key: cord-0034409-ntuptc1j authors: Pérez-López, Briza; Merkoçi, Arben title: Nanomaterials-Based (Bio)Sensing Systems for Safety and Security Applications date: 2012-01-05 journal: Portable Chemical Sensors DOI: 10.1007/978-94-007-2872-1_3 sha: 61c1468397b7de9962956e6fe69b08925a6a880c doc_id: 34409 cord_uid: ntuptc1j The development of new nanomaterials and nanotechnologies has ­provided many new opportunities for (bio)sensing systems. The introduction of nanomaterials, such as magnetic nanoparticles, gold nanoparticles, graphene, quantum dots, etc. is bringing advantages in terms of improving the selectivity and sensitivity of these systems. These nanomaterials also offer advantages in biosensors owing to their nanometric size, shape, composition, physical properties, ability to manipulate their surface chemistry and the property that they have in terms of adsorbing biological molecules and the change of their physical properties. In recent years, several ­bacterial pathogens, toxins, viruses, parasites and explosives have been considered as potential threats for bioterrorism, among which can find Escherichia coli, Salmonella, Bacillus anthracis, Clostridium botulinum, Botulinum Neurotoxin, Vaccinia, Plasmodium falciparum, Trinitrotoluene, etc. Bioterrorism is extremely complex to tackle but the science and technology are fundamental ­elements to reduce its threat. For this reason, monitoring systems for quick identification of biomolecules are the core of much of the basic research activities in combating bioterrorism. In this chapter we discuss the research efforts by using nanobiotechnologies with the aim of developing accurate, easy, cheap, portable and ultrasensitive assays for agents that pose a biologic threat. Some ­nanomaterial-based (bio)sensing systems used to detect agents related with bioterrorism for safety and security applications in agriculture, food, forensic, biomedical are also given. agents that pose a biologic threat. Some nanomaterial-based (bio)sensing systems used to detect agents related with bioterrorism for safety and security applications in agriculture, food, forensic, biomedical are also given. The discovery and study of nanomaterials has enabled the development of ultrasensitive (bio)sensing systems. This is due to their high surface area, favorable electronic and optical properties and electrocatalytic activity as well as good biocompatibility induced by nanometer size and specifi c physicochemical characteristics [1] [2] [3] [4] [5] . In recent years, the advent of nanomaterial-based (bio)sensing systems for safety and security applications is offering key researches and developments. In this context, of special interest are the 'nanosized' and nanomaterials based biosensors, called also nanobiosensors -a modern and effi cient class of detection systems [ 1, 3, [6] [7] [8] [9] [10] . The application of these devices in food industry, environmental monitoring and clinic diagnostics could lead great improvements in safety and security against bioterror agents. Nowadays, laboratories and institutions related with the nano-biotechnology are working together to increase the capabilities to detect and respond to an attack by biological or chemical weapons [ 11 ] . Examples of nanomaterial-based bioterrosist agents for safety and security applications are given in the Table 3 .1 . The magnetic nanoparticles (M NPs), graphene (G), quantum dots (QDs), and more extensively gold nanoparticles (Au NPs) are being invaluable nanomaterials to detect bioterrorist analytes in macro-to nano-scale, including bacterial pathogens, toxins, viruses, parasites, explosives, etc. Biological agents such as bacteria, viruses, biological toxins, and genetically altered organisms are contagious, and during this lag time, infected persons could continue to spread the disease, further increasing its reach. Hundreds or even thousands of people could become sick or die if a biological attack were to occur in a major metropolitan area [ 11 ] . For this reason, problems related to the risks of human health and environmental need to be carefully considered, mainly assays that involve a variety of safety and security applications. In this chapter will be given some important strategies used in (bio)sensing systems with different nanomaterials to detect bioterrorist agents. In addition, this chapter will give opinions about the importance that these systems have for safety and security applications in food, environmental and other related fi elds. Numerous laboratories and public and private research institutions during decades have been designing and developing technologies to safeguard from biological attacks provoked by terrorists. Nowadays, they are