Transcriptomic analysis of Bombyx mori in its early larval stage (2nd instar) of development upon Nosema bombycis transovarial infection.

The infection caused by Nosema bombycis often known as pebrine, is a devastating sericulture disease. The infection can be transmitted to the next generation through eggs laid by infected female Bombyx mori moths (transovarial) as well as with N. bombycis contaminated food (horizontal). Most diagnoses were carried out in the advanced stages of infection until the time that infection might spread to other healthy insects. Hence, early diagnosis of pebrine is of utmost importance to quarantine infected larvae from uninfected silkworm batches and stop further spread of the infection. The findings of our study provide an insight into how the silkworm larval host defence system was activated against early N. bombycis transovarial infection. The results obtained from transcriptome analysis of infected 2nd instar larvae revealed significant (adjusted P-value < 0.05) expression of 1888 genes of which 801 genes were found to be upregulated and 1087 genes were downregulated when compared with the control. Pathway analysis indicated activation of the immune deficiency (IMD) pathway, which shows a potential immune defence response against pebrine infection as well as suppression of the melanin synthesis pathway due to lower expression of prophenoloxidase activating enzyme (PPAE). Liquid chromatography mass spectrometry (LC-MS/MS) analysis of haemolymph from infected larvae shows the secretion of serpin binding protein of N. bombycis which might be involved in the suppression of the melanization pathway. Moreover, among the differentially expressed genes, we found that LPMC-61, yellow-y, gasp and osiris 9 can be utilised as potential markers for early diagnosis of transovarial pebrine infection in B. mori. Physiological as well as biochemical roles and functions of many of the essential genes are yet to be established, and enlightened research will be required to characterize the products of these genes.

Mechanism of carbendazim in treating pebrine disease of Bombyx mori based on GC/MS-based metabonomics.

Pebrine disease is caused by microporidia (Nosema bombycis) and is destructive to sericulture production. A carbendazim-based drug FangWeiLing (FWL) has a significant control effect on the disease, which is a successful example of drug treatment of microsporidia. In this study, the therapeutic effect and critical action time of FWL were investigated by silkworm rearing biological test. Besides, the hemolymph samples from silkworms in the control group, model group, and FWL group were analyzed by metabonomics based on gas chromatography-mass spectrometry (GC/MS). The results showed that FWL had a significant therapeutic effect on pebrine disease, and the critical action time was 24 ~ 48 h post inoculation. Forty-seven different metabolites related to pebrine disease were screened out, and correlated with starch and sucrose metabolism; aminoacyl-tRNA biosynthesis; arginine biosynthesis; glycine, serine, and threonine metabolism; and phenylalanine, tyrosine, and tryptophan biosynthesis. After pretreatment with FWL, the metabolites were all effectively regulated, indicating productive intervention. Principal component analysis (PCA) also showed that the overall metabolic profile of the FWL group tended toward the control group. Compared with the control group, 16 different metabolites were obtained from the hemolymph of B.mori in FWL group, mainly involving aminoacyl-tRNA biosynthesis and taurine and hypotaurine metabolism. It indicated that FWL had some effect on silkworm metabolism, which might be related to the decrease in cocoon quality. In conclusion, combined with the life cycle of N. bombycis, the mechanism of carbendazim in the treatment of pebrine disease can be fully revealed. Carbendazim can effectively reduce the destruction of amino acid metabolism and carbohydrate metabolism by N. Bombycis infection by inhibiting the proliferation of the meronts in silkworms, thus maintaining the normal physiological state of B. mori and achieve therapeutic effects. GC/MS-based metabonomics is a valuable and promising strategy to understand the disease mechanism and drug treatment of pebrine disease.

Rapid detection of silkworm microsporidia by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Microsporidia cause the disease pébrine in silkworm and are known to be detrimental to sericulture and beekeeping. The microsporidian species Nosema bombycis was rapidly identified in silkworm (Bombyx mori) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Four types of microsporidian spores purified from infected silkworm could be distinguished based on the differences in their mass fingerprints. Microsporidia growing in a silkworm larva were also identified based on their mass spectra after rapid separation using filtration and centrifugation for 30 min.

