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21 Century Nanoscience – A Handbook
Bioinspired Systems and Methods (Volume Seven)
Klaus D. Sattler
Bacterial Detection with Magnetic
Nanoparticles
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https://www.routledgehandbooks.com/doi/10.1201/9780429351525-3
Nayeem A. Mulla, Raghvendra A. Bohara, Shivaji H. Pawar
Published online on: 22 Apr 2020
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Nayeem A. Mulla, Raghvendra A. Bohara, Shivaji H. Pawar. 22 Apr 2020,
Bacterial
Detection with Magnetic
Nanoparticles from: 21 Century Nanoscience – A Handbook, Bioinspired Systems and
Methods (Volume Seven) CRC Press
Accessed on: 14 Jun 2021
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3
Bacterial Detection with Magnetic
Nanoparticles
Nayeem A. Mulla and Raghvendra A. Bohara
D. Y. Patil University
Shivaji H. Pawar
D. Y. Patil University Anekant Education Society
3.1 Introduction to Bacteria. . . . 3-1 3.2 Bacteria and Their Detection. . . . 3-2 3.3 Biomagnetism. . . . 3-2 3.4 Magnetic Nanoparticles. . . . 3-3 3.5 Role of MNPs in Microbiology and Biotechnology. . . . 3-3 3.6 Methods to Generate Bioconjugated MNPs. . . . 3-4 3.7 Rapid and Real-Time Detection of Bacteria Using MNP. . . . 3-4 3.8 Role of MNPs in Bacterial Detection. . . . 3-4 3.9 Synthesis of MNPS for Bacterial Detection. . . . 3-6
Thermal Decomposition•Hydrothermal Synthesis•Microwave-Assisted
Synthesis•Template-Assisted Fabrication•Coprecipitation•Microemulsion
3.10 Innovative Techniques for Bacterial Detection by Using MNPs. . . . 3-7
Recognition Moieties Used for Enrichment of Bacteria•Use of Antibiotics for
Enrichment of Bacteria•Use of Antibodies for Enrichment of Bacteria•Use
of Other Biomolecules for Enrichment of Bacteria
3.11 Conclusion . . . . 3-9 References. . . . 3-9
3.1
Introduction to Bacteria
Bacteria are microscopic single-celled organisms that are all around us. They come in many different sizes and shapes, and a common way to classify them is by their morphology, or shape and appearance [1]. The three basic shapes of bacteria are spherical, rod shaped, and spiral. Spiral-shaped bacteria can be further categorized depending in part on how much spiraling they show. Not all bacteria are capable of causing disease, but each morphology-based group has at least some disease-causing representatives [2].
Bacteria are the most abundant and diverse life forms on the earth. A bacterium belongs to the prokaryote group, which means they are unicellular organism without nucleus. A typical bacteria is only several micrometers in size, and because they are such small organisms light microscopes only demonstrate their morphology. The reproduction rate of bacteria is the fastest among all living organisms [3]. They have a rapid adaptive nature not only morphologically and metabolically but also genetically. Therefore bacterial popu-lations can adapt to new environments very quickly, and they evolve to meet the demands of any new localized stres-sors. They can change so that they can use other substances as their food and can develop resistance to antibiotics [4,5]. Certain bacteria, like actinomycetes, produce antibi-otics such as streptomycin and nocardicin; others live
symbiotically in the gut of other animals (including humans) or elsewhere in their bodies, or in the roots of certain plants, converting nitrogen into a usable form [6]. Bacteria put the tang in yogurt and the sour in sourdough bread; bacteria help to break down dead organic matter; bacteria make up the base of the food web in many environments. Bacteria are of such immense importance because of their extreme flexibility, capacity for rapid growth and reproduction, and their age—the oldest fossils known, nearly 3.5 billion years old, are fossils of bacteria-like organisms [7].
A bacterium is a single-celled, unicellular microorganism that does not have a nucleus or any other membrane-bound organelles. Bacteria are sometimes called “prokary-otes”. In Greek, “prokaryote” literally means “before the nut”. Bacteria adapt to become well suited to their envi-ronments, and therefore come in many shapes and forms [8]. However, they all have a few features in common (Figure 3.1).
1. Capsule: A protective, often slimy, coating mainly made up of sugars that help to protect the bacterium. It also makes bacteria virulent. This means the bacteria are more likely to cause disease, since it aids the cell survive against immunological attack. For example, the bacteria may survive an attack from the human body’s immune system.
