Detection Of Biological Warfare Agents Research Articles

A cell-free biosensor for multiplexed and sensitive detection of biological warfare agents

The rapid and precise detection of pathogenic agents is critical for public health and societal stability. The detection of biological warfare agents (BWAs) is especially vital within military and counter-terrorism contexts, essential in defending against biological threats. Traditional methods, such as polymerase chain reaction (PCR), are limited by their need for specific settings, impacting their adaptability and versatility. This study introduces a cell-free biosensor for BWA detection by converting the 16S rRNA of targeted pathogens into detectable functional protein molecules. The modular nature of this approach allows for the flexible configuration of pathogen detection, enabling the simultaneous identification of multiple pathogenic 16S rRNAs through customized reporter proteins for each targeted sequence. Furthermore, we demonstrate how this method integrates with techniques utilizing retroreflective Janus particles (RJPs) for facile and highly sensitive pathogen detection. The cell-free biosensor, employing RJPs to measure the reflection of non-chromatic white light, can detect 16S rRNA from BWAs at femtomolar levels, corresponding to tens of colony-forming units per milliliter of pathogenic bacteria. These findings represent a significant advancement in pathogen detection, offering a more efficient and accessible alternative to conventional methodologies.

Standoff detection of bacterial spores by field deployable coherent Raman spectroscopy

Vibrational spectroscopies offer great potential for standoff detection of chemical and biological warfare agents, avoiding contamination to the operator and equipment. Among them, particularly promising is Coherent anti-Stokes Raman scattering (CARS) spectroscopy, using synchronized pump/Stokes laser pulses to set up a vibrational coherence of target molecules at a laser focus, which is read by further interaction with a probe pulse, resulting in the emission of a coherent beam detectable at a distance. CARS has previously demonstrated the capability to detect bacterial spores based on the Raman spectrum of the characteristic molecule calcium dipicolinate (CaDPA); however, a complex and bulky laser technology, which is only suitable for a laboratory environment, was employed. Here we develop a broadband CARS setup based on a compact, industrial grade ytterbium laser system. We demonstrate high signal-to-noise ratio detection of Bacillus atrophaeus spores at a concentration of 105 cfu/mm2, at a standoff distance of 1 m, and an acquisition time of 1 s. Our system, which combines chemical specificity and sensitivity along with improved ruggedness and portability, paves the way to a new generation of instruments for real-world standoff detection of chemical and biological threats.

Development of novel electrochemical sensor for the detection of biological warfare agents: enzyme, antibody, and DNA free

There is an urgent need to develop a novel sensor that can quickly and accurately detect biological warfare agents (BWAs). A novel electrochemical sensor was specially designed and developed for Staphylococcus aureus as a potential BWAs without using any enzyme, antibody, or genetic material. Cyclic voltammetry (CV), linear resistance polarization (LRP), chronoamperometry, impedance spectroscopy, and Differential Pulse Voltammetry (DPV) techniques were used for fast and accurate detection of BWAs. Co-precipitation technique was used for the synthesis of CuO nanoparticles (NPs) for their use in the electrochemical sensor. The effect of temperature, stirring and addition of CuO NPs was thoroughly investigated. The storage, stability, and performance of electrochemical sensor was tested for 45 days. The current response of the electrochemical sensor was found linear with the correlation coefficient, R2 = 0.991. The major advantage of this novel electrochemical sensor is that immobilization of enzyme, antibody, or DNA is not required in sensing technology as the immobilization process makes biosensors a highly specific. The novel electrochemical sensor was very quick, accurate, non-specific, and provide reliable results in the qualitative and quantitative estimation of pathogenic bacteria as a potential BWAs. A novel electrochemical sensor was specially designed and developed for the detection of Staphylococcus aureus pathogenic bacteria as a potential BWAs without using any enzyme, antibody, or genetic material. Cyclic voltammetry, linear resistance polarization, chronoamperometry, impedance spectroscopy, and Differential Pulse Voltammetry (DPV) experiments were carried out for fast and accurate detection of BWAs. Storage, stability, and reusability of the electrochemical sensor was tested for continuous 45 days. The current response of the electrochemical sensor was found to be linear with the correlation coefficient, R2 = 0.991. The major advantage of this novel sensor is that immobilization of enzyme, antibody, or DNA is not required in sensing technology as the immobilization process makes biosensor a highly specific. The novel electrochemical sensor was proved to be very quick, accurate, non-specific, and provide reliable results in the qualitative and quantitative estimation of pathogenic bacteria as a potential BWAs.