Isolation and Molecular Identification of Microsporidian Pathogen Causing Nosemosis in Muga Silkworm, Antheraea assamensis Helfer (Lepidoptera: Saturniidae).

Microsporidia are intracellular fungal parasites and they are the most common pathogens for sericulture. Microsporidian sp. can cause pebrine, a dreadful disease and lead to destructive disorder in Muga silkworm, Antheraea assamensis Helfer by vertical and horizontal transmission. This disease is the key factor obstructing the developmental progress of Muga culture in India. Nevertheless, molecular identification and characterization of pathogen associated with pebrine disease in A. assamensis is not yet established. Insect bioassay studies revealed that microsporidian infection in Muga silkworm, A. assamensis Helfer significantly reduced (P < 0.005) cocoon weight, pupal weight, shell weight and silk ratios. A new set of PCR primers suitable for amplification of small subunit ribosomal RNA (SSU-rRNA) of microsporidia infecting A. assamensis have been designed. The amplicon was cloned, sequenced and analysed. Microsporidia pathogen of wild silk moth A. assamensis has been identified at genus level as Nosema sp. AA1. Phylogeny of Nosema sp. AA1 was constructed on the basis of SSU-rRNA sequence and it has a close evolutionary relationship with microsporidian pathogens of other wild silkmoths. The arrangement and organization of the rRNA genes inferred that Nosema sp. AA1 belongs to true Nosema group and not to members of the Nosema/Vairimorpha group.

Molecular characterization and phylogenetic analyses of MetAP2 gene and protein of Nosema bombycis isolated from Guangdong, China.

Background: Pebrine, caused by microsporidium Nosema bombycis, is a devastating disease that causes serious economic damages to the sericulture industry. Studies on development of therapeutic and diagnostic options for managing pebrine in silkworms are very limited. Methionine aminopeptidase type 2 (MetAP2) of microsporidia is an essential gene for their survival and has been exploited as the cellular target of drugs such as fumagillin and its analogues in several microsporidia spp., including Nosema of honeybees. Methods: In the present study, using molecular and bioinformatics tools, we performed in-depth characterization and phylogenetic analyses of MetAP2 of Nosema bombycis isolated from Guangdong province of China. Results: The full length of MetAP2 gene sequence of Nosema bombycis (Guangdong isolate) was found to be 1278 base pairs (bp), including an open reading frame of 1,077 bp, encoding a total of 358 amino acids. The bioinformatics analyses predicted the presence of typical alpha-helix structural elements, and absence of transmembrane domains and signal peptides. Additionally, other characteristics of a stable protein were also predicted. The homology-based 3D models of MetAP2 of Nosema bombycis (Guangdong isolate) with high accuracy and reliability were developed. The MetAP2 protein was expressed and purified. The observed molecular weight of MetAP2 protein was found to be ~43-45 kDa. The phylogenetic analyses showed that MetAP2 gene and amino acids sequences of Nosema bombycis (Guangdong isolate) shared a close evolutionary relationship with Nosema spp. of wild silkworms, but it was divergent from microsporidian spp. of other insects, Aspergillus spp., Saccharomyces cerevisiae, and higher animals including humans. These analyses indicated that the conservation and evolutionary relationships of MetAP2 are closely linked to the species relationships. Conclusion: This study provides solid foundational information that could be helpful in optimization and development of diagnostic and treatment options for managing the threat of Nosema bombycis infection in sericulture industry of China.

Strategies for diagnosing Nosema bombycis (Microsporidia: Nosematidae); the agent of pebrine disease.