FIGURE 3.1 Anatomy of a typical bacterium.
2. Cell wall: In bacteria, the cell wall is usually made of peptidoglycan, a protein and sugar compound. This structure gives the cell some rigidity and protection.
3. Plasma membrane: As in most cells, the bacterium’s plasma membrane acts to coordinate the passage of molecules into and out of the cell. 4. Cytoplasm: Again, as in many cells, the
cyto-plasm serves as a medium through which molecules are transported as well as a system to maintain homeostatic conditions (like temperature and pH) that are best for the cell.
5. Ribosomes: These are the main site for the bacterium’s protein synthesis. They are scattered throughout the cell cytoplasm.
6. Plasmid: It is an extrachromosomal, small, circular double-stranded DNA. It replicates itself independently of the bacterial genome and many times contains important genes.
7. Nucleoid: This is the region where the bacterium’s DNA is located. It is not the same as a nucleus in eukaryotic cells because it is not surrounded by a membrane.
8. Flagellum: In many bacteria, a flagellum is present, and it is the means by which the cell moves around.
9. Pili: Many species of the bacterium contains hair-like structure on the cell surface called pili. They are an important organ in the bacterial reproduc-tion process.
3.2
Bacteria and Their Detection
Fundamentals of morphology, molecular chemistry, and surface physiochemical are important considerations while detecting bacterial species. With respect to conventional sense of biology, bacteria are known as microscopic organisms, ranging a few micrometers in length and of
various different kinds. They are unicellular, prokaryotic in nature with no internal organization, and multiply by under-going fission. They have different shapes like rod, spher-ical, and cuboidal. They can be found either singly or in pairs or even as chains or clusters. They have a single chromosome with a closed circle of double-stranded DNA [9]. Sometimes, they possess characteristic appendages like flagella. The cell wall is rigid and made up of phospho-lipid bilayers. Bacteria can be categorized on different bases such as by staining (e.g., Gram-positive, Gram-negative), culturing requirements (aerobic, anaerobic), etc. Mostly, they are recognized based on Gram staining. Gram-positive bacteria possess a thick cell wall containing many layers of peptidoglycan and teichoic acid. In contrast, Gram-negative bacteria have a relatively thin cell wall consisting few layers of peptidoglycan surrounded by a second lipid layer containing lipopolysaccharides and lipoproteins [10].
3.3
Biomagnetism
Magnetoreception is a sense that allows an organism to detect a magnetic field to perceive direction, altitude, or location. This sensory modality is used by a range of animals for orientation and navigation [11] and as a method for animals to develop regional maps. For the purpose of navigation, magnetoreception deals with the detection of the earth’s magnetic field. Magnetoreception is present in bacteria, arthropods, molluscs, and members of all major taxonomic groups of vertebrates. Humans are not thought to have a magnetic sense, but there is a protein (a cryp-tochrome) in the eye, which could serve this function. Unequivocal demonstration of the use of magnetic fields for orientation within an organism is seen in a class of bacteria known as magnetotactic bacteria. These bacteria demonstrate a behavioral phenomenon known as magneto-taxis, in which the bacterium orients itself and migrates in the direction that follows the earth’s magnetic field lines. The bacteria contain magnetosomes, which are nanometer-sized particles of magnetite or iron sulfide enclosed within the bacterial cells [12]. The magnetosomes are surrounded by a membrane composed of phospholipids and fatty acids and contain at least 20 different proteins. They form as chains where the magnetic moments of each magneto-some align in parallel, causing each singular bacterium to act as a magnetic dipole, giving the bacteria their permanent-magnet characteristics. In animals, the mecha-nism for magnetoreception is unknown, but there are two main hypotheses to explain this phenomenon [13]. According to one model, magnetoreception is possible via the radical-pair mechanism. The radical-radical-pair mechanism is well estab-lished in spin chemistry and was speculated to be applicable to magnetoreception [14]. In year 2000, cryptochrome was proposed as the “magnetic molecule”, so to speak, that could harbor magnetically sensitive radical-pairs. Cryptochrome, a flavoprotein found in the eyes of European robins and other animal species, is the only protein known to form photo induced radical-pairs in animals. The function of
cryptochrome is diverse across species; however, the photo induction of radical-pairs occurs by exposure to blue light, which excites an electron in a chromophore. The Earth’s magnetic field is only 0.5 Gauss, so it is difficult to conceive of a mechanism other than phase shift, by which such a field could lead to any chemical changes other than those affecting the weak magnetic fields between radical pairs [14]. Cryptochromes are therefore thought to be essential for the light-dependent ability of the fruit fly Drosophila