Remote Sensing of Biochemicals in Aerosol Form Using Fluorescence Sensor for Defence and Security

The use of biological warfare agents by terror groups is one of the biggest concerns in today’s scenario. The present work provides a solution for early detection of biological warfare agents from standoff distances of up to 1.0 km to mitigate the threat. The experimental results reported in the paper are a part of development of Light Detection and Ranging (LiDAR) system. The system utilizes UV Laser (266 nm) induced fluorescence (LIF) spectroscopy for the detection of bio-molecules in aerosol form. The technology has been demonstrated and the signals from bio aerosols have been detected from distances of up to 1.0 km in day time with detection limit of the order of 106 bacteria per litre.

Detection of Protein Toxin Simulants from Contaminated Surfaces by Paper Spray Mass Spectrometry.

Proteinaceous toxins are harmful proteins derived from plants, bacteria, and other natural sources. They pose a risk to human health due to infection and also as possible biological warfare agents. Paper spray mass spectrometry (PS-MS) with wipe sampling was used to detect proteins from surfaces as a potential tool for identifying the presence of these toxins. Proteins ranging in mass between 12.4 and 66.5kDa were tested, including a biological toxin simulant/vaccine for Staphylococcal enterotoxin B (SEBv). Various substrates were tested for these representative proteins, including a laboratory bench, a notebook cover, steel, glass, plant leaf and vinyl flooring. Carbon sputtered porous polyethylene (CSPP) was found to outperform typical chromatography paper used for paper spray, as well as carbon nanotube (CNT)-coated paper and polyethylene (PE), which have been previously shown to be well-suited for protein analysis. Low microgram quantities of the protein toxin simulant and other test proteins were successfully detected with good signal-to-noise from surfaces using a porous wipe. These applications demonstrate that PS-MS can potentially be used for rapid, sample preparation-free detection of proteins and biological warfare agents, which would be beneficial to first responders and warfighters.

Field-deployable rapid multiple biosensing system for detection of chemical and biological warfare agents

The threat of biological and chemical terror acts remains a growing worldwide concern. There is therefore a need to develop appropriate technology for the detection of chemical and biological warfare agents, with early identification intended for use by first responders. Here, we disclose the developed autonomous air sampling and detection system for evaluation of the presence of chemical and biological warfare agents that can be harmful to the population. The current device utilizes the designed mist generator-assisted air collection system (338 l min−1) and biosensing chip technologies, such as electrochemical measurement, Au nanoparticle-based localized surface plasmon resonance, and rapid microfluidic chip PCR for detection of minute concentrations lower than the mean lethal dose (LD50) of nerve gases (sarin and VX), toxic proteins (BTX/A/Hc and ricin), and pathogens (anthrax simulant). An operation time of only 5–15 min is needed for the collection and detection; sample preparation is already integrated into the system without the need for direct human intervention. In addition to the system’s sensitivity and ease of use, its portability makes it highly beneficial for first responders, which could aid in immediate risk assessment and mitigation of on-site events.

Frequency-Based Analysis of Gramicidin A Nanopores Enabling Detection of Small Molecules with Picomolar Sensitivity.

Methods to detect low concentrations of small molecules are useful for a wide range of analytical problems including the development of clinical assays, the study of complex biological systems, and the detection of biological warfare agents. This paper describes a semisynthetic ion channel platform capable of detecting small molecule analytes with picomolar sensitivity. Our methodology exploits the transient nature of ion channels formed from gramicidin A (gA) nanopores and the frequency of observed single channel events as a function of concentration of free gA molecules that reversibly dimerize in a bilayer membrane. We initially use a protein (here, a monoclonal antibody) to sequester the ion channel activity of a C-terminally modified gA derivative. When a small molecule analyte is introduced to the electrical recording medium, it competitively binds to the protein and liberates the gA derivative, restoring its single ion channel activity. We found that monitoring the frequency of gA channel events makes it possible to detect picomolar concentrations of small molecule in solution. In part, due to the digital on/off nature of frequency-based analysis, this approach is 103 times more sensitive than measuring macroscopic membrane ion flux through gA channels as a basis for detection. This novel methodology, therefore, significantly improves the limit of detection of nanopore-based sensors for small molecule analytes, which has the potential for incorporation into miniaturized and low cost devices that could complement current established assays.