Pebrine disease, caused by Nosema bombycis (N. bombycis), is the most important pathogen known to the silk industry. Historical evidence from several countries shows that the outbreaks of pebrine disease have largely caused the decline of the sericulture industry. Prevention is the first line to combat pebrine as a deadly disease in silkworm; however, no effective treatment has yet been presented to treat the disease. Many different methods have been used for detection of pebrine disease agent. This review focuses on the explanation and comparison of these methods, and describes their advantages and/or disadvantages. Also, it highlights the ongoing advances in diagnostic methods for N. bombycis that could enable efforts to halt this microsporidia infection. The detection methods are categorized as microscopic, immunological and nucleic acid-based approaches, each with priorities over the other methods; however, the suitability of each method depends on the available equipment in the laboratory, the mass of infection, and the speed and sensitivity of detection. The accessibility and economic efficiency are compared as well as the speed and the sensitivity for each method. Although, the light microscopy is the most common method for detection of N. bombycis, qPCR is the most preferred method for large data based on speed and sensitivity as well as early detection ability.

Comparison of the Germination Conditions of Two Large-Spore Microsporidia Using Potassium and Sodium Ion Solutions.

The germination of a microsporidian polar tube generally occurs under alkaline conditions. Typically, microsporidian spores can be stored in physiological salt solution for short periods. However, because of differences in the lodging area, the requirements may not always be uniform. In fact, Trachipleistophora sp. OSL-2012-10 (nomen nudum Trachipleistophora haruka) germinated when preserved in physiological salt solution. In this study, the germination characteristics of the large-spore microsporidia Trachipleistophora sp. FOA-2014-10 and Vavraia sp. YGSL-2015-13 were compared with those of Trachipleistophora sp. OSL-2012-10; moreover, we investigated whether these characteristics are specific to these microsporidia. We found that both microsporidia germinated in physiological salt solution. These differences in germination rates were affected by the preservation solution and temperature.

Diagnosis of Pebrine Disease in Silkworm Using Molecular Methods.

Since pebrine disease, as the most important and dangerous disease in silkworms, spreads horizontally through the spores and vertically through the eggs, combating the disease and eliminating it completely from livestock production has been associated with numerous problems. This project aimed to identify the molecular cause of pebrine disease in silkworms using a sensitive, specific, and accurate method. To this purpose, a 136 bp fragment was selected based on the Nosema bombycis partial SSU rDNA sequence, and a pair of primers was designed. Afterward, using the conventional polymerase chain reaction (PCR) method, the target fragment was amplified and sequenced. After that, to determine the detection sensitivity, using the Real-Time PCR method, 5-fold serial dilutions of N. bombycis DNA were prepared, and the last dilution that produced a fluorescent signal was considered the minimum detection limit. All tests were performed in duplicates. Based on the results of the sensitivity test, the standard curve including Ct values ​​and DNA concentration was used for analysis. Moreover, 80 unknown samples examined by light microscope were evaluated using conventional PCR and Real-Time PCR. Both PCR results showed no amplification for the negative control samples. The findings demonstrated that the lowest detection limit for N. bombycis was less than 6 pg of DNA, while, this amount was 8 ng for conventional PCR. Out of 80 samples examined, 55, 60, and 62 samples were positive for light microscope, conventional PCR, and Real-Time PCR methods, respectively. The findings suggested that the Real-Time PCR method had a higher ability to detect the causative agent of pebrine disease than the conventional PCR method, and both methods were superior to light microscopy. Therefore, due to the fewer steps and higher accuracy of Real-Time PCR, it can be introduced as a suitable method for diagnosing pebrine disease.

Generation of Resistance to Nosema bombycis (Dissociodihaplophasida: Nosematidae) by Degrading NbSWP12 Using the Ubiquitin-Proteasome Pathway in Sf9-III Cells.