melanogaster to sense magnetic fields. The second proposed
model for magnetoreception relies on Fe3O4, also referred
to as iron (II, III) oxide or magnetite, a natural oxide with strong magnetism. Iron (II, III) oxide remains perma-nently magnetized when its length is larger than 50 nm and becomes magnetized when exposed to a magnetic field if its length is less than 50 nm. In both of these situations, the earth’s magnetic field leads to a transducible signal via a physical effect on this magnetically sensitive oxide. The movement of charged particles causes magnetism. To under-stand the magnetic properties of a substance, one would need to look at the motion of electrons within the mate-rial [15]. Without the force of magnetism, or the knowledge of it, we would not be able to navigate without the sun or stars. In addition, we would not be able to run most electronics, from a loudspeaker to a car or a plane without magnetism. The medical field would not be advanced enough to diagnose diseases within hours, or to detect cancerous tumors. Without magnetism, stores and libraries would not be able to have anti-theft security systems. Similarly, detector systems such as those in airports would not be able to scan people for weapons with metal detectors. Migrating animals use magnetism to find their proper habi-tats; without it, entire species could die. In fact, without the Earth’s magnetic field, the entire planet would erode away. Magnetism is clearly an unseen force that our world depends on [16].
3.4
Magnetic Nanoparticles
Magnetic nanoparticles (MNPs) have shown great promise in many fields. MNPs are nanoparticles which can be manip-ulated by using magnetic field gradients. When the size of the nanoparticles becomes smaller, from a few nanome-ters to a couple of tenths of nanomenanome-ters, depending on the material, superparamagnetism appears. Superparam-agnetic nanoparticles are single-domain particles with all their magnetic moments aligned in the same direction, and with a short relaxation time. These particles have unique properties such as nearly instantaneous change of magne-tization in the applied magnetic field, which allows them to be directed toward a target using an external magnetic field and heating in alternating magnetic fields [17]. Owing to their unique properties, superparamagnetic nanoparticles have been intensively developed and have found numerous applications in biomedical, optical and electronic fields such as biosensing [18], targeted drug delivery [19], destruction of cancer tissues through hyperthermia, magnetic resonance
imaging, jet printing, cell separation, DNA separation [20,21], pathogen detection [22] immunoassay, and tissue repair [23].
The two main forms of nanoparticles with superparamag-netic properties are magnetite (Fe3O4) and its oxidized form
maghemite (c-Fe2O3). As the superparamagnetic behavior
of iron oxide nanoparticles strongly depends upon the dimension of the nanoparticles, control of uniform size distri-bution is very important in synthesis. The size and shape of the nanoparticles, however, depend on many factors such as pH, ionic strength, temperature, nature of the salts, and the Fe(II)/Fe(III) concentration ratio [24].
Various procedures have been described to synthesize MNPs, and among these methods, chemical coprecipita-tion of Fe2+ and Fe3+ ions by an alkali such as NH
4OH
or ammonia, in an aqueous solution, is the most commonly used solution-phase procedure. Nucleation and growth of nanocrystals are the two main steps that are involved in this bottom–up method [25]. The advantage of the precipitation method is that comparatively large quantities of nanoparti-cles can be prepared. Nevertheless, there are several draw-backs for this method such as difficulty in controlling the size distribution and the formation of impurities such as goethite and maghemite [26]. The main factors affecting the forma-tion of impurities during coprecipitaforma-tion are the initial and final pH of the solution and the reaction temperature [27]. It is reported that iron oxide nanoparticles with sizes below 20 nm cannot be labeled as “magnetite”. When the particle size is less than 10 nm, increase in surface area/volume ratio results in a large number of surface atoms, which would oxidize readily to Fe3+, thereby leading to the formation of
maghemite on the surface of the magnetic particle. Longer storage periods (6 months) and exposure to high tempera-tures (>180◦C) would also cause the Fe2+ ions to oxidize
to Fe3+ ions leading to the formation of maghemite. The
disadvantage is that the maghemite has slightly less satura-tion magnetizasatura-tion than magnetite. However, when it comes to the technological application of the nanoparticles, both magnetite and maghemite are suitable, as both of them are ferromagnetic in nature with superparamagnetism. In contrast, goethite is antiferromagnetic in nature. The forma-tion of goethite can increase with an increase in pH above 4.7 and, thus in co-precipitation, the ideal ratio of Fe2+/Fe3+
is about 1:2 [28].