developing monitoring technologies for bioterrorism agents with fast analysis, low limit of detection, and high accuracy to prevent false negatives and positives and maintain confi dence in the monitoring system [ 30 ] . Among the highest-risk threat agents in bioterrorism can be considered: (1) Bacteria, they are prokaryotes, and their distribution is ubiquitous (humans, animals, and environment). They are the oldest living organisms in the history of this planet and play an important role in maintaining the ecosystem [ 11 ] . Escherichia coli ( E. coli ) is a family of naturally occurring bacteria. Some of these cause sickness and even death. Most E. coli infections come from eating undercooked (i.e. contaminated ground beef). Terrorists could use E. coli 0157:H7 bacteria as a weapon to strike many people at one time, also the Bacillus anthracis ( B. anthracis ) is considered a high pathogenic agent that could deliberate contamination events causing anthrax [ 31 ] ; (2) Neurotoxins produced by Clostridium botulinum are among the most known poisonous substances [ 32 ] . For example, botulinum toxin is the most toxic substance known and is extremely poisonous by the oral route (estimated lethal oral dose, 10-70 m g for a 70 kg human) and potentially toxic by inhalation [ 33 ] , for this reason it is used as bioterrorism agent (3) Infl uenza A (H1N1) virus also could be considered a bioterrorist agent, it belongs to the Orthomyxoviridae family and corresponds to the specifi c combination of glycol-protein hemagglutinin (HA) and neuraminidase (NA) variants, which are present on the surface of the enveloped RNA virus [ 24 ] . (4) Malaria is the most prevalent parasitic disease in the worldcaused by the apicomplex protozoan of the Plasmodium genus . Malaria is present over the tropics, where four species, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale are transmitted to humans by the bites of the female mosquito vector of the Anopheles genus [ 34 ] and although the use of parasites as bioterrorism agents has not received so much attention. Parasites could contribute to the installation of fear in human population upon intentional addition to their food and water supplies, which makes malaria suitable for being used by terrorists (5) . In recent years, the detection and quantifi cation of nitroaromatic explosives such as 2,4,6-trinitrotoluene (TNT) have also received considerable attention due to their environmental, security against terrorists and health related concerns [ 35, 36 ] . Various methods have been developed for the detection of these bioterrorist agents and many more still are in development phase. Most of the assays are based on detecting the (a) whole organism, (b) bacterial antigens, and/or (c) the related nucleic acid. In the Table 3.2 are described some detection methods more commonly used for bioterrorist agents with safety and security applications. Conventional methods such as the culture and colony counting methods that involve counting of bacteria, immunology-based methods that use antigen-antibody interactions and the polymerase chain reaction (PCR) method which involves DNA analysis are being used. These methods can be sensitive, inexpensive and give qualitative and quantitative information, however, a pre-treatment of the samples is needed; furthermore they are greatly restricted by assay time [ 37 ] . The electrochemical detection methods possess several advantages such as easy operation, low cost, high sensitivity, simple instrument and suitability for portable devices. However, to improve their performance one of the most popular ways is to use nanomaterials with a high surface area to functionalize the electrode [ 4, 5, 10 ] . In this context, nanomaterial-based methods have demonstrated improvement of the sensitivity, but due to reproducibility problems, as well as interferences, their application in real samples is still limited. The prospect of nanomaterials is promising for rapid and sensitive pathogen detection [ 12, [14] [15] [16] . Current literature shows numerous applications of different nanostructures in biosensor devices for the detection of pathogenic microorganisms that are of importance to food and environmental safety, biosecurity, and medical diagnostics. In this section, some aspects related to the detection of Salmonella , E. Coli, B. anthracis and Clostridium botulinum by using metal nanoparticles such as Au NPs, Cu@Au NPs, M NPs and QDs will be described. Nanotechnology gives new approaches in order to detect microorganisms through the use of nanomaterials. This fi eld has been explored by Lin et al. [ 12 ] using screen-printed carbon electrodes (SPCEs) modifi ed with Au NPs (13-nm diameter) and