Nanobiosensors for Pathogenic Agents Detection

Conventional methods to detect pathogenic microorganisms can be wasteful, expensive, and not very sensitive. Due to these limitations, approaches based on nanotechnology and the development of new diagnostic tests are emerging as alternative methods. Resulting from the unique electrical, magnetic, luminescent, and catalytic properties of nanomaterials, rapid and cost-effective diagnostic tests have been developed to recognize the presence of microbial pathogens or some of their components in different samples. The purpose of this review is to provide an overview and an update regarding the use of several nanomaterials as biosensors, among which are gold nanoparticles, carbon nanotubes, magnetic nanoparticles, and quantum dots. The application of these biosensors has opened a new interdisciplinary frontier between biological detection and materials science.


Introduction
Pathogens are responsible for the death of a large number of people in both rural and urban settings. Unfortunately, the inaccurate diagnosis of their presence leads to improper handling of its consequences. Additionally, pathogen detection is based on methods that require extended analysis time and are limited by laboratory-based setups, such as microscopy and culture-based techniques, making them even more unsuitable for field application. Therefore, it is necessary to develop highly efficient pathogen detection systems, that are easy to use, low cost, and easy to implement. 1 Conventional methods for the identification and detection of pathogens (Table 1) are primarily based on (i) culture and colony counting methodologies (involving the counting of bacteria); (ii) immunology-based methods (including antigen-antibody interactions); and (iii) the polymerase chain reaction (PCR) procedure (involving deoxyribonucleic acid (DNA) analysis). However, there are some limitations given that culture-dependent methods are costly. Additionally, pathogens in the sample being analyzed are present in very low concentrations, making their detection very difficult. Besides, a requirement of most of these detection methods is knowledge of the structural and biochemical properties of the bacterial species, limiting the application of these techniques when the pathogen is unknown. [9][10][11] The development of immunoassays as a sensitive method for detecting microorganisms shows some disadvantages due to the use of antibodies that are easily denatured. On the other hand, molecular approaches for the detection of markers associated with the virulence of the pathogen provide satisfactory results but require instrumentation and trained personnel, and such analyses cannot be executed at the field level. 4,12 Therefore, new analysis techniques have been developed involving biosensors as detection tools that, due to the incorporation of nanomaterials, have increased sensitivity, sample performance, specificity, and limits of detection of the assay, reducing both its complexity and cost. 4 The benefits of using nanomaterials for biosensor applications are remarkable. Due to their size, quantum confinement effects, reactivity, magnetic properties, electrical properties, and optical properties, nanomaterials, when incorporated into biosensors, can substantially increase the sensitivity of detections. 13 The use of nanomaterials in transducers leads to the generation of large surface areas, making possible their functionalization with various biomolecules such as nucleic acids, toxins, or proteins. As a consequence, the number of available sites for the reaction with the target molecule increases. 10 Also, the excellent optical, catalytic, magnetic, and electronic properties of nanomaterials facilitate their use in the manufacturing of nanobiosensors with high sensitivity and faster response times as compared to conventional methods. 12 The two main challenges for detecting a single bacterial cell are rapid real-time detection and ultrasensitivity in the analysis. Due to the significant developments in the last few decades, nanobiosensors are now considered as practical alternatives to the conventional methods for pathogen detection. 14 Furthermore, advances in pathogen detection could reduce unnecessary drug use and prevent the spread of diseases. 11 That being said, this review aims to present the characteristics of the nanomaterials used for the development of nanobiosensors and the usefulness of these instruments to detect pathogenic microorganisms.