3.5
Role of MNPs in Microbiology
and Biotechnology
MNPs possess certain remarkable physicochemical prop-erties such as superparamagnetic behavior, the ability to become heated under alternating magnetic field, high surface/volume ratio, and versatility in synthesis [29]. These particles are used to kill/inhibit growth of bacteria or to act as carriers of antibiotic and other antibacterial nanomaterials to help in the prevention, diagnosis, and treatment of infectious disease. At the same time, these
FIGURE 3.2 Role of MNPs in biological applications.
characteristics can also be used in microbial biotechnology to make or modify products or processes using micro-bial systems. The bioapplications of MNPs are shown in Figure 3.2.
3.6
Methods to Generate
Bioconjugated MNPs
Many strategies have been developed to immobilize specific molecules with high affinity for bacteria onto the surface of nanoparticle substrates. These methods include both covalent and noncovalent attachment strategies. A detailed discussion is provided (vide infra). A popular coupling method to bind molecules or materials to biomolecules is the use of the N -hydroxysuccinimide ester (NHS-ester) functionalization group. The NHS-ester can readily react with primary amines, resulting in an amide bond cova-lent linkage [30,31]. For example, NHS-ester functional-ized MNPs can be conjugated to the amino groups of biomolecules (see Figure 3.3a). Another approach is the use of a dual NHS ester reagent, bis-(N-hydroxysuccinimide ester) (DSS), which can link amino groups of biomolecules to amino-functionalized MNPs (see Figure 3.3b). Indeed, biomolecules with amide bond linkages to nanoparticles can also be created by first converting the amino group of amino-functionalized MNPs to a carboxylic acid by reaction with succinic anhydride. The amino group of the biomolecule can then react with the carbonyl group forming an amide bond linkage (Figure 3.3c) [32,33]. A click chemistry approach to connect biomolecules and MNPs can be realized by employingtrans-cyclooctene (TCO) and tetrazine (Tz) functional groups (Figure 3.3d). Another common method to tether molecules to proteins utilizes the streptavidin–biotin interaction. The streptavidin–biotin interaction is a well-known recognition event with extremely high binding affinity (Figure 3.3e). The hydroxyl group on the surface of the Fe4O3 can be
converted into an epoxide by silanization in the presence of 3-glycidyloxypropyltrimethoxysilan (GOPTS) and ethanol.
The amino group of the biomolecule can then react with the epoxy group to form bioconjugated MNPs (Figure 3.3f) [33].
3.7
Rapid and Real-Time Detection
of Bacteria Using MNP
Pathogenic bacteria are major concerns when it comes to human health, food industry, and water facilities. Accu-rate and definitive microorganism identification, including bacterial identification and detection, is essential for correct disease diagnosis, treatment of infection, and trace-back of disease outbreaks associated with microbial infections. For this, innovative, rapid, sophisticated, and highly sensitive detection methods are necessary. Successful attempts have been made in the development of molecular analyzing tech-niques like ELISA, PCR, ribotyping, microarray, etc. Aside from high sensitivity and reliability, these techniques suffer from high cost of performance, sample pretreatment, and lower limit of detection [17,18]. The drawbacks of conven-tional and current molecular diagnostics can be overcome with the help of nanoscience. Nanotechnology is a multidis-ciplinary branch of science which that deals with technology relating to nanosized materials. It has a huge significance in biomedical, pharmaceutical, agricultural, environmental, and many more branches of science. Nanobiotechnology is the branch of science that deals with the fabrication and use of nanomaterials for biological and biochemical appli-cations. It demonstrates all facets of research of biology assisted with nanotechnology [19]. As regards the properties of sub 100-nm materials and devices, their surface modi-fication has contributed significantly to biomedical fields such as cellular repair, drug delivery, therapeutic appli-cations, and diagnostic aids [20]. Knowledge and applica-tion of nanomaterials enable in-depth understanding of all bimolecular processes easy. Although many techniques are still in the nascent stage of development, some are actually being employed in daily practices [21]. Use of nanobiotech-nology extend the limits of current molecular diagnostics, allows point-of-care diagnostics, and integrates diagnostics with therapeutics. It enables diagnosis at the single-cell and molecular level [22]. MNPs possessing nanoscale size ranges are examples of bionanomaterials that mimic the size of molecules in nature and possess favorable characteris-tics making them multifunctional for bio-nanoapplications. The use of MNP’s high surface area and superparamagnetic property provides a promising and sophisticated platform for detection techniques so that conventional and molec-ular diagnostics become much easier and more accurate [23] (Figure 3.4).