ferrocenedicarboxylic acid (FeDC). The detection method consists of a sensitive detection of horseradish peroxidise (HRP) activity coupled with Au NPs and FeDC to amplify the amperometric effect. This has the potential for further applications in the rapid pathogen detection. One important advantage of these amperometric immunosensing strips is that approximately 50 CFU of E. coli O157:H7 in milk samples can be detected in 1 h. In this same context, Zhang and co-workers [ 13 ] described a sensitive electrochemical immunoassay for rapid detection of E. coli by ASV based on core-shell Cu@Au nanoparticles (NPs) as anti-E. coli antibody labels. M NPs due to their high surface/volume ratio offer more contact surface area for attaching carbohydrates and for capturing pathogens. Based on this, E. coli detection using epifl uorescent microscopy has been performed by functionalizing the surface of M NPs with D-manosse sugar (man-M NPs) through an amide linkage [ 14 ] , subsequently incubations with fl uorescein-labeled concanavalin A (Con A) and E. coli cells in phosphate buffer solution (PBS). After that, a magnetic fi eld was applied for separating M NPs/ E. coli aggregates (see Fig. 3 .1a ). The supernatants were removed and the remaining aggregates were washed thoroughly, stained with a fl uorescent dye (PicoGreen), transferred to a glass slide, and imaged. Fluorescent microscopic imaging showed that E. coli can be detected (see Fig. 3 .1b ) with a limit of detection 10 4 cells/mL by obtaining a high capture of bacteria. Metal nanoshells based nanomaterials that exhibit a surface plasmon resonance (SPR) are also used for E. coli detection. Metal nanoshells are thin coatings (a few tens of nanometers thick) on large particles (a few hundreds of nanometers in diameter) which form the cores. Researchers have demonstrated a rapid and reliable test for the detection of E. coli , by using the SPR band associated with the coupling of the antibodies to the silver nanoshells [ 15 ] . This detection method has shown that the E. coli antibody interaction is extremely specifi c and that the presence of other microorganisms could not produce changes in the SPR band. Furthermore, it could help to shorten the testing time of drinking water used as sample with interest in possible terrorist attacks beside other applications related to the human health. Electrochemical immunoassays based on Au NPs have also attracted considerable interest for Salmonella determination due to its simplicity, high sensitivity, inexpensive instrumentation, and miniaturization. For example, a highly sensitive strategy based on Au NPs for detecting Salmonella typhi (S. typhi) has been studied by Dungchaia et al. [ 17 ] . They immobilized monoclonal antibodies (McAbs) on polystyrene microwells and captured S. typhi bacteria by using a copper-enhanced Au NPs label coupled with anodic stripping voltammetry (ASV). The amount of [ 14 ] with permission) deposited copper was related to the amount of Au NP tag present, which was controlled by the amount of S. typhi attached to the polyclonal antibody-colloidal gold conjugate. Therefore, the anodic stripping peak current was linearly dependent on the S. typhi concentration with a detection limit as low as 98.9 CFU/mL with interest for real samples analysis with a low detection limit, high accuracy, and fast analysis time. Au NPs modifi ed with Salmonella spp McAbs can also be evaluated using the electrochemical impedance spectroscopy (EIS) technique as an effi cient method for fabricating a capacitive immunosensor for the detection of Salmonella spp. in real samples [ 16 ] . B. anthracis is another of the biggest threats special because of its potential use in bioterrorism. B. anthracis spores can be transmitted easily to humans. These spores are highly resistant to normally destructive environmental factors to living cells, such as heat, toxic chemicals, desiccation, and physical damages. These properties make them suitable for a potential biological warfare [ 49 ] . For this reason, the rapid and accurate detection of B. anthracis spores in the environment prior to infection is very important for human safety and national security. However, few technologies have been widely evaluated under fi eld conditions [ 46 ] . Accordingly, nanomaterial-based biosensors are evolving as promising alternatives to meet this challenge in terms of sensitivity, specifi city, time-and costeffi ciency [ 30 ] . For this, label-free (bio)sensing systems based on nanomaterials present certain advantages in detection. Among label-free assays, the quartz crystal microbalance (QCM) piezoelectric sensor has proved to be a useful platform in the effi cient detection of pathogens including