Biological Warfare Agents and their Detection and Monitoring Techniques (Review Paper)

Recently, threat from biological warfare agents (BWAs) has emerged as the foremost national and global security challenge because of their simple and cheap production, easy dispersal, complicated detection, expensive protection and psychological, economical and social impact. Early detection and identification of BWAs during intentional biological event is essential to initiate corrective emergency responses for management of such incidents. Efforts are being made across the globe for development of state of the art technologies and systems for detection and identification of BWAs. However, till date there is no single system which can detect all the biothreat agents. In the present review, we describe the currently available techniques and systems for detection and identification of these agents. The basic identification techniques including biological culture, immunological methods, nucleic acid based detection, MALDI-TOF MS, cellular fatty acid profiling and flow cytometry based detection are presented. Detection of BWAs with biosensors, surface plasmon resonance, biological detectors, and stand-off detection systems is also summarized. However, despite of availability of several techniques and tools, no full proof system is available for detection/identification of all the BWAs.

Mass spectrometry for the detection of bioterrorism agents: from environmental to clinical applications.

In the current context of international conflicts and localized terrorist actions, there is unfortunately a permanent threat of attacks with unconventional warfare agents. Among these, biological agents such as toxins, microorganisms, and viruses deserve particular attention owing to their ease of production and dissemination. Mass spectrometry (MS)-based techniques for the detection and quantification of biological agents have a decisive role to play for countermeasures in a scenario of biological attacks. The application of MS to every field of both organic and macromolecular species has in recent years been revolutionized by the development of soft ionization techniques (MALDI and ESI), and by the continuous development of MS technologies (high resolution, accurate mass HR/AM instruments, novel analyzers, hybrid configurations). New possibilities have emerged for exquisite specific and sensitive detection of biological warfare agents. MS-based strategies for clinical application can now address a wide range of analytical questions mainly including issues related to the complexity of biological samples and their available volume. Multiplexed toxin detection, discovery of new markers through omics approaches, and identification of untargeted microbiological or of novel molecular targets are examples of applications. In this paper, we will present these technological advances along with the novel perspectives offered by omics approaches to clinical detection and follow-up.

Decontamination by Persteril 36 may affect the reliability of DNA-based detection of biological warfare agents-short communication.

Persteril 36 is a disinfectant with a broad spectrum of antimicrobial activity. Because of its bactericidal, virucidal, fungicidal, and sporicidal effectiveness, it is used as a disinfectant against biological warfare agents in the emergency and army services. In case of an attack with potentially harmful biological agents, a person's gear or afflicted skin is sprayed with a diluted solution of Persteril 36 as a precaution. Subsequently, the remains of the biological agents are analyzed. However, the question remains concerning whether DNA can be successfully analyzed from Persteril 36-treated dead bacterial cells. Spore-forming Bacillus subtilis and Gram-negative Pseudomonas aeruginosa and Xanthomonas campestris were splattered on a camouflage suit and treated with 2 or 0.2% Persteril 36. After the disinfectant vaporized, the bacterial DNA was extracted and quantified by real-time PCR. A sufficient amount of DNA was recovered for downstream analysis only in the case of spore-forming B. subtilis treated with a 0.2% solution of Persteril 36. The bacterial DNA was almost completely destroyed in Gram-negative bacteria or after treatment with the more concentrated solution in B. subtilis. This phenomenon can lead to false-negative results during the identification of harmful microorganisms.

Ultrasensitive electrochemical immunoassay for surface array protein, a Bacillus anthracis biomarker using Au–Pd nanocrystals loaded on boron-nitride nanosheets as catalytic labels

Bacillus anthracis, the causative agent of anthrax, is a well known bioterrorism agent. The determination of surface array protein (Sap), a unique biomarker for B. anthracis can offer an opportunity for specific detection of B. anthracis in culture broth. In this study, we designed a new catalytic bionanolabel and fabricated a novel electrochemical immunosensor for ultrasensitive detection of B. anthracis Sap antigen. Bimetallic gold–palladium nanoparticles were in-situ grown on poly (diallyldimethylammonium chloride) functionalized boron nitride nanosheets (Au–Pd NPs@BNNSs) and conjugated with the mouse anti-B. anthracis Sap antibodies (Ab2); named Au–Pd NPs@BNNSs/Ab2. The resulting Au–Pd NPs@BNNSs/Ab2 bionanolabel demonstrated high catalytic activity towards reduction of 4-nitrophenol. The sensitivity of the electrochemical immunosensor along with redox cycling of 4-aminophenol to 4-quinoneimine was improved to a great extent. Under optimal conditions, the proposed immunosensor exhibited a wide working range from 5pg/mL to 100ng/mL with a minimum detection limit of 1pg/mL B. anthracis Sap antigen. The practical applicability of the immunosensor was demonstrated by specific detection of Sap secreted by the B. anthracis in culture broth just after 1h of growth. These labels open a new direction for the ultrasensitive detection of different biological warfare agents and their markers in different matrices.

