New approach for the rational selection of markers to identify botulinum toxins.

The application of mass spectrometry (MS) to detect unique peptide markers has been widely employed as a means of identifying bacterial proteins. Botulinum neurotoxins (BoNTs) are bacterial proteins that cause the life-threatening disease botulism. BoNTs are divided into several antigenically distinct serotypes and several dozen subtypes. The toxins' molecular heterogeneity makes their detection highly challenging. In this study, we describe a new LC-MS/MS-based platform for the direct identification of proteins derived from various species and subspecies in a single assay, as exemplified by BoNTs. The platform employs a rational down-selection process through several steps based on a combination of bioinformatics, tryptic digestion, and LC-MS, each leads to the final panel of markers. This approach has been demonstrated for all 8 subtypes of botulinum serotype A (BoNT/A). Ab-independent and Ab-dependent assays were developed based on the identification of 4 rationally selected markers or a combination of some of them, which enables full selectivity coverage. The Ab-independent assay, which is highly simple and rapid, has a sample-to-result turnaround time of approximately 40 min and enables the identification of 500 MsLD50/mL (5 ng/mL) BoNT/A in complex environmental matrices. The Ab-dependent assay, which is based on toxin's specific enrichment, has a turnaround time of 100 min, but enables improved sensitivity (50 MsLD50/mL, 0.5 ng/mL). Both assays were verified and validated using various environmental samples. This approach can easily be expanded to other botulinum serotypes and exhibits the potential for even further extension as a highly multiplexed assay for protein-based toxins, viruses, and organisms.

High-resolution crystal structures of the botulinum neurotoxin binding domains from subtypes A5 and A6.

Clostridium botulinum neurotoxins (BoNTs) cause flaccid paralysis through inhibition of acetylcholine release from motor neurons; however, at tiny doses, this property is exploited for use as a therapeutic. Each member of the BoNT family of proteins consists of three distinct domains: a binding domain that targets neuronal cell membranes (HC ), a translocation domain (HN ) and a catalytic domain (LC). Here, we present high-resolution crystal structures of the binding domains of BoNT subtypes/A5 (HC /A5) and/A6 (HC /A6). These structures show that the core fold identified in other subtypes is maintained, but with subtle differences at the expected receptor-binding sites.

A viral-fusion-peptide-like molecular switch drives membrane insertion of botulinum neurotoxin A1.

Botulinum neurotoxin (BoNT) delivers its protease domain across the vesicle membrane to enter the neuronal cytosol upon vesicle acidification. This process is mediated by its translocation domain (HN), but the molecular mechanism underlying membrane insertion of HN remains poorly understood. Here, we report two crystal structures of BoNT/A1 HN that reveal a novel molecular switch (termed BoNT-switch) in HN, where buried α-helices transform into surface-exposed hydrophobic ß-hairpins triggered by acidic pH. Locking the BoNT-switch by disulfide trapping inhibited the association of HN with anionic liposomes, blocked channel formation by HN, and reduced the neurotoxicity of BoNT/A1 by up to ~180-fold. Single particle counting studies showed that an acidic environment tends to promote BoNT/A1 self-association on liposomes, which is partly regulated by the BoNT-switch. These findings suggest that the BoNT-switch flips out upon exposure to the acidic endosomal pH, which enables membrane insertion of HN that subsequently leads to LC delivery.

Isolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 6.