Nanomaterials
Nanomaterials are defined as those materials with sizes less than 100 nm in one of their dimensions. 15 Characterized by a large surface area per unit mass and several orders of magnitude larger than macroscopic materials, nanomaterials play a fundamental role in both present and future technologies. 16 Several nanomaterials properties are due to the atomic and molecular constitution of their surfaces, chemical composition, wettability, and electrical charge. 17 Some examples of nanomaterials used in the manufacture of nanosensors are presented in Table 2.
In nanomaterials, the physicochemical and photophysical properties such as molecular weight, purity, stability, solubility, and catalytic activity rely on their size/shape relationship. Interestingly, many nanomaterial properties can be modified by manipulating the parameters  Loop-mediated isothermal amplification (LAMP) the reaction mixture is incubated at a specific temperature using a single tube containing a buffer, target DNA, DNA polymerase, and primers; the detection of the amplified product occurs by the presence of a precipitate 10  polyacrylamide (0.6 g); ferric chloride (FeCl 3 ), iron(II) chloride (FeCl 2 ) and aqueous hydrochloric acid (HCl, 2 mol L -1 ); ferric chloride (FeCl 3 , 1 mol L -1 ), iron sulfate (II) (FeSO 4 , 1 mol L -1 ) and aqueous hydrochloric acid (HCl, 2 mol L -1 ); iron(III) acetylacetonate and benzyl alcohol 10,12,27,28 Quantum dots (QDs) particles formed by a semiconductor core material (mixtures of cadmium-selenium or cadmiumtellurium) covered by an additional semiconductor layer, usually zinc sulfide colloidal and fluorescent semiconductors with unique optical properties; diameters of 2-8 nm; by coating with an organic material layer, surface functionalization for the binding of biomolecules is achieved; bright with high photostability cadmium oxide (0.2 mol L -1 ), selenium (2 mmol L -1 ), sulfur solution (0.04 mol L -1 ), trioctylphosphine oxide (0.5 g), tributylphosphine (0.472 g), 1-octadecene (2 g), oleic acid (1.5 g), octadecylamine (1.5 g) and stearic acid (0.8 mmol L -1 ); cadmium selenide (CdSe) nanocrystals, and zinc sulfide (ZnS); mercaptoacetic acid liquid, aqueous CdCl 2 (0.01 mol L -1 ), aqueous Na 2 S (5 mmol L -1 ), and chlorophyll 10,12,28,29 of the synthesis, such as the precursors' temperature and concentration, and through the nanomaterial surface's functionalization. 32 In the biological environment, properties such as biodistribution, transport, retention at the tissue level, and degradation of the nanomaterial are also determined by its shape and chemical composition, being the charge relevant in the union to receptors and the penetration of physiological barriers. 17 When nanomaterials are exposed to living systems, they are quickly surrounded by proteins, lipids, and other biomolecules, increasing their biocompatibility. This is a determining factor in bioapplications, since it avoids nanoparticle detection as a foreign element and evades rejection by natural defense systems. This phenomenon is known as the formation of the bio-crown, which gives a new biological identity as their surface changes. 33