Nosema bombycis Naegeli (Dissociodihaplophasida: Nosematidae), an obligate intracellular parasite of the silkworm Bombyx mori, causes a devastating disease called pébrine. Every year pébrine will cause huge losses to the sericulture industry worldwide. Until now, there are no effective methods to inhibit the N. bombycis infection in silkworms. In this study, we first applied both the novel protein degradation Trim-Away technology and NSlmb (F-box domain-containing in the N-terminal part of supernumerary limbs from Drosophila melanogaster) to lepidopteran Sf9-III cells to check for specific degradation of a target protein in combination with a single-chain Fv fragment (scFv). Our results showed that the Trim-Away and NSlmb systems are both amenable to Sf9-III cells. We then created transgenic cell lines that overexpressed the protein degradation system and N. bombycis chimeric scFv targeting spore wall protein NbSWP12 and evaluated the effects of the insect transgenic cell lines on the proliferation of N. bombycis. Both methods could be applied to cell lines and both Trim-Away and NSlmb ubiquitin degradation systems effectively inhibited the proliferation of N. bombycis. Further, either of these degradation systems could be applied to individual silkworms through a transgenic platform, which would yield individual silkworms with high resistance to N. bombycis, thus greatly speeding up the process of acquiring resistant strains.

Detection and Characterization of Nosema bombycis Using TEM and SEM Techniques.

Pebrine disease is the most important and dangerous disease of silkworm caused by Nosema bombycis as an obligate intracellular parasitic fungus. It has caused tremendous economic losses in the silk industry in recent years. Given the fact that light microscopy method (with low accuracy) is the only method for diagnosing pebrine disease in the country, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) methods were adopted in this study for accurate morphological identification of the spores causing pebrine disease. Infected larvae and mother moth samples were collected from several farms (Parand, Parnian, Shaft, and Iran Silk Research Center in Gilan province, Iran). The spores were then purified using the sucrose gradient method. From each region, 20 and 10 samples were prepared for SEM and TEM analysis, respectively. In addition, an experiment was performed to evaluate the symptoms of pebrine disease by treating fourth instars with the spores purified for the present study, along with a control group. The results of SEM analysis showed that the mean±SD length and width of spores were 1.99±0.25 to 2.81±0.32 µm, respectively. Based on the obtained results, the size of spores was smaller than the Nosema bombycis (N. bombycis) as the classic species that cause pebrine disease. In addition, transmission electron microscopy (TEM) pictures showed that the grooves of the adult spores were deeper than those of other Nosema species, Vairomorpha, and Pleistophora, and resembled N. bombycis in other studies. Examination of pathogenicity of the studied spores indicated that the disease symptoms in controlled conditions were similar to those in the sampled farms. The most important symptom in fourth and fifth instrars were the small size and no growth in the treatment group compared with the control group. Findings of SEM and TEM analysis showed better morphological and structural details of parasite compared with light microscopy, and demonstrated that the studied species were a native strain of N. bombycis specific to Iran, whose size and other characteristics were unique and introduced for the first time in this study.

Molecular Characterization of a New Nosema bombycis Strain Detected in Iranian Silkworm Farms.

PURPOSE: Pebrine as the most dangerous disease of silkworm mostly caused by Nosema species has caused huge economic losses. There is no information on the species and the genomic sequences of the pebrine-causing microsporidia in Iran. METHODS: In the present research, we tried to determine the sequences of two regions of rDNA using molecular methods. First, infected larvae and mother moths were collected from several farms in the north of Iran for identification and molecular characterization of microsporidian isolates. After extracting the spores and genomic DNA from the collected samples, two fragments of internal transcribed spacer (ITS) rDNA and small subunit (SSU) rDNA were amplified and sequenced, and registered in NCBI database and then, the phylogenetic tree was drawn. RESULTS: Results showed the obtained sequences (ITS rDNA: Accession No. MZ322002 and SSU rDNA: Accession No. MZ314703) represent a new strain of Nosema bombycis, which differs from the sequences deposited in the NCBI. CONCLUSION: The new N. bombycis strain identified in our study will help in control and management of the pebrine disease by specific detection of the infectious agent.

Silkworm, science worm.