3.8
Role of MNPs in Bacterial
Detection
Nanoparticles are the key focus of research for a variety of novel applications, not only because of their wonderful properties but also due to their nanosize, smaller compared
FIGURE 3.3 A summary of methods to synthesize bioconjugated MNPs. (a) The amino group of a biomolecule reacts with the NHS-ester coated nanoparticles forming an amide linkage. (b) Using the dual NHS-NHS-ester reagent (DSS), amino-functionalized nanoparticles are linked to biomolecules. (c) By conversion of amino-functionalized nanoparticles to carboxyl-modified particles, the amide bond linkage can be achieved by reaction with the amino group of the biomolecule. (d) Teatrazine-modified nanoparticles are attached to trans-cyclooctene modified biomolecules through a cycloaddition reaction. (e) Streptavidin-coated nanoparticles can bind to biomolecules via the biotin–streptavidin interaction. (f) Fe3O4 nanoparticles are converted to an epoxy-functionalized nanoparticle in the presence of
3-glycidyoxypropyltrimethoxysilan and ethanol. The antibody amino groups can then react with the epoxy on the nanoparticles forming antibody-modified nanoparticles.
FIGURE 3.4 Rapid and real-time detection of bacteria by using MNPs.
with their bulk counterparts. Nanoparticles are intermediate between atomic- and bulk-level particles. At the nano level, the properties greatly change as the size of the particles change owing to their large surface to volume ratio. Owing to their widespread applications, much research has been carried on the synthesis various nanostructures. For decades,
MNPs have been in focus as they show high potential in a variety of different application fields, ranging from chem-istry, biology, and medicine to physics. Magnetic nanoparti-cles have a wide range of applications ranging from magnetic fluids recording, catalysis [34], and biomedicine [35] to mate-rial sciences, photocatalysis [36,37], and so on. Nowadays,
many novel binary and ternary magnetic nanocomposites have also been synthesized with various core–shell structures including grapheme [38], carbon nanotube [39], conducting polymer [40], metal oxide, and other inorganic materials. Owing to its unique and creative applications in every field of life, researchers are highly focused in developing a number of synthetic ways to synthesize MNPs with different sizes, morphologies, and compositions, but the successful applica-tion of MNPs for the abovemenapplica-tioned applicaapplica-tions is highly dependent on the stability of the particles under a plethora of different conditions. The significance of size for control-ling the various properties is obvious, because in most of the cases, the properties of the magnetic nanoparticles are dependent on their dimension and morphology. Therefore, the synthesis of magnetic nanoparticles with controlled size and exposed facets is of core importance. The main problem associated with magnetic particles is their agglomeration, which tends to reduce the energy associated with the high surface area to volume ratio of the nanosized particles. In addition, the magnetic nanoparticles are highly chemically active. Therefore, it is of utmost importance to protect these magnetic nanoparticles against oxidation, which may involve functionalization and coating with certain protec-tive layers to form a core–shell structure that completely modifies the MNPs. Various methods have been used for the synthesis of different kinds of magnetic nanostructures including iron oxide and different metal alloys. MNPs of core–shell nature and composites structures have also been synthesized for various applications with suitable modifi-cations. Throughout the last decades, synthesis methods for MNPs using different techniques have been evolved and specialized for fine-tuning of the nanostructures [41].
3.9
Synthesis of MNPS for
Bacterial Detection
Here, we will give a short description of only those methods that offer excellent size and shape control (Figure 3.5).
FIGURE 3.5 Different synthesis methods opted for develop-ment of MNPs for bacterial detection.