Nanobiosensors for Pathogenic Agents Detection
Biosensors were reported for the first time in the 1960s; since then, they have been used mainly in environmental monitoring and biological detection. 34 As analytical instruments, biosensors combine biological recognition, transforming this phenomenon into digital signals that are interpreted through software. 35 Depending on the operating principle of the biosensor, the substance of interest present in living or non-living systems, the analyte, can be sensed by taking advantage of some of its properties (electrical, chemical, electrochemical, optical, magnetic, or vibratory, Table 2). 3 Generally, these devices are made up of a biorecognition unit or bioreceptor and a transducer ( Figure 1). The analyte-bioreceptor interaction will produce an effect measurable by the transducer, generating, for example, an electronic signal. 34 This is achieved by the immobilization of the biorecognition elements through encapsulation, covalent interaction, adsorption, or a combination of these. 3,36 These biorecognition units, or bioreceptors, present in cells (such as glycoproteins, glycopeptides, lipoproteins, carbohydrates, lipids, and receptor proteins) fulfill multiple functions, participating in infection processes, adhesion to cell surfaces and non-cellular substrates, evasion of the immune system, nutrient intake and transport thereof. Since they are exposed to the extracellular environment, bioreceptors have one feature in common; they serve as biorecognition elements during the bisosensors' assembly. 12 By using nanomaterials in their construction, the limit of detection of biosensors is greatly improved. This is attributed to their large surface area, high electronic conductivity, and plasmonic properties, such as the ability to store light in confined spaces. 3,36 Additionally, nanomaterials as biosensors allow the use of new optical or mechanical signals, mainly as transduction methods. 36 As defined above and applied to biosensors, a material with a size below 100 nm is considered a nanobiosensor. 28 In these, the fundamentals of spectroscopy, optics, and mechanics, among others, are used as the operating principle (Table 3). These dimensions imply that nanobiosensors, due to the small area available for detection, demand less analyte to register a measurable response. Smaller spaces generally allow higher density arrays, which maximize the number of analytes detected in a single test. Additionally, the complexity of the pathogen detection tests and their costs can be reduced by using nanobiosensors through eliminating some of the sample processing stages normally present in conventional methods, such as DNA amplification or fluorochrome staining. 11 Generally, nanobiosensors are based on the interaction between nucleic acids, enzymes-substrate, cells-bacteria, or antigen-antibody interactions, using biomimetic materials that replicate biological processes and materials such as synthetic bioreceptors ( Figure 2). 12,34,44 Several pathogens have been detected using these interactions, especially in vitro, and on different food samples such as milk and drinking water (Table 4). Escherichia coli stands out as the most studied target bacteria using nanobiosensors devices.

Biosensors with gold nanoparticles (AuNPs)
AuNPs as biosensors are very attractive, mainly due to their high electrical conductivity that allows them to transfer electrons easily. 3 Other benefits include their efficient optical properties and facile surface functionalization. This has led to their widespread use in biological detection processes such as DNA hybridization and DNA-protein interactions. 90 These features, and the fact that the surface of AuNPs displays an excellent affinity for sulfhydryl groups (-SH), allow the nanoparticles' functionalization by conjugating them with molecules or polymers containing these functional groups through covalent bonds. This facilitates their application in several tests using antibodies and nucleic acids. 12 Moreover, the fluorescence in AuNPs has been used in quenching processes, where one side of the nanoparticle has oligonucleotide molecules marked with a sulfhydryl group, while the other end is attached to a fluorophore. For example, optical biosensors for specific DNA sequences show an increase in the deactivated fluorophore's fluorescence after the biosensor-analyte  Operating principle Reference Based on spectroscopy using SRP (surface plasmon resonance) as a direct means to measure the binding of a target molecule to a surface ligand, light is reflected from the detection surface when binding events occur; this method measures reflected light changes as an interaction between the target molecule and receptors occurs on the detection surface; the SRP-based sensors detect the binding of an analyte to the bioreceptor by monitoring the change in the wavelength necessary to excite the SRP

22,37-42
Optical usually based on semiconductor particles that fluoresce when excited at different wavelengths by a light source; when the analyte of interest (yellow sphere) interacts with the sensor, fluorescence is emitted due to the semiconductor particles' excitation; an optical detection unit captures the fluorescence that evidences the union 14,18,19,[25][26][27]41,[43][44][45][46][47][48][49][50][51][52][53][54][55] Operating principle Reference Mechanics based on atomic force microscopy: high sensitivity requires small sample volumes for which no marking is necessary; this technique uses a nanometer-sized tip-shaped structure to scan the surface to which the pathogen binds; then, a detailed map of the surface is generated, revealing the presence of the agent; this technique has the advantage of allowing the simultaneous scanning of several pathogens microcantilevers: biosensors in the form of scaffolds with a section for selective recognition of either antibody-antigens or DNA fragments with their complementary probes; the analytes bind to the device in a dynamic manner in which the binding of the target molecule generates a change in the resonance frequency or in a static way where the deflection of the scaffold occurs by the binding of the target molecule 44,52,53,56 Adapted from Driskell and Tripp. 11 DNA: deoxyribonucleic acid.  interaction due to a conformational change in the system (Table 5). 36 In 2008, Nath et al. 19 used AuNPs functionalized with dextran to detect E. coli in three hours, in the absence of carbohydrate intake by E. coli or sterile conditions. The presence of free carbohydrates can be detected by the addition of concanavalin A, which produces a change in the plasmon band due to nanostructure formation ( Figure 3). However, DNA-based biosensors have the disadvantage of requiring isolation of the microorganism, cell lysis, and bacterial DNA extraction, limiting its application as a diagnostic technique at the field level in developing countries. 12 Fullerene nanoparticles/ nitrogen-doped graphene (nano-C60/NGS)