Botulinum neurotoxins (BoNTs), the most potent toxins known to humans and the causative agent of botulism, exert their effect by entering motor neurons and cleaving and inactivating SNARE proteins, which are essential for neurotransmitter release. BoNTs are proven, valuable pharmaceuticals used to treat more than 200 neuronal disorders. BoNTs comprise 7 serotypes and more than 40 isoforms (subtypes). BoNT/A1 is the only A-subtype used clinically due to its high potency and long duration of action. While other BoNT/A subtypes have been purified and described, only BoNT/A2 is being investigated as an alternative to BoNT/A1. Here we describe subtype BoNT/A6 with improved pharmacological properties compared to BoNT/A1. It was isolated from Clostridium botulinum CDC41370, which produces both BoNT/B2 and BoNT/A6. The gene encoding BoNT/B2 was genetically inactivated, and A6 was isolated to greater than 95% purity. A6 was highly potent in cultured primary rodent neuronal cultures and in human induced pluripotent stem cell-derived neurons, requiring 20-fold less toxin to cause 50% SNAP-25 cleavage than A1. Second, A6 entered hiPSCs faster and more efficiently than A1 and yet had a long duration of action similar to BoNT/A1. Third, BoNT/A6 had similar LD50 as BoNT/A1 after intraperitoneal injection in mice; however, local intramuscular injection resulted in less systemic toxicity than BoNT/A1 and a higher (i.m.) LD50, indicating its potential as a safer pharmaceutical. These data suggest novel characteristics of BoNT/A6 and its potential as an improved pharmaceutical due to more efficient neuronal cell entry, greater ability to remain localized at the injection site, and a long duration.IMPORTANCE Botulinum neurotoxins (BoNTs) have proved to be an effective treatment for a large number of neuropathic conditions. BoNTs comprise a large family of zinc metalloproteases, but BoNT/A1 is used nearly exclusively for pharmaceutical purposes. The genetic inactivation of a second BoNT gene in the native strain enabled expression and isolation of a single BoNT/A6 from cultures. Its characterization indicated that BoNT/A subtype A6 has a long duration of action comparable to A1, while it enters neurons faster and more efficiently and remains more localized after intramuscular injection. These characteristics of BoNT/A6 are of interest for potential use of BoNT/A6 as a novel BoNT-based therapeutic that is effective and has a fast onset, an improved safety profile, and a long duration of action. Use of BoNT/A6 as a pharmaceutical also has the potential to reveal novel treatment motifs compared to currently used treatments.

Yesterday and Today: The Impact of Research Conducted at Camp Detrick on Botulinum Toxin.

Introduction: This review summarizes the research conducted on botulinum toxin (BoTx) from 1943 to 1956 by a small group of Camp Detrick investigators and their staff. A systematic, cross-disciplinary approach was used to develop effective vaccines against this biological warfare threat agent. In response to the potential need for medical countermeasures against BoTx during World War II, the refinement of isolation and purification techniques for BoTx successfully led to the large-scale production of botulinum toxoid vaccines. In addition, the work at Camp Detrick provided the foundation for the subsequent use of BoTx as a tool for studying the trophic regulation of skeletal muscle within motor neuron terminals and, more recently, for elucidation of the intricate details of neurotransmitter release at the molecular level. Indirectly, Camp Detrick investigators also played a significant role in studies that culminated in the use of BoTx as a pharmaceutical product that has been approved by the U.S. Food and Drug Administration for treating movement disorders, autonomic dysfunctions, and other conditions. Methods: Online literature searches were performed with Google, Google Scholar, PubMed, the bibliography from the Camp Detrick technical library, and at the Defense Technical Information Center. Reference lists in some of the primary research publications and reviews also provided source material. Search terms included botulinum, botulinus, and Camp Detrick. References related to the subsequent impacts of the Camp Detrick results were selected and cited from reviews and primary references in the more recent literature. Notes on toxin nomenclature and potential sources of error in this study are presented. Results: The literature searches returned 27 citations of Camp Detrick authors, 24 of which were articles in peer-reviewed journals. The publications by these investigators included several disciplines such as biochemistry, immunology, pharmacology, physiology, and toxicology. A fundamental finding was the identification of critical nutritional components for improved growth of Clostridium botulinum and the increased production of BoTx serotype A. The purification processes that were developed at Camp Detrick allowed for the production of crystalline material to be scaled up for the manufacture of toxoid vaccine. Based on the research by Camp Detrick scientists, a toxoid supply of over 1 million units was available to vaccinate ~300,000 troops before the large-scale operations of D-Day. Conclusions: BoTx research during the period 1943 to 1956 resulted in refinements in the techniques for isolating and purifying the crystalline BoTx type A. These results led to the development and manufacture of a toxoid vaccine that was available in a sufficient quantity to protect ~300,000 warfighters in a large-scale military operation. One of the most important long-term consequences derived from the knowledge gained by the efforts at Camp Detrick was the development in the 1980s of safe and effective therapeutic uses for BoTx type A, the most lethal biological substance known.

Foodborne botulism due to ingestion of home-canned green beans: two case reports.