3.9.1
Thermal Decomposition
This method of synthesis based on the chemical decomposition of the substance at elevated tempera-ture involves breaking of the chemical bonds. It mostly uses organometallic compounds such as acetylacetonates in organic solvents with surfactants. In this method, the composition of various precursors that are involved in the reaction determine the final size and morphology of the magnetic nanostructures. Using this method, nanocrystals with very narrow-sized distribution (4–45 nm) could be synthesized along with excellent control of morphology [42].
3.9.2
Hydrothermal Synthesis
Another important chemical synthesis technique that involves the use of liquid–solid–solution (LSS) reaction and gives excellent control over the size and shape of the magnetic nanoparticles is hydrothermal synthesis. This method involves the synthesis of magnetic nanoparticles from high boiling point aqueous solution at high vapor pres-sure. It is a unique approach for the fabrication of metal, metal oxide, rare earth transition metal magnetic nanocrys-tals, semi-conducting, dielectric, rare-earth fluorescent, and polymeric nanomaterials [43]. This synthetic technique involve the fabrication of magnetic metallic nanocrystals under different reactions conditions. The reaction strategy is based upon phase separation which occurs at the interface of the solid–liquid–solution phases present in the reaction. For example, the fabrication of monodisperse (6, 10, and 12 nm) Fe3O4and MFe2O4nanocrystals has been demonstrated by
Sun et al [44]. The synthesized magnetic nanoparticles were used for photocatalytic degradation of organic dyes, and it was observed that truncated nanocubes possess much higher photocatalytic degradation activity as compared to oblique nanocubes [45].
3.9.3
Microwave-Assisted Synthesis
The microwave-assisted method is a chemical method that use microwave radiation for heating materials containing electrical charges, for instance polar molecule in the solvent or charged ions in the solid. As compared to the other heating methods, microwave-assisted solution fabrication methods get more research attention because of rapid processing, high reaction rate, reduced reaction time and high yield of product [46].
3.9.4
Template-Assisted Fabrication
Another fabrication method used for the synthesis of MNPs is the assisted fabrication [47]. Active template-based synthesis involves the growth of the nuclei in the holes and defects of the template. Subsequently, the growth of the nuclei at the preformed template yields the desired morphology of the nanostructures. So, through proper selec-tion of a base template, the size and shape of the magnetic
nanoparticles can be controlled. This technique has two important advantages over the chemical routes:
• Template use in the fabrication process determines the final size and morphology of the nanostruc-tures.
• Complex nanostructures such as nano-barcode (segmented nanorods); nanoprism; nanocube – hexagon, and -octahedron MNPs can be fabricated in an easy manner, with full control of size and morphology.
However, this method has also some drawbacks. It is a multi-step process that first of all requires the fabrication of base templates followed by the subsequent deposition of magnetic material within the template [48].
3.9.5
Coprecipitation
Coprecipitation is a facile and convenient way to synthe-size of MNPs from aqueous salt solutions by the addition of a base under inert atmosphere at room temperature or at elevated temperature. The size, shape, and composition of the MNPs very much depends on the type of salts used, the salt ratio, the reaction temperature, the pH value, and ionic strength of the media. With this synthesis, once the synthetic conditions are fixed, the quality of the magnetite nanoparticles is fully reproducible [49].
3.9.6
Microemulsion
A microemulsion is a thermodynamically stable isotropic dispersion of two immiscible liquids, where the microdomain of either or both liquids is stabilized by an interfacial film of surfactant molecules. In water-in-oil microemulsions, the aqueous phase is dispersed as microdroplets (typically 1–50 nm in diameter) surrounded by a monolayer of surfac-tant molecules in the continuous hydrocarbon phase. The size of the reverse micelle is determined by the molar ratio of water to surfactant ratio [50]. By mixing two identical water-in-oil microemulsions containing the desired reactants, the microdroplets will continuously collide, coalesce, and break again, and finally a precipitate forms in the micelles [51]. By the addition of a solvent, such as acetone or ethanol, to the microemulsions, the precipitate can be extracted by filtering or centrifuging the mixture. In this case, a microemulsion can be used as reactor for the formation of nanoparticles. Using the microemulsion technique, nanoparticles can be prepared as spheroids [52]. Although many types of MNPs have been synthesized in a controlled manner using the microemulsion method, the particle size and shapes usually vary over a relative wide range. Moreover, the working window for synthesis of microemulsions is usually quite narrow, and the yield of nanoparticles is low compared to other methods, such as thermal decomposition and copre-cipitation. Large amounts of solvent are necessary to synthe-size appreciable amounts of material. It is thus not a very efficient process and also rather difficult to scale up [53].