Mycobacterium tuberculosis
Au-nano-C60/NGS acting as a new signal tag produce a response in the absence of redox molecules; subsequently, to achieve signal amplification, it is labeled with signal probes creating a tracer label Polyaniline (PANI) Pseudomonas aeruginosa the PANI/AuNP prepared show to be a highly sensitive pyocyanin sensor in a pathogen culture, which uses electron microscopy and cyclic voltammetry to study morphology and electrical conductivity of the constructed electrode 500 nmol L -1 24 95 Oligonucleotide primers

Biosensors with silver nanoparticles (AgNPs)
Silver has been widely used within the metallic elements to manufacture NPs and has received considerable attention due to its particular physicochemical properties. Among these are the ability to interact with visible light, the presence of a characteristic band of superficial plasmons, and the absence of toxicity to humans and animals. 47 Silver nanoparticles' unique properties make them highly attractive commercially and scientifically, especially in technologies such as disinfectants, antimicrobials, cryogenic superconductors, composite fibers, cosmetics, electronics, and particularly as biosensors. Among the many methods for synthesizing silver nanoparticles, techniques such as electrochemistry, radiolysis, and chemical reduction stand out. The latter is one of the most widely used synthetic procedures, using a wide variety of inorganic and organic reducing agents. More recently, the synthesis of nanoparticles with an ecological vision has become a fascinating research area. This green chemistry perspective has included the use of mixedvalence polyoxometalates, polysaccharides, irradiation, and the inclusion of methods from biology, which have shown advantages over conventional methods that involve chemical agents associated with toxicity (Table 6). 24 From the spectroscopic point of view, AgNPs have a single absorption band in the UV-Vis range, in contrast to the bulk material. The absorption occurs when the excitation frequency alters the electrons, generating a phenomenon called localized surface plasmon resonance (LSPR). Studies have shown that the absorption wavelength in AgNPs is extremely sensitive to its local refractive index, particle size, and shape. 11 Since the resonance band of the surface plasmons is susceptible to several surface-level processes such as the absorption of molecules or nanoparticles' aggregation, AgNPs have shown fascinating results in numerous applications as biosensors. 36 This, and the ability to functionalize its surface using various biopolymers, have allowed AgNPs to have a high impact on the detection of proteins, antibodies, peptides, and oligonucleotides. 12,25 Zhou et al. 22 developed a SERS (surface enhance Raman scattering) based detection technique that uses metal nanoparticles, given the electromagnetic field present on their surface. Raman signals are a spectroscopic fingerprint to monitor microorganisms in vivo, making it possible to detect Escherichia coli and Staphylococcus epidermidis in drinking water. For this, a layer of AgNPs coated with the bacterial cells was synthesized, and then the bacteria were analyzed by SERS. It was found that the intensity of the Raman signals increases after the nanoparticle synthesis, which mainly depends on the zeta potential of the bacterial cell wall. Interestingly, the total time required for the test was only ten minutes, and the sample volume required for the analysis just 1 mL, detecting up to 2.5 × 10 2 cells per mL. With a very similar approach, Chen et al. 74 used positively charged silver nanoparticles (AgNPs + ) and Raman scattering as a method for the identification, in one hour, of methicillin-resistant Staphylococcus aureus (Figure 4).