BACKGROUND: Foodborne botulism is a life-threatening, rapidly progressive disease. It has an incidence of less than 10 cases per year in Germany and mostly affects several previously healthy people at the same time. The only specific treatment is the administration of botulism antitoxin. According to the German guidelines administration of antitoxin is recommended only in the first 24 hours after oral ingestion of the toxin. CASE PRESENTATION: A 47-year-old white woman and her 51-year-old white husband presented with paralysis of multiple cranial nerves and rapidly descending paralysis approximately 72 hours after ingestion of home-canned beans. The disease was complicated by autonomic changes like hypertension, febrile temperatures, and a paralytic ileus. The diagnosis was confirmed by identification of botulinum neurotoxin type A in the serum of the woman. In accordance with the German guidelines, antitoxin was not given due to the prolonged time interval at diagnosis. Both patients had a long intensive care unit course requiring ventilation for approximately 5 months. Finally they recovered completely. CONCLUSIONS: A full recovery from foodborne botulism is possible even in patients with intensive care lasting several months. There are only case reports indicating that administration of antitoxin may shorten the course of the disease, even if given later than 24 hours after intoxication. Due to the rarity of the disease and its rapid course there are no randomized controlled trials. Thus, evidence of the superiority of this treatment is lacking. However, the prevailing view according to the German guidelines to administer antitoxin only within 24 hours after ingestion of the toxin should be questioned in the case of progression of the disease with proof of remaining toxin in the blood.

High Yield Preparation of Functionally Active Catalytic-Translocation Domain Module of Botulinum Neurotoxin Type A That Exhibits Uniquely Different Enzyme Kinetics.

Botulinum neurotoxins (BoNTs) are the most toxic proteins known to cause flaccid muscle paralysis as a result of inhibition of neurotransmitter release from peripheral cholinergic synapses. BoNT type A (BoNT/A) is a 150 kDa protein consisting of two major subunits: light chain (LC) and heavy chain (HC). The LC is required for the catalytic activity of neurotoxin, whereas the C and N terminal domains of the HC are required for cell binding, and translocation of LC across the endosome membranes, respectively. To better understand the structural and functional aspects of BoNT/A intoxication we report here the development of high yield Escherichia coli expression system (2-20-fold higher yield than the value reported in the literature) for the production of recombinant light chain-translocation domain (rLC-TD/A) module of BoNT/A which is catalytically active and translocation competent. The open reading frame of rLC-TD/A was PCR amplified from deactivated recombinant BoNT/A gene (a non-select agent reagent), and was cloned using pET45b (+) vector to express in E. coli cells. The purification procedure included a sequential order of affinity chromatography, trypsinization, and anion exchange column chromatography. We were able to purify > 95% pure, catalytically active and structurally well-folded protein. Comparison of enzyme kinetics of purified LC-TD/A to full-length toxin and recombinant light chain A suggest that the affinity for the substrate is in between endopeptidase domain and botulinum toxin. The potential application of the purified protein has been discussed in toxicity and translocation assays.

A childhood-onset intestinal toxemia botulism during chemotherapy for relapsed acute leukemia.

BACKGROUND: Botulism is a potentially fatal infection characterized by progressive muscle weakness, bulbar paralysis, constipation and other autonomic dysfunctions. A recent report suggested that cancer chemotherapy might increase the risk for the intestinal toxemia botulism in both adults and children. CASE PRESENTATION: We report a 5-year-old boy, who developed general muscle weakness, constipation, ptosis and mydriasis during the third induction therapy for relapsed acute myeloid leukemia. He had recent histories of multiple antibiotic therapy for bacteremia and intake of well water at home. Repeated bacterial cultures identified Clostridium botulinum producing botulinum neurotoxin A. Botulinum toxin A was isolated from his stools at 17, 21, and 23 days after the onset. Symptoms were self-limiting, and were fully recovered without anti-botulinum toxin globulin therapy. CONCLUSION: This is the second report of a pediatric case with cancer chemotherapy-associated intestinal toxemia botulism. Our case provides further evidence that the immunocompromised status due to anti-cancer treatments increases the risk for the development of botulism at all ages in childhood.

Impedimetric immunosensor for the label-free and direct detection of botulinum neurotoxin serotype A using Au nanoparticles/graphene-chitosan composite.