From 1865 to 1869, on a "government order", Louis Pasteur tackled a silkworm disease, the pebrine, which was ruining the economy of southern France. Well beyond the scientific results-he was going to highlight a second disease, the flacherie-and the operational results-he installed techniques to limit the progression of one disease and protected the farms from the other-, this sequence opened the door to what would become Pasteur's working method: a science involved in practice, a great importance given to the team of collaborators and to innovations of all kinds, in this case, microphotography. It also establishes the characteristics of the socialization of the Pasteurian approach: diffusion of methods among all the social actors concerned, networking of scientists and internationalization of research.

De 1865 à 1869, sur une « commande de l'État ¼, Louis Pasteur s'attaque à une maladie du ver à soie, la pébrine, qui ruine l'économie du sud de la France. Bien au-delà des résultats scientifiques ­ il va mettre en évidence une deuxième maladie, la flacherie ­ et opérationnels ­ il installe des techniques pour limiter la progression de l'une et protéger les élevages de l'autre ­, cette séquence ouvre la porte à ce qui deviendra la méthode de travail de Pasteur : une science impliquée dans la pratique, une grande importance donnée à l'équipe de collaborateurs et à des innovations de toute sorte, ici la microphotographie. Elle installe aussi les caractéristiques de la mise en société de la démarche pasteurienne : diffusion des méthodes parmi tous les acteurs sociaux concernés, mise en réseau de scientifiques et internationalisation des recherches.

Cloning, Expression and Characterization of Spore Wall Protein 5 (SWP5) of Indian Isolate NIK-1S of Nosema bombycis.

SWPs are the major virulence component of microsporidian spores. In microsporidia, SWPs can be found either in exospore or endospore to serve as a putative virulence factor for host cell invasion. SWP5 is a vital protein that involves in exospore localization and supports the structural integrity of the spore wall and this action potentially modulates the course of infection in N. bombycis. Here we report recombinant SWP5 purification using Ni-NTA IMAC and SEC. GFC analysis reveals SWP5 to be a monomer which correlates with the predicted theoretical weight and overlaps with ovalbumin peak in the chromatogram. The raised polyclonal anti-SWP5 antibodies was confirmed using blotting and enterokinase cleavage experiments. The resultant fusion SWP5 and SWP5 in infected silkworm samples positively reacts to anti-SWP5 antibodies is shown in ELISA. Immunoassays and Bioinformatic analysis reveal SWP5 is found to be localized on exospore and this action could indicate the probable role of SWP5 in host pathogen interactions during spore germination and its contribution to microsporidian pathogenesis. This study will support development of a field-based diagnostic kit for the detection N. bombycis NIK-1S infecting silkworms. The analysis will also be useful for the formulation of drugs against microsporidia and pebrine disease.

Supplementation of Lactobacillus casei reduces the mortality of Bombyx mori larvae challenged by Nosema bombycis.

OBJECTIVE: Pebrine, caused by the microsporidium Nosema bombycis, is one of the severe diseases in Thai polyvoltine strains of the silkworm Bombyx mori. Studies showing the presence of Lactobacillus species in the silkworm gut, where the Nosema parasites enter, suggests that these bacteria may have a protective effect. The aim of this study was to investigate the effect of supplementation of Lactobacillus casei on the survival ratio of silkworm larvae challenged with N. bombycis. RESULTS: A group of silkworm larvae of the commercial Thai polyvoltine hybrid strain DokBua was supplemented with L. casei on the second day of the 2nd, 3rd, 4th, and 5th instar. When a control group of silkworm larvae were challenged with N. bombycis on the second day of the 4th instar, the survival rate was 68%, but it was 91% for larvae supplemented with L. casei. For those larvae that survived the treatments until pupation, we determined the growth characters larval weight, cocooning ratio, and pupation ratio, and the economic characters cocoon weight and cocoon shell weight. When infected with N. Bombycis, growth characters were significantly higher in larvae also receiving L. casei.