Biosensors with silica nanoparticles
Fluorescent silica nanoparticles (SiNP) have attracted particular attention because of their potential in applications such as generating diagnostic images at the clinical level and the detection of pathogens or ligand-receptor interactions (Table 7). 101 Also, SiNP can be manufactured in various stable fluorescence colors and are biocompatible.
Like other nanoparticles, SiNP can be functionalized with several chemical groups such as amines, carboxyls, sulfhydryls, and organic compounds such as methacrylate. 26 Zhao et al. 14 developed a bioassay based on in situ detection of Escherichia coli O157:H7 using bioconjugated SiNP in a fluorescence-based immunoassay, using antibodies that accurately detect the bacteria. Of note, Escherichia coli O157:H7 is one of the agents responsible for transmitting food-borne diseases. The technique showed high effectiveness, identifying from 1 to 400 cells of the  In this AgNP biosensor, nanoparticles interact with the bacterial surface due to electrostatic forces when they are in contact with a silica-chip, generating changes in the SERS spectrum obtained using a Raman micro-spectrometer (adapted from Chen et al. 74 ).  60 107 Organic dyes (dark blue, reaction green, reaction blue, bright red, and reactive purple) Pseudomonas aeruginosa monodisperse-colored SiNPs as agglutination test vehicles were prepared using reverse microemulsion; the SiNPs were sensitized with eleven types of monospecific antibodies against Pseudomonas aeruginosa (IgG-colored SiNPs); the agglutination test was able to identify different serotypes of P. aeruginosa  pathogenic agent Escherichia coli O157:H7 in beef samples with the help of a spectrofluorometer. Chitra and Annadurai 26 used a similar immunoassay for detecting Escherichia coli using antibodies conjugated with SiNP functionalized with carboxyl groups and fluorescent dyes, the latter based on the reactive amino dyes dimethylformamide and 3-amino propyltriethoxysilane ( Figure 5). The results showed nanoparticle emission at 536 nm and the presence of nanoparticle-bacteria aggregates, suggesting that several antibodies could bind to the cell surface. This method provides a fast, accurate, and straightforward way for the detection of pathogens using fluorescence-based immunoassays.

Biosensors with magnetic nanoparticles
Magnetic nanoparticles (MNPs) are biocompatible structures with adjustable sizes, making them ideal for a wide range of biomedical or biotechnological applications, especially for magnetic resonance imaging, magnetic labeling of secreted bioactive molecules, nanosorbents in environmental decontamination technologies, and biosensors (Table 8). 109 Among the most innovative methods for detecting pathogens are those based on magnetic nanoparticles coupled to biorecognition elements such as antibodies, proteins, viruses, bacteria, or nucleic acids. This type of nanoparticles has been used to detect immobilized targets by applying a magnetic field to the magnetic nanoparticles or using conductometry, due to its excellent conductive properties, as part of the biosensor. 2,10 As an example, a methodology that allows the identification and quantification of DNA and messenger ribonucleic acid (mRNA) employs iron oxide with various ligands such as peptides, antibodies, and nucleic acids on the nanoparticle surface. 2 Also, nanoparticles that use magnetism can allow the isolation of the analyte of interest. This methodology is known as immunocapture of pathogenic bacteria, a variant of the conventional enzyme-linked immunoassay (ELISA) technique, with capture efficiencies of up to 97%.
Joo et al. 77 reported a straightforward and sensitive analytical method for detecting bacteria such as Salmonella in milk. After forming an interaction between the bacteria and antibody-conjugated magnetic nanoparticle, the bacteria were separated from the sample by an external magnetic field. Subsequently, the MNP-Salmonella complexes were dispersed in a buffer solution and exposed  reaching limits of detection in milk as low as 100 colonyforming unit (CFU) mL -1 ( Figure 6). Finally, it