In this work, a novel nanocomposite film consisting of the Au nanoparticles/graphene-chitosan has been designed to construct an impedimetric immunosensor for a rapid and sensitive immunoassay of botulinum neurotoxin A (BoNT/A). BoNT/A antibody was immobilized on glassy carbon electrode modified with Au nanoparticles/graphene-chitosan for the signal amplification. The fabrication of immunosensor was extensively characterized by using transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray diffraction (XRD), Fourier transform infrared (FTIR), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). The impedance changes, due to the specific immuno-interactions at the immunosensor surface that efficiently restricted the electron transfer of redox probe Fe(CN)64-/3- were utilized to detect BoNT/A. The measurements were highly targeted specific and linear with logarithmic BoNT/A concentrations in PBS, milk and human serum across a 0.27-268pgmL-1 range and associated with a detection limit of 0.11pgmL-1.

Detection of Clostridium botulinum neurotoxin genes (A-F) in dairy farms from Northern Germany using PCR: A case-control study.

Classical botulism in cattle mainly occurs after ingestion of feed contaminated with preformed toxin. In 2001 a form of botulism ("visceral botulism") was postulated to occur after ingestion of Clostridium (C.) botulinum cells or spores, followed by colonization of the intestine, and local production of botulinum neurotoxin (BoNT) causing chronic generalized disease. To verify the potential role of C. botulinum in the described syndrome, a case-control study was conducted, including 139 farms. Fecal samples, rumen content, water and silage samples were collected on each farm. Real time BoNT gene PCR assays were conducted after enrichment in RCM (Reinforced Clostridial Medium) at 37 °C and conventional PCRs after enrichment in MCM (Modified Cooked Meat Medium) at 30 °C. Furthermore, a direct detection of BoNT genes without prior enrichment was attempted. BoNT A, B, C, D, E and F genes were detected in animal samples from 25 (17.99%), 3 (2.16%), 0 (0.0%), 2 (1.44%), 1 (0.72%), and 3 (2.16%) farms, respectively. Eleven feed samples were positive for BoNT A gene. By enrichment a significant increase in sensitivity was achieved. Therefore, this should be an essential part of any protocol. No significant differences regarding BoNT gene occurrence could be observed between Case and Control farms or chronically diseased and clinically healthy animals within the particular category. Thus, the postulated form of chronic botulism in cows could not be confirmed. This study supports the general opinion that C. botulinum can occasionally be found in the rumen and intestine of cows without causing disease.

Outbreak of Foodborne Botulism Associated with Improperly Jarred Pesto--Ohio and California, 2014.

On July 28, 2014, the Cincinnati Health Department was notified of suspected cases of foodborne botulism in two women admitted to the same hospital 12 days apart. Patient A had been treated for 12 days for suspected autoimmune disease. When patient B, the roommate of patient A, was evaluated at the same medical center for similar symptoms, it was learned that on July 13, patient A and patient B had shared a meal that included prepackaged pesto from a jar; clinicians suspected botulism and notified the local health department. The pesto had been purchased from company A's farm stand in San Clemente, California. Laboratory testing detected botulinum toxin type B by enzyme-linked immunosorbent assay (ELISA) in leftovers of pasta with pesto. A culture of these food samples yielded Clostridium spp. that produced botulinum toxin type B; polymerase chain reaction (PCR) testing also was positive for type B toxin gene. Environmental assessment of company A identified improper acidification and pressurization practices and lack of licensure to sell canned products commercially, including products in hermetically-sealed jars. On July 30, the vendor voluntarily recalled all jarred products, and the California Department of Public Health (CDPH) warned the public not to consume company A's jarred foods. This report describes the two cases and the public health investigation that traced the source of the outbreak.

Outbreak of Botulism After Consumption of Illicit Prison-Brewed Alcohol in a Maximum Security Prison--Arizona, 2012.

The authors investigated the second botulism outbreak to occur in a maximum security prison in Arizona within a 4-month period. Botulism was confirmed in eight men aged 20 to 35 years who reported sharing a single batch of pruno made with potatoes. Initial symptoms included blurred vision, slurred speech, muscle weakness, ptosis, and dysphagia. All patients received heptavalent botulinum antitoxin, seven required mechanical ventilation, and all survived. The median incubation period was 29 hours. Sera from all patients and leftover pruno tested positive for botulinum toxin type A. Botulism should be considered among prisoners with cranial nerve palsies and descending, symmetric flaccid paralysis. Prison-brewed alcohol, particularly when made with potatoes, can be a vehicle for botulism and is associated with outbreaks of botulism in prisons.