Development and optimization of a TaqMan assay for Nosema bombycis, causative agent of pébrine disease in Bombyx mori silkworm, based on the ß-tubulin gene.

"Pébrine" is a devastating disease of Bombyx mori silkworms that is highly contagious and can completely destroy an entire crop of silkworms and is thus a serious threat for the viability and profitability of sericulture. The disease is most commonly attributed to microsporidians of the genus Nosema, which are obligate intracellular parasites that are transmitted through spores. Nosema infections in silkworms are diagnosed primarily through light microscopy, which is labour intensive and less reliable, sensitive, and specific than PCR-based techniques. Here, we present the development and optimization of a new TaqMan based assay targeting the ß-tubulin gene in the pébrine disease causing agent Nosema bombycis in silkworms. The assay displayed excellent quantification linearity over multiple orders of magnitude of target amounts and a limit of detection (LOD) of 6.9 × 102 copies of target per reaction. The method is highly specific to N. bombycis with no cross-reactivity to other Nosema species commonly infecting wild silkworms. This specificity was due to three nucleotides in the probe-binding region unique to N. bombycis. The assay demonstrated a high reliability with a Coefficient of variation (CV) <5% for both intra-assay and inter-assay variability. The assay was used to trace experimental N. bombycis infection of silkworm larvae, in the fat body, midgut and ovary tissues, through pupation and metamorphosis to the emerging female moth, and her larval off-spring, confirming the vertical transmission of N. bombycis in silkworms. The TaqMan assay revealed a gradual increase in infection levels in the post-infection samples. The assay is reliable and simple to implement and can be a suitable complement to microscopy for routine diagnostics and surveillance in silkworm egg production centres with appropriate infrastructure.

Pebrine diagnosis using quantitative phase imaging and machine learning.

Pebrine is the most dreaded infectious disease of the silkworm and has devastated sericulture in Europe during the 18th century. Thereafter, if it is detected, the crop is burned to prevent further dissemination. The conventional microscopic examination of moth's body fluid is erroneous and it exacerbates on Metarhizium anisopliae (MA) contaminated test samples. This is due to the resemblance of pebrine and MA spores in the microscopic examination. Therefore, this study aims to demonstrate an efficient pebrine detection technique. In the proposed method, a motorised brightfield microscope is custom-made to acquire focused and defocused images of test spores. These images are used to produce quantitative phase images of the spores by the transport of intensity equation method. The phase images' histogram of oriented gradients feature is used by a machine learning classifier to categorise the spores. This system classified 92 pebrine and 185 MA spores with an accuracy of 97% at 0.04 seconds/spore. The duration taken for image acquisition is 2.5 minutes per sample (10 fields of view covering an area of 302 × 260 µm2 ). The proposed method shows reliable results in pebrine diagnosis and would be an efficient alternative for current approaches.

Quantitative PCR for detection of Nosema bombycis in single silkworm eggs and newly hatched larvae.

Pebrine disease is the only mandatory quarantine item in sericultural production due to its destructive consequences. So far, the mother moth microscopic examination method established by Pasteur (1870) remains the only detection method for screening for the causative agent Nosema bombycis (N. bombycis). Because pebrine is a horizontal and vertical transmission disease, it is better to inspect silkworm eggs and newly hatched larvae to investigate the infection rate, vertical transmission rate and spore load of the progenies. There is a rising demand for a more direct, effective and accurate detection approach in the sericultural industry. Here, we developed a molecular detection approach based on real-time quantitative PCR (qPCR) for pebrine inspection in single silkworm eggs and newly hatched larvae. Targeting the small-subunit rRNA gene of N. bombycis, this assay showed high sensitivity and reproducibility. Ten spores in a whole sample or 0.1 spore DNA (1 spore DNA represents the DNA content of one N. bombycis spore) in a reaction system was estimated as the detection limit of the isolation and real-time qPCR procedure. Silkworm egg tissues impact the detection sensitivity but are not significant in single silkworm egg detection. Of 400 samples produced by infected moths, 167 and 195 were scored positive by light microscopy and real-time qPCR analysis, respectively. With higher accuracy and the potential capability of high-throughput screening, this method is anticipated to be adaptable for pebrine inspection and surveillance in the sericultural industry. In addition, this method can be applied to ecology studies of N. bombycis-silkworm interactions due to its quantitative function.