Lateral Flow Assay - A New Platform for Diagnosis of Livestock Disease

The livestock sector of India contributes the largest livestock populations in the world and plays an important role in Indian economy. Many bacterial, viral, and fungal diseases impact a great loss to the production and productive potential of livestock’s of India, which currently, a major concern in both small and large ruminants. Proper differential diagnosis might play an elite role in prevention of these above mentioned diseases. Therefore the diagnosis should be very accurate, rapid and sensitive enough to confirm the occurrence of these diseases. Albeit various sophisticated diagnostic techniques like molecular, biochemical and microbiological are still existing however these techniques are mainly lab based method which requires highly sophisticated environment. Thus we should change our traditional diagnostic methods to a new concept for which it can be performed at pen side with low cost and high sensitivity. For this purpose, immuno-chromatographic based lateral flow assay may be the major concern intended to detect the presence (or absence) of a target analyte in sample without any need of sophisticated equipment. The lateral flow assay will provide a potential biological tool not only in diagnosis and control of emerging livestock diseases but also in detection of biological warfare agents. So this review article is a brief overview based on the salient aspects of this assay which could be extensively employed in the livestock sector.

MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis.

Currently microorganisms are best identified using 16S rRNA and 18S rRNA gene sequencing. However, in recent years matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a potential tool for microbial identification and diagnosis. During the MALDI-TOF MS process, microbes are identified using either intact cells or cell extracts. The process is rapid, sensitive, and economical in terms of both labor and costs involved. The technology has been readily imbibed by microbiologists who have reported usage of MALDI-TOF MS for a number of purposes like, microbial identification and strain typing, epidemiological studies, detection of biological warfare agents, detection of water- and food-borne pathogens, detection of antibiotic resistance and detection of blood and urinary tract pathogens etc. The limitation of the technology is that identification of new isolates is possible only if the spectral database contains peptide mass fingerprints of the type strains of specific genera/species/subspecies/strains. This review provides an overview of the status and recent applications of mass spectrometry for microbial identification. It also explores the usefulness of this exciting new technology for diagnosis of diseases caused by bacteria, viruses, and fungi.

A novel nanoprobe for the sensitive detection of Francisella tularensis

Francisella tularensis is a human zoonotic pathogen and the causative agent of tularemia, a severe infectious disease. Given the extreme infectivity of F. tularensis and its potential to be used as a biological warfare agent, a fast and sensitive detection method is highly desirable. Herein, we construct a novel detection platform composed of two units: (1) Magnetic beads conjugated with multiple capturing antibodies against F. tularensis for its simple and rapid separation and (2) Genetically-engineered apoferritin protein constructs conjugated with multiple quantum dots and a detection antibody against F. tularensis for the amplification of signal. We demonstrate a 10-fold increase in the sensitivity relative to traditional lateral flow devices that utilize enzyme-based detection methods. We ultimately envision the use of our novel nanoprobe detection platform in future applications that require the highly-sensitive on-site detection of high-risk pathogens.

Development and Applications of Portable Biosensors.

The significance of microfluidics-based and microelectromechanical systems-based biosensors has been widely acknowledged, and many reviews have explored their potential applications in clinical diagnostics, personalized medicine, global health, drug discovery, food safety, and forensics. Because health care costs are increasing, there is an increasing need to remotely monitor the health condition of patients by point-of-care-testing. The demand for biosensors for detection of biological warfare agents has increased, and research is focused on ways of producing small portable devices that would allow fast, accurate, and on-site detection. In the past decade, the demand for rapid and accurate on-site detection of plant disease diagnosis has increased due to emerging pathogens with resistance to pesticides, increased human mobility, and regulations limiting the application of toxic chemicals to prevent spread of diseases. The portability of biosensors for on-site diagnosis is limited due to various issues, including sample preparation techniques, fluid-handling techniques, the limited lifetime of biological reagents, device packaging, integrating electronics for data collection/analysis, and the requirement of external accessories and power. Many microfluidic, electronic, and biological design strategies, such as handling liquids in biosensors without pumps/valves, the application of droplet-based microfluidics, paper-based microfluidic devices, and wireless networking capabilities for data transmission, are being explored.

