effects of dietary administration of Lactococcus lactis HNL12 on growth, innate immune response, and disease resistance of humpback grouper (Cromileptes altivelis).

Lactic acid bacteria are a common group of probiotics that have been widely studied and used in aquaculture. In the present study, we isolated Lactococcus lactis HNL12 from the gut of wild humpback grouper (Cromileptes altivelis) and explored its probiotic properties. For this purpose, L. lactis HNL12 was added to the commercial fish feed. The results showed that HNL12 had high auto-aggregation ability and strong tolerance to simulated gastrointestinal stress. When C. altivelis consumed a diet containing 0 (control), 106, 108, or 1010 CFU/g HNL12 for four weeks, all of the groupers fed a diet with HNL12 had significantly increased percent weight gain (PWG), especially those fed with 108 CFU/g, which had a PWG of 231.45%. Compared to the control, fish fed with L. lactis HNL12 exhibited significantly increased survival rates following injection with Vibrio harveyi after one month. Immunological analysis showed that C. altivelis fed with HNL12 had (i) enhanced respiratory burst activity of head kidney macrophages, superoxide dismutase, acid phosphatase, and lysozyme activities of serum; (ii) an improved survival rate from 36% to 70%; and (iii) upregulated expression of a broad spectrum of immunity. Meanwhile, de novo transcriptome assembly yielded 89,314 unigenes, which were annotated by at least one of the reference databases (Nr, Swiss-Prot, GO, COG and KEGG). A total of 307 genes showed significantly different expression between the groups fed with or without added HNL12. GO and KEGG enrichment analyses of the significantly different expression gene categories and pathways were related to infectious diseases, antigen processing and presentation, and other immune system responses. These results indicate that L. lactis HNL12 is effective for enhancing the growth, immunity, and disease resistance of C. altivelis; this study also provides insight into the use of probiotics for commercial applications.