B. anthracis . Based on this platform, electrically active magnetic (EAM) nanoparticles are being used as concentrator of DNA targets as well as electrochemical transducers for detection of the B. anthracis protective antigen A (pag A) gene [ 18 ] . More details can be seen in the Fig. 3.2 . Another biosensor based on QCM has been developed by R. Hao and coworkers for B. anthracis spores detection by an anti-B. anthracis monoclonal antibody designated to 8 G3 (mAb 8 G3, IgG) functionalized QCM sensor [ 50 ] . Neurotoxins produced by Clostridium botulinum , serotypes A through G (BoNT A-G), are considered the most potent toxins known to humans who cause neuroparalytic disease [ 51 ] . The "Class A agents" are listed as one of the six highest risk threat agents for bioterrorism [ 22 ] . Food-borne botulism is the most common intoxication form due to the ingestion of pre-formed Botulimun Neurotoxin (BoNT) in food. For this reason, the development of nanomaterial-based rapid methods that can help to detect terrorist agents, such as anthrax, BoNT, etc. is very important for safety and security purposes. Colloidal gold has emerged as the preferred label [ 52 ] for toxin detection. Alternative rapid methods, as colloidal gold-based immunochromatographic assays (ICA) (also called lateral fl ow (LF) or strip assay) have been developed for detection of botulinum neurotoxin type B (BoNT/B). This class of assays was based on the sandwich format using polyclonal antibody (Pab). A thiophilic gel purifi ed anti-BoNT/B Pab was immobilized in a defi ned detection zone on a porous nitrocellulose membrane and conjugated to colloidal gold particles as a detection reagent. The BoNT/B-containing sample was added to the membrane to react with Pab-coated particles, thus, a change of colour was given in the detection zone with an intensity of red colour proportional to BoNT/B concentration. The strip assay exhibits potential as a rapid and simple assay method for the detection of toxin in biological fl uid that requires no separation steps. Moreover, in function of these characteristics the assay can be considered superior to other immunoassay, such as radioimmunoassay and enzyme-linked immunosorbent assay [ 20 ] . Another test based on ICA has been reported by Klewitz et al. [ 21 ] to detect botulism neurotoxin D (BoNT/D). The test was based on double sandwich format using a gold-anti BoNT/D monoclonal antibody conjugates (detector reagent) and an anti-BoNT/D chicken polyclonal antibody. Feacal samples or standard samples spiked with various concentrations of BoNT/D were treated in gelatine-phosphate buffer, vortexed and stored at temperature ranging 5-8°C overnight. Polyclonal chicken anti-BoNT/D IgG and gold conjugated monoclonal antibody were added to the sample extracts and incubated at 37°C for 3 h. Then, 50 m L of pre-treated samples were applied to the test strip. The intensity of colour, of the red test line (signal intensity), is directly related with the concentration of BoNT/D in the standard or spiked horse faecal samples. Thus, toxin concentrations were determined within 3.5 h down to 50 pg/mL. In the last years, several researchers have used luminescence methods instead of colorimetric methods for (bio)sensing applications. A novel class of luminescence nanomaterial is emerging for botulinum toxin assays [ 22 ] . This is the case of colloidal semiconductor QDs, which are single crystals of few nanometers of diameter whose size and shape can be precisely controlled by the duration, temperature, and ligand molecules used in their synthesis [ 53 ] . These nanomaterials have unique optical properties such as high extinction coeffi cients over a wide wavelength range, size dependent optical emission (due to quantum confi nement effects on the electronic structure of the QDs) and relatively high quantum yields, which makes them suitable for biological applications such as fl uorescence immunoassays, DNA array technology, fl uorescence labeling of cells and tissues, and in the detection of chemical and biological agents. In Fig. 3 .3 is shown an example of a fl uorescence sandwich immunoassay using high-affi nity antibodies and quantum dot (QD) reporters for detection of BoNT serotype A (BoNT/A) using a nontoxic recombinant fragment of the holotoxin (BoNT/A-H C -fragment) as a structurally valid simulant for the full toxin molecule [ 22 ] . Recently, a detection method based on the use of semiconductor QD-peptide Förster resonance energy transfer (FRET) assemblies to monitor the activity of the BoNT serotype A light chain protease (LcA) has been reported by Sapsford et al. [ 48 ] .They evaluated the ability to self-assemble dye-labeled LcA peptide substrates by using a