Point of Care Test

India’s livestock sector is one of the largest in the world and plays an important role in economy of India. The major problem in health sector is diseases and the lack of rapid detection systems for their diagnosis. Treatment of a disease condition is based on the diagnosis of the etiology /cause; hence diagnosis plays a pivotal role in devising a control strategy. Syndromic management as a first line of therapeutic practice have shown to be effective in various approaches but may inevitably results in overtreatment, resulting in wasted resources and, potentially, increased antimicrobial resistance. So the next generation of therapeutic management depends largely on evolving rapid point of care techniques for targeted management of diseases. The point of care test will prove to be a potential tool not only in diagnosis and control of emerging and dreaded diseases of human and animal significance but also detection of biological warfare agents. The current era in the field of point of care has come way far beyond detection of non infectious conditions like blood glucose monitoring, pregnancy diagnosis etc and has plunged into a sea of avenues. So this review is a brief overview on salient aspects on the transformation taken in this field for benefit of both the sectors.

Rapid detection of Bacillus spore aerosol particles by direct in situ analysis using MALDI-TOF mass spectrometry

Accurate and rapid analytical methods are essential for the detection and identification of biological warfare agents (BWA). Although various studies have investigated the use of MALDI-TOF MS for bacterial classification, only a few studies have examined the applicability of method for the identification of BWAs. This study aimed to generate, collect and analyse Bacillus spore aerosol particles of 2-10μm, the optimal size of a BWA. Bacillus spore aerosol particles of 2-10μm were rapidly analysed using direct in situ MALDI-TOF MS without pretreatment processes. A mass spectrum of Bacillus spore aerosol particles was successfully detected. For real-time detection and identification, a mass spectral database of Bacillus spores was constructed, and an algorithm was developed and applied. Bacillus spore particles were rapidly detected and identified by MS, which can be used for the detection and inspection of BWAs. Methods that permit accurate and fast detection and identification are essential in the case of pathogens such as Bacillus anthracis, which can be used as biological warfare agents or bioterrorism materials. Here, direct in situ MALDI-TOF MS allowed the high-throughput detection and identification of aerosolized Bacillus spore particles, and this method could be effectively applied for the detection and inspection of BWAs. In this context, we believe that this method may contribute to the realization of real-time and on-site identification of BWAs.

Intact cell/intact spore mass spectrometry (IC/ISMS) on polymer-based, nano-coated disposable targets

Identification and differentiation of microorganisms has and still is a long arduous task, involving culturing of the organism in question on different growth media. This procedure, which is still commonly applied, is an established method, but takes a lot of time, up to several days or even longer. It has thus been a great achievement when other analytical tools like matrix-assisted laser desorption/ionization (MALDI) mass spectrometry were introduced for faster analysis based on the surface protein pattern. Differentiation and identification of human pathogens as well as plant/animal pathogens is of increasing importance in medical care (e.g. infection, sepsis, and antibiotics resistance), biotechnology, food sciences and detection of biological warfare agents. A distinction between microorganisms on the species and strain level was made by comparing peptide/protein profiles to patterns already stored in databases. These profiles and patterns were obtained from the surface of vegetative forms of microorganisms or even their spores by MALDI MS. Thus, an unknown sample can be compared against a database of known pathogens or microorganisms of interest.To benefit from newly available, metal-based disposable microscope-slide format MALDI targets that promise a clean and even surface at a fraction of the cost from full metal targets or MTP (microtiter plate) format targets, IC/ISMS analysis was performed on these and the data evaluated. Various types of bacteria as well as fungal spores were identified unambiguously on this disposable new type of metal nano-coated targets. The method even allowed differentiation between strains of the same species. The results were compared with those gained from using full metal standard targets and found to be equal or even better in several aspects, making the use of disposable MALDI targets a viable option for use in IC/ISMS, especially e.g. for large sample throughput and highly pathogenic species.

Development of biosensors for the detection of biological warfare agents: its issues and challenges.

This review discusses current development in biosensors for the detection of biological warfare agents (BWAs). BWAs include bacteria, virus and toxins that are added deliberately into air water and food to spread terrorism and cause disease or death. The rapid and unambiguous detection and identification of BWAs with early warning signals for detecting possible biological attack is a major challenge for government agencies particularly military and health. The detection devices--biosensors--can be classified (according to their physicochemical transducers) into four types: electrochemical, nucleic acid, optical and piezoelectric. Advantages and limitations of biosensors are discussed in this review followed by an assessment of the current state of development of different types of biosensors. The research and development in biosensors for biological warfare agent detection is of great interest for the public as well as for governments.

Effective Collection and Detection of Airborne Species Using SERS‐Based Detection and Localized Electrodynamic Precipitation

Three different delivery concepts (standard diffusion, global electrodynamic precipitation, and localized nanolens-based precipitation) and three different SERS enhancement layers (a silver film, a nanolens-based localized silver nanoparticle film, and the standard AgFON) are compared. The nanolens concept is applied to increase the SERS signal: a factor of 633, when compared to a standard mechanism of diffusion, is observed.