3.10
Innovative Techniques for
Bacterial Detection by Using
MNPs
The integration of bioconjugate MNPs with different analyt-ical methods has paved a new path for bacteria, protein, and cancer cell sensing, purification, and quantitative analysis. The scope of superparamagnetic nanoparticles in many technological applications like magnetic storage media, biosensing applications, and medical applications caused this field to develop intensively [54]. In the absence of an external magnetic field, the overall magnetization value of superparamagnetic nanoparticles is randomized to zero. Such fluctuations in magnetization direction result in minimization of the magnetic interactions between any two NPs in the dispersion, making the dispersion stable in physiological solutions and facilitating NP coupling with biological agents [55]. When exposed to an external magnetic field, these MNPs align along the direction of magnetic field, achieving magnetic saturation at a magni-tude that far exceeds any of the known biological enti-ties. Due to this unique property of MNPs, detection of the MNP-containing biological samples is enhanced on manipulation of these biological samples with an external magnetic field [56].
3.10.1
Recognition Moieties Used for
Enrichment of Bacteria
Surface modification of MNPs with recognition moieties such as antibodies, antibiotics (vancomycin, daptomycin, etc.), and carbohydrate enables its use for bacterial detec-tion. These recognition moieties help to detect the bacteria selectively and at low concentration. Different approaches have been used to isolate bacteria using MNPs, such as those described in the following (Figure 3.6).
FIGURE 3.6 Strategies to develop MNPs for efficient capture and detection of bacteria.
3.10.2
Use of Antibiotics for Enrichment
of Bacteria
Vancomycin belongs to the glycopeptide group of antibi-otics, which is known to interact strongly with a broad range of Gram-positive bacteria. Vancomycin kills bacteria by inhibiting bacterial cell wall synthesis. This interaction is mediated via five hydrogen bond motifs between the heptapeptide backbone of vancomycin and the D-alanyl-D-alanine dipeptide from the cell wall [57]. As a result, vancomycin-functionalized MNPs are capable of recognizing the cell surfaces of different bacteria. It has been demonstrated that vancomycin offers less speci-ficity when compared with a monoclonal antibody but can bind to different Gram-positive bacteria such as
Entero-coccus faecalis, StreptoEntero-coccus pneumoniae, and Staphylo-coccus aureus but not against Gram-negative bacteria. Gu
et al. reported a strategy to use vancomycin-conjugated FePt MNPs of around 4 nm that are water soluble in nature to detect Gram-negative as well as Gram-positive bacteria at low concentrations [58]. As a control experiment, they have used FePt nanoparticles capped with an amine group (FePt–NH2), which failed to capture the bacteria because of the lack of specific molecular recognition. Lin et al. reported vancomycin-immobilized iron oxide nanopar-ticles that can be used to trap Gram-positive bacteria such as Staphylococcus saprophyticus, S. aureus, and E.
faecalis selectively from urine samples, followed by
detec-tion with matrix-assisted laser desorpdetec-tion/ionizadetec-tion mass spectrometry (MALDI-MS). Their result suggests that this method is capable of rapidly identifying trace pathogens in urine samples [59]. Kell et al. have reported a series of vancomycin-modified Fe3O4 MNPs used in magnetic
confinement assay to isolate different Gram-positive and Gram-negative bacteria at a low concentration. Their results demonstrate that small moieties are an excellent alterna-tive to antibody-mediated detection of bacteria, where more precaution is required as compared to small moieties like vancomycin [60]. In 2011,Chung et al. reported that the bio-orthogonal modification of vancomycin and daptomycin, which is lipopeptide in nature and binds to the cell wall of Gram-positive bacteria via its hydrophobic tail, resulted in the depolarization of the bacterial cell membrane. Primarily, they have synthesized trans-cyclooctene derivatives of these antibiotics, which are attached to tetrazine-decorated Fe3O4
fluorescent MNPs. Their result shows that using a two-step labeling procedure, their assay is superior to using direct antibiotic–nanoparticle conjugates [61]. Recently, Chen et al. synthesized fluorescent MNPs with a core–shell structure followed by conjugation of gentamycin, which is a FDA-approved thermal-resistant antibiotic belonging to the aminoglycoside group and used for the treatment of infection caused by Gram-negative bacteria. Their results demon-strate that gentamicin-bioconjugated fluorescent MNPs can capture Gram-negative bacteria, i.e., Escherichia coli (1 × 107 CFU/mL) within 20 min from 10 mL of solution.