number of different QD materials displaying charged or PEGylated surface-coatings by monitoring FRET interactions. Furthermore, Grate and co-authors [ 47 ] have developed for botulinum toxin detection an electrochemiluminescence (ECL) method based on a sandwich complex with paramagnetic beads and capture of antibodies bound to the beads by a streptavidin-biotin linkage, and the detection of antibodies labeled with a ruthenium chelate. Historically, nanoparticles have been used as label biomolecules. Their properties coupled with the ability to attach nearly any biologic recognition element to the particle surfaces, facilitates their application in multi-target assays. These nanomaterials can be conjugated to DNA sequences or proteins so that, using size-dependent properties as just one example, fl uorescence or light scattering can be used as an output signal, respectively. For example, a method for analyzing combinatorial DNA arrays using oligonucleotide-modifi ed gold nanoparticle probes and a conventional fl atbed scanner has been studied by the Mirkin's group [ 54 ] . Labeling oligonucleotide targets (based on the anthrax lethal factor sequence) with nanoparticles rather than fl uorophore probes substantially alters the melting profi les of the targets from an array substrate. This difference permits the discrimination of an oligonucleotide sequence from targets with single nucleotide mismatches with a selectivity that is over three times that observed for fl uorophore-labeled targets. In addition, when coupled with a signal amplifi cation method in which silver ions are reduced by hydroquinone to silver metal at the surfaces of the Au NPs, the sensitivity of this scanometric array detection system exceeds that of the analogous fl uorophore system by two orders of magnitude. Labeling technology based on highly fl uorescent europium (Eu + ) NPs could provide a rapid and sensitive testing platform for sensing bioterrorist agents. Lately a europium-nanoparticle based immunoassay (ENIA) for the sensitive detection of anthrax protective antigen has been reported [ 19 ] . The use of Eu + NPs further permits to the assay to be adapted to an ELISA format that is already in place in testing laboratories because the antibody-antigen sandwich complex bound to Eu + NPs coated with streptavidin (SA) can be directly measured with a fl uorescence reader. This ultrasensitive NP-based assay for the detection of anthrax toxin could provide a useful new tool for infectious agents and chemical contaminants. The detection of infectious viral diseases is very important for the public health. In recent years, a number of viral outbreaks, such as severe acute respiratory syndrome (SARS), infl uenza A (H1N1 fl u) and avian infl uenza A (H5N1 fl u) are emerging. These have raised signifi cant fears due to that could rapidly spread and turn into a pandemic similar to 1918 Spanish fl u that killed more than 50 million people [ 55 ] . For this, rapid and sensitive diagnostic techniques using different nanomaterials are being developed for recognizing and controlling future epidemics. An example of this is the recent development of immunochromatographic strip for rapid detection of H9 subtype avian infl uenza viruses (H9 AIVs) [ 23 ] . The assay is based on a colloidal gold anti-hemagglutin monoclonal antibody conjugate (detection antibody) and an anti-Nucleocapside protein monoclonal antibody used as a precipitation reagent on the test line of a nitrocellulose membrane. This detection method is rapid and easy to operate without the requirement of special skills and equipment, which makes it a strip suitable for fi eld detection. In addition, this generation of ICA allows doing multiple assays that can help to diagnostic of common poultry diseases, such as Newcastle disease, avian infectious bronchitis, avian infectious laryngotracheitis, etc. Another similar study has been reported by the same authors to detect IgG antibodies against the nucleocapsid protein of AIV subtypes (H5, H7 and H9) in chicken sera [ 56 ] . The use of immunosensors based on the identifi cation of virus glycoproteins, [ 57, 58 ] or genosensors based on the detection of specifi c DNA sequences correlated to the virus RNA [ 59, 60 ] , has been performed as an alternative to more expensive classical methods that consume time. In Mexico and USA, in March-April 2009, there was an outbreak of human H1N1 fl u virus that created a pandemic concern [ 61 ] . At the time more than 207 countries worldwide reported cases of pandemic H1N1, including at least 8,768 deaths [ 62 ] . For this reason developing of accurate, rapid and low cost sensing methods for the early detection of this kind of virus is of great importance. An