Botulinum neurotoxin dose-dependently inhibits release of neurosecretory vesicle-targeted luciferase from neuronal cells.

Botulinum toxin is a bacterial toxin that inhibits neurotransmitter release from neurons and thereby causes a flaccid paralysis. It is used as drug to treat a number of serious ailments and, more frequently, for aesthetic medical interventions. Botulinum toxin for pharmacological applications is isolated from bacterial cultures. Due to partial denaturation of the protein, the specific activity of these preparations shows large variations.Because of its extreme potential toxicity, pharmacological preparations must be carefully tested for their activity. For the current gold standard, the mouse lethality assay, several hundred thousand mice are killed per year. Alternative methods have been developed that suffer from one or more of the following deficits: In vitro enzyme assays test only the activity of the catalytic subunit of the toxin. Enzymatic and cell based immunological assays are specific for just one of the different serotypes. The current study takes a completely different approach that overcomes these limitations: Neuronal cell lines were stably transfected with plasmids coding for luciferases of different species, which were N-terminally tagged with leader sequences that redirect the luciferase into neuro-secretory vesicles. From these vesicles, luciferases were released upon depolarization of the cells. The depolarization-dependent release was efficiently inhibited by of botulinum toxin in a concentration range (1 to 100 pM) that is used in pharmacological preparations. The new assay might thus be an alternative to the mouse lethality assay and the immunological assays already in use.

Alcohol Production, Prevention Strategies, and Inmate Knowledge About the Risk for Botulism From Pruno Consumption in a Correctional Facility--Arizona, 2013.

During July to November 2012, two botulism outbreaks (12 cases total) occurred in one all-male prison; both were associated with illicitly brewed alcohol (pruno) consumption. Inmate surveys were conducted to evaluate and develop prevention and education strategies. Qualitative surveys with open-ended questions were performed among inmates from rooms where outbreaks occurred to learn about pruno consumption. Quantitative surveys assessed knowledge gained after the outbreaks and preferred information sources. For the quantitative surveys, 250 inmates were randomly selected by bed from across the correctional facility and 164 inmates were interviewed. Only 24% of inmates reported any botulism knowledge before the outbreaks and education outreach, whereas 73% reported knowledge after the outbreaks (p < .01). Preferred information sources included handouts/fliers (52%) and the prison television channel (32%).

Large Outbreak of Botulism Associated with a Church Potluck Meal--Ohio, 2015.

On April 21, 2015, the Fairfield Medical Center (FMC) and Fairfield Department of Health contacted the Ohio Department of Health (ODH) about a patient suspected of having botulism in Fairfield County, Ohio. Botulism is a severe, potentially fatal neuroparalytic illness.* A single case is a public health emergency, because it can signal an outbreak. Within 2 hours of health department notification, four more patients with similar clinical features arrived at FMC's emergency department. Later that afternoon, one patient died of respiratory failure shortly after arriving at the emergency department. All affected persons had eaten at the same widely attended church potluck meal on April 19. CDC's Strategic National Stockpile sent 50 doses of botulinum antitoxin to Ohio. FMC, the Fairfield Department of Health, ODH, and CDC rapidly responded to confirm the diagnosis, identify and treat additional patients, and determine the source.

A Double-Sandwich ELISA for Identification of Monoclonal Antibodies Suitable for Sandwich Immunoassays.

The sandwich immunoassay (sELISA) is an invaluable technique for concentrating, detecting, and quantifying target antigens. The two critical components required are a capture antibody and a detection antibody, each binding a different epitope on the target antigen. The specific antibodies incorporated into the test define most of the performance parameters of any subsequent immunoassay regardless of the assay format: traditional ELISA, lateral-flow immunoassay, various bead-based assays, antibody-based biosensors, or the reporting label. Here we describe an approach for identifying monoclonal antibodies (mAbs) suitable for use as capture antibodies and detector antibodies in a sELISA targeting bacterial protein toxins. The approach was designed for early identification of monoclonal antibodies (mAbs), in the initial hybridoma screen.