In addition to this, these gentamycin-modified MNPs are
also able to detect diluted E. coli cells at a concentration as low as 1 × 103 CFU/mL [62]. Several such approaches are
reported [63].
3.10.3
Use of Antibodies for Enrichment of
Bacteria
Antibody-conjugated MNPs can selectively capture target bacteria from the given biological sample. Here, application of a magnetic field separates the particle–bacteria complexes from the solution, thereby enriching the concentration of the bacteria and enabling the detection of target bacteria without a culturing process. The use of this approach seems to be more specific in nature. Recently, Tran et al. reported the use of protein A-conjugated chitosan-modified Fe3O4
MNPs for separation Vibrio cholerae at low concentration. In their study, they have prepared a conjugation of chitosan-coated MNPs and protein A. This conjugate was incubated with specific IgG antibodies against V. cholera that could be detected by a conventional diagnostic method as well as immune chromatographic strip test. This method serves as a convenient stage for enrichment and separation of various pathogens from different liquid samples, after incubation with specific IgG antibodies [32]. Several such immunomag-netic approaches have been developed for the enrichment of MNPs with bacteria and used for detection at a low concen-tration [64,65].
3.10.4
Use of Other Biomolecules for
Enrichment of Bacteria
Biomolecules such as carbohydrate, protein, and nucleic acid are used for enrichment of MNPs. It is known that many bacteria use mammalian cell surface carbohydrates as anchors for attachment, which subsequently results in infection [66]. The unique combination of MNPs and carbo-hydrate group helps to enrich MNPs with bacteria, which can thus be detected [67]. Pigeon ovalbumin (POA), a phos-phoprotein, contains high levels of terminal Gal α(1/4) Gal units. Thus, MNPs with immobilized POA can be used as affinity potential probes for bacteria enrichment [66]. Lee et al. have recently developed a magneto-DNA nanopar-ticle system for rapid detection of bacteria. In their work, they have used oligonucleotide probes to detect specifi-cally targeted nucleic acids, particularly 16S rRNAs, from the pathogen. Furthermore, the assay is rapid in nature and able to simultaneously detect 13 bacterial specimens within 2 h [68]. A study conducted by Huang et al. has reported the use of amine-functionalized MNPs for capturing bacteria from water, food, and urine samples. This developed method does not require the use of affinity molecules on the surface and is able to detect different Gram-positive and Gram-negative bacteria. The detection is based upon the positive charge present on the surface of the MNPs and negatively charged bacterial cell, which promotes a strong electrostatic interaction that results in
FIGURE 3.7 Schematic representation showing the surface-modified MNPs for detection of pathogens.
efficient adsorptive ability [69]. Schematic representation for the detection and isolation of pathogenic bacteria is presented in Figure 3.7.
3.11
Conclusion
MNPs gained much interest due to their ability to be manipulated upon application of a magnetic field. MNPs are now being used extensively for multiple functions that are much needed for biological applications. Factors such as biocompatibility, toxicity, in vivo and in vitro targeting efficiency, and long-term stability of MNPs should always be addressed with respect to their use in bioapplications. The increasing demands and versatile requirements imposed on MNPs intended for bioapplications require close moni-toring of the size, structure, and surface properties of MNPs. Although functionalized MNPs have great potential appli-cations for bacterial detection, it can be difficult to detect organisms in real-life samples when the bacterial concen-tration is low. In addition, attempts need to be made to detect the microbes in the presence of other microbes of different other genus and in the presence of contaminants. Optimum surface modification of nanoparticles is hard to control. Hence, strategies that are more consistent have to be developed to enable precise composition and uniform surface modification with reproducible functionalization. It is also seen that much work is being carried on Fe3O4MNPs.
Other ferrites should be explored in this direction. Future research should focus more on sensitivity, reproducibility, and improving the ability of the technique, so that it can be used for normal samples.
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