impedimetric detection method of a DNA sequence correlated to H1N1 virus using carbon nanotubes platform and Au NPs amplifi cation could be considered a good possibility for improving the sensitivity and rapidity of analysis [ 24 ] . Such systems are based on the use of colloidal gold for the labelling of DNA oligonucleotides, and the electrochemical signal of Au NPs onto screen printed carbon nanotubes electrode is measured and correlated to the DNA target concentration. The use of parasites as bioterrorism agents has not received so much attention. Parasites could contribute to the installation of fear in human population upon intentional addition to their food and water supplies. In the last years, biosecurity issues are gaining importance as a consequence of globalization. Surveillance is critical in maintaining biosecurity and early detection of infectious disease agents is essential [ 63 ] . Infectious diseases, like malaria, are being one of the greatest health challenges worldwide. Nanotechnology is one of the promising strategies for malaria treatment. The identifi cation of new Plasmodium or infected cell targets can be used to modify existing drug delivery systems employing nanotechnology to more effi ciently deliver antimalarial drug molecules to the newly-targeted sites of action. Electrochemical immunosensors offer several advantages compared to alternative detection methods, including the ability to analyze the direct blood samples, high sensitivity, require low sample volume and can be used in fi eld application [ 64 ] . For example, nanosized carriers are receiving special attention with the goal of minimizing the side effects of drug therapy, such as poor bioavailability and the selectivity of drugs. Several nanosized delivery systems have already proved their effectiveness in animal models for the treatment and prophylaxis of malaria [ 34 ] . A number of strategies for the detection of Plasmodium falciparum are being developed by using rapid diagnostic tests based on nanomaterials. Magneto immunoassay-based strategies for the detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) related to malaria have been described by using M NPs [ 25 ] . The immunological reaction for the protein HRP2 was performed in a sandwich assay on M NPs by using a second monoclonal antibody labeled with horseradish peroxidase (HRP) enzyme. The modifi ed M NPs were captured on the graphite-epoxy composite electrode surface using a magnet inside of the composite electrode which was used as transducer in the electrochemical detection. This magneto immunoassay based on magnetic nanoparticles has shown a limit of detection (LOD) of 0.36 ng mL −1 , which makes it a suitable method for Plasmodium falciparum histidine-rich Protein 2 detection related to malaria. Recently, electrochemical immunosensors based on screen printed electrodes are (SPEs) attracting great interest [ 65 ] due to ease of fabrication, ability to mass produce, disposability and portability. Sharma et al. [ 26 ] have developed an amperometric immunosensor based on Au NPs/alumina sol-gel modifi ed SPEs for antibodies to Plasmodium falciparum histidine rich protein-2 ( Pf HRP-2) by dripping Al 2 O 3 sol-gel on SPE followed by electrochemical deposition of Au NPs. The antibodies in rabbit serum sample were allowed to react with the Pf HRP-2 protein which was immobilized on the modifi ed SPE to form antigen-antibody immune complex ( Pf HRP-2/anti-Pf HRP-2). The bound antibodies were quantifi ed by alkaline phosphatase (AP) enzyme labeled secondary antibodies (anti-rabbit immunoglobulins-AP conjugate). Enzymatic substrate, 1-naphthyl phosphate was converted to 1-naphthol by AP and an electroactive product was quantifi ed using amperometric technique. Figure 3 .4 shows some results of this electrochemical detection. Although the electrochemical immunosensors together with the nanotechnology are providing certain advantages, in terms of improving the selectivity and sensitivity of the detection systems in the fi eld applied to parasites detection, it is a domain that is still under development. The conventional methods to detect explosives are restricted by disadvantages such as expensive instrument usage and time-consuming processes. Therefore, it is important to develop methods for trinitrotoluene (TNT) assay with simplicity, sensitivity, rapidity and cost-effi ciency. Lately, methods for TNT assay have been developed by using Au NPs and QDs. For example, Jiang and co-workers [ 66 ] reported a simple and sensitive method for colorimetric visualization of TNT at picomolar levels based on color changes of Au NPs in the presence of TNT via the electron-donor-acceptor interaction between TNT and primary