An amplified electrochemical proximity immunoassay for the total protein of Nosema bombycis based on the catalytic activity of Fe3O4NPs towards methylene blue.

A simple electrochemical proximity immunoassay (ECPA) system for the total protein of Nosema bombycis (TP N.b) detection has been developed on the basis of a new amplification strategy combined with target-induced proximity hybridization. The desirable ECPA system was achieved through following process: firstly, the methylene blue (MB) labeled hairpin DNA (MB-DNA) were immobilized on electrode through Au-S bonding. Then, the antibody labeled complementary single-stranded DNA probe (Ab1-S1) hybridized with MB-DNA to open its hairpin structure, which led to the labeled MB far away from electrode surface. After that, the presence of target biomarker (TP N.b) and antibody labeled single-stranded DNA (Ab2-S2) triggered the typical sandwich reaction and proximity hybridization, which resulted in the dissociation of Ab1-S1 from electrode and the transformation of the MB-DNA into a hairpin structure with MB approaching to electrode surface. In consequence, the hairpin-closed MB was electrocatalyzed by the modified magnetic nanoparticles (Fe3O4NPs), leading to an increased and amplified electrochemical signal for the quantitative detection of TP N.b. In the present work, Fe3O4NPs were acted as catalyst to electrocatalyze the reduction of electron mediator MB for signal amplification, which could not only overcome the drawbacks of protein enzyme in electrocatalytic signal amplification but also shorten the interaction distance between catalyst and substance. Under optimal condition, the proposed ECPA system exhibited a wide linear range from 0.001ngmL(-1) to 100ngmL(-)(1) with a detection limit (LOD) of 0.54pgmL(-1). Considering the desirable sensitivity and specificity, as well as the novel and simple features, this signal amplified ECPA system opened an opportunity for quantitative analysis of many other kinds of protein biomarker.

Electrochemical immunosensor for detecting the spore wall protein of Nosema bombycis based on the amplification of hemin/G-quadruplex DNAzyme concatamers functionalized Pt@Pd nanowires.

In this work, an ultrasensitive electrochemical immunosensor for detecting the Pebrine disease related spore wall protein of Nosema bombycis (SWP N.b) was fabricated based on the amplification of hemin/G-quadruplex functionalized Pt@Pd nanowires (Pt@PdNWs). The synthesized Pt@PdNWs possessed large surface area, which could effectively improve the immobilization amount of hemin/G-quadruplex DNAzyme concatamers produced via hybridization chain reaction (HCR). In the presence of SWP N.b, the hemin/G-quadruplex labeled Pt@PdNWs bioconjugations was captured on electrode surface and thus obtained electrochemical signal. After the addition of NADH into the electrolytic cell, hemin/G-quadruplex firstly acted as an NADH oxidase to locally produce H2O2 in the presence of dissolved O2. Then, the generated H2O2 would be quickly reduced via hemin/G-quadruplex as a horseradish peroxidase mimicking (HRP-mimicking) DNAzyme, which finally promoted the self-redox reaction of hemin/G-quadruplex and a greatly enhanced electrochemical signal was obtained. Furthermore, Pt@PdNWs with excellent electrocatalytic performance could also amplify electrochemical signal. With these amplification factors, the electrochemical immunosensor exhibited a wide linear range from 0.001 ng mL(-1) to 100 ng mL(-1) with a detection limit (LOD) of 0.24 pg mL(-1), providing a new promise for the diagnosis of Pebrine disease.