Expression, purification and characterization of the receptor-binding domain of botulinum neurotoxin serotype B as a vaccine candidate.

The receptor-binding domain of botulinum neurotoxins (the HC fragment) is a promising vaccine candidate. Among the HC fragments of the seven BoNT serotypes, the expression of HC/B in Escherichia coli is considered especially challenging due to its accumulation as a non-soluble protein aggregate. In this study, the effects of different parameters on the expression of soluble HC/B were evaluated using a screening assay that included growing the bacterium at a small scale, a chemical cell lysis step, and a specific ELISA. The highest soluble HC/B expression levels were obtained when the bacterium E. coli BL21(DE3)+pET-9a-HC/B was grown in terrific broth media at 18°C without induction. Under these conditions, the yield was an order of magnitude higher than previously reported. Standard purification of the protein using a nickel column resulted in a low purity of HC/B. However, the addition of an acidic wash step prior to protein elution released a major protein contaminant and significantly increased the purity level. Mass spectrometry analysis identified the contaminant as ArnA, an E. coli protein that often contaminates recombinant His-tagged protein preparations. The purified HC/B was highly immunogenic, protecting mice from a 10(6) LD50 challenge after a single vaccination and generating a neutralizing titer of 50IU/ml after three immunizations. Moreover, the functionality of the protein was preserved, as it inhibited BoNT/B intoxication in vivo, presumably due to blockade of the neurotoxin protein receptor synaptotagmin.

Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System.

Clostridium botulinum subtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producing C. botulinum strain 657Ba at 100× lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenic C. botulinum expression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided in trans by the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed in Escherichia coli has indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of the in vitro, cellular, and in vivo activities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin.

Elimination of bioweapons agents from forensic samples during extraction of human DNA.

Collection of DNA for genetic profiling is a powerful means for the identification of individuals responsible for crimes and terrorist acts. Biologic hazards, such as bacteria, endospores, toxins, and viruses, could contaminate sites of terrorist activities and thus could be present in samples collected for profiling. The fate of these hazards during DNA isolation has not been thoroughly examined. Our goals were to determine whether the DNA extraction process used by the Royal Canadian Mounted Police eliminates or neutralizes these agents and if not, to establish methods that render samples safe without compromising the human DNA. Our results show that bacteria, viruses, and toxins were reduced to undetectable levels during DNA extraction, but endospores remained viable. Filtration of samples after DNA isolation eliminated viable spores from the samples but left DNA intact. We also demonstrated that contamination of samples with some bacteria, endospores, and toxins for longer than 1 h compromised the ability to complete genetic profiling.

Combination of biobarcode assay with on-chip capillary electrophoresis for ultrasensitive and multiplex biological agent detection.

Early diagnosis of biological agents is of paramount importance to prevent the casualties and fatal disease in human during bioterrorism or biological warfare. In this study, we reported an efficient and sensitive multiplex biological agent detection method based on the DNA biobarcode assay and the micro-capillary electrophoresis (µCE) technology. Monoplex as well as multiplex pathogen identification was performed using five targets including Bacillus anthracis, Francisella tularensis, Yersinia pestis, Vaccinia virus and Botulinum toxin A. Through the DNA biobarcode assay process, the magnetic microparticle-pathogen-polystyrene microbead complexes were formed, and the FAM labeled single stranded barcode DNA could be released from the complexes upon denaturation. Different lengths of a barcode DNA were designed to designate each pathogen, so that the specific peak elution time in the capillary electrophoresis on a chip allows us to distinguish the target with high accuracy within 3 min. We improved the assignment accuracy of the peak in the electropherogram by adding two bracket ladders. Owing to the abundant amount of barcode DNAs, the presence of B. anthracis, F. tularensis, Y. pestis, Vaccinia virus was confirmed with a limit of detection of 50CFU/mL, while Botulinum toxin A was analyzed even at a concentration of 12.5 ag/mL. Multiple pathogen detection was also successfully conducted in a phosphate buffered saline (PBS) as well as a serum medium with background of other pathogens. Thus, our analytical platform based on the biobarcode assay and on-chip CE analysis provides rapid, sensitive, multiplex, and accurate biological agent identification.