amines. New methods have been tested for the determination of TNT based on fl uorescence quenching of QDs. Chen et al. [ 27 ] have reported a sensitive method with high selectivity for TNT detection by using water-soluble L-cysteine-capped CdTe QDs as fl uorescence probe. L-cysteine is used as stabilizer of QDs and as primary amine provider. Intermediate complexes between TNT and cysteine are formed, resulting in the fl uorescence quenching of the QDs. This method can be adapted for the detection of TNT. The LOD was 1.1 nM and specifi city detection was achieved. More details are observed in the Fig. 3 .5 . Other methods based on fluorescence detection have been reported by Wang and co-authors where the fluorescence of oleic-acid-covered CdSe QDs Recently, graphene owing to its unique properties, such as remarkable electronic conductivity, incredibly large electroactive surface area, high affi nity and electrocatalytic activity [ 69 ] is considered another of the interesting nanomaterials to be used for nitroaromatic explosives detection. Guo et al. [ 28 ] have evaluated a new ionic liquid (IL)-graphene composite prepared by combining IL and graphene with large specifi c surface area and pronounced mesoporosity for ultratrace explosive trinitrotoluene detection. On the other hand, the determination of 2,4-Dinitrotoluene (2,4-DNT) by means of electrochemically reduced graphene on glassy carbon electrode (GCE) has been analyzed by using electrochemical detection, which gave a low detection limit of 42 nmol L −1 (S/N = 3) [ 29 ] . Nanotechnology could provide unlimited opportunities for improving the effi cacy of (bio)sensing systems for bioterrorist agents. In this chapter are presented some of the detection methods more commonly used for bioterrorist agents as well as for other safety and security applications. However, the domain that nanotechnology has in the detection of bacteria, toxins, parasites, viruses and explosives is still in development phase. To overcome the challenges of nanomaterial-based (bio)sensing strategies for safety and security applications a more detailed study related to interferences for real sample analysis as well as technological aspects related to fi nal application need to be addressed. Point strategies to overcome the challenges should be (a) In-fi eld applications . In-fi eld applications of nano-biosensing still need a big effort so as to overcome problems related to applications in real samples. Avoidance of interferences coming from sample matrix is the key point for success; (b) Low detection limits. Reaching of low detection limits (detection of few molecules, bacteria, cells, etc.) in a relatively high volume of samples (i.e. 1 molecule or 1 cell in 1 mL food sample) needs the development of fast and effi cient preconcentration tools/routes based on nano & microfabrication. Electrochemical analysis with nanoparticle-based biosystems Merkoçi A (ed) Biosensing using nanomaterials -Bionano Nanomaterials for electroanalysis Nanoparticles for the development of improved (bio)sensing systems Nanomaterials based biosensors for food analysis applications Development of an immunoassay-based lateral fl ow dipstick for the rapid detection of afl atoxin B1 in pig feed Nanoparticle-based biosensors for detection of pathogenic bacteria Improvement of the electrochemical detection of catechol by the use of a carbon nanotube based biosensor Carbon nanofi ber vs. carbon microparticles as modifi ers of glassy carbon and gold electrodes applied in electrochemical sensing of NADH Magnetic nanoparticles modifi ed with carbon nanotubes for electrocatalytic magnetoswitchable biosensing applications Principle of bacterial detection: biosensors, recognition receptors and microsystems Disposable amperometric immunosensing strips fabricated by Au nanoparticles-modifi ed screen-printed carbon electrodes for the detection of foodborne pathogen Escherichia coli O157:H7 Development of an electrochemical immunoassay for rapid detection of E. coli using anodic stripping voltammetry based on Cu@Au nanoparticles as antibody labels Magnetic glyco-nanoparticles: a unique tool for rapid pathogen detection, decontamination, and strain differentiation Rapid detection of Escherichia coli by using antibody-conjugated silver nanoshells A reusable capacitive immunosensor for detection of Salmonella spp. based on grafted ethylene diamine and self-assembled gold nanoparticle monolayers Salmonella typhi determination using voltammetric amplifi cation of nanoparticles: a highly sensitive strategy for metalloimmunoassay based on a copper-enhanced gold label Electrically active magnetic nanoparticles as novel concentrator and electrochemical redox transducer in Bacillus anthracis DNA detection Detection of anthrax toxin by an ultrasensitive immunoassay using europium nanoparticles Colloidal gold-based immunochromatographic assay for detection of botulinum neurotoxin type B Immunochromatographic assay for determination of botulinum neurotoxin type D Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fl uorescence detection of botulinum neurotoxin using high-affi nity antibodies Development of an immunochromatographic strip for rapid detection of H9 subtype avian infl uenza viruses Impedimetric detection of infl uenza A (H1N1) DNA sequence using carbon nanotubes platform and gold nanoparticles amplifi cation Magneto immunoassays for Plasmodium falciparum histidine-rich protein 2 related to malaria based on magnetic nanoparticles Amperometric immunosensor based on gold nanoparticles/alumina sol-gel modifi ed screen-printed electrodes for antibodies to Plasmodium falciparum histidine rich protein-2 L-cysteine-capped CdTe QD-based sensor for simple and selective detection of trinitrotoluene Ionic liquid-graphene composite for ultratrace explosive trinitrotoluene detection Determination of explosives using electrochemically reduced graphene Current and developing technologies for monitoring agents of bioterrorism and biowarfare Bacillus anthracis , Francisella tularensis and Yersinia pestis . The most important bacterial warfare agents -review The use of phages and aptamers as alternatives to antibodies in medical and food diagnostics Clostridium botulinum neurotoxins -applications in medicine and potential agents of bioterrorism Nanotechnology applied to the treatment of malaria Integrated explosive preconcentrator and electrochemical detection system for 2,4,6-trinitrotoluene (TNT) vapor Ultrasensitive detection of TNT in soil, water, using enhanced electrogenerated chemiluminescence An overview of foodborne pathogen detection: in the perspective of biosensors Rapid assay for microbially reducible ferric iron in aquatic sediments Patterned protein microarrays for bacterial detection Immunobiosensor chips for detection of Escherichia coli O157:H7 using electrochemical impedance spectroscopy Antibodies and immunoassays for detection of bacterial pathogens An optofl uidic nanoplasmonic biosensor for direct detection of live viruses from biological media Rapid detection of bovine viral diarrhea virus as surrogate of bioterrorism agents Development and application of lateral fl ow test strip technology for detection of infectious agents and chemical contaminants: a review Electrochemical detection of ultratrace nitroaromatic explosives using ordered mesoporous carbon Detection technologies for Bacillus anthracis : prospects and challenges Advances in assays and analytical approaches for botulinum-toxin detection Monitoring botulinum neurotoxin a activity with peptidefunctionalized quantum dot resonance energy transfer sensors Rapid and accurate detection of Bacillus anthracis spores using peptide-quantum dot conjugates Rapid detection of Bacillus anthracis using monoclonal antibody functionalized QCM sensor Receptor and substrate interactions of clostridial neurotoxins Lateral fl ow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey Semiconductor clusters, nanocrystals, and quantum dots Scanometric DNA array detection with nanoparticle probes A history of infl uenza Comparison of a new gold-immunochromatographic assay for the detection of antibodies against avian infl uenza virus with hemagglutination inhibition and agar gel immunodiffusion assays Attomole detection of hemagglutinin molecule of infl uenza virus by combining an electrochemiluminescence sensor with an immunoliposome that encapsulates a Ru complex Microgravimetric immunosensor for direct detection of aerosolized infl uenza A virus particles Label-free electrical detection of DNA hybridization for the example of infl uenza virus gene sequences Microfl uidic device architecture for electrochemical patterning and detection of multiple DNA sequences Swine infl uenza Pandemic (H1N1) 2009 -update 77 Bioterrorism, parasites as potential bioterrorism agents and biosecurity studies Highly sensitive amperometric immunosensor for detection of Plasmodium falciparum histidine-rich protein 2 in serum of humans with malaria: comparison with a commercial kit Size-dependent direct electrochemical detection of gold nanoparticles: application in magnetoimmunoassays A simple assay for direct colorimetric visualization of trinitrotoluene at picomolar levels using gold nanoparticles Fluorescence quenching of CdSe quantum dots by nitroaromatic explosives and their relative compounds Fluorescence detection of trace TNT explosive Chemical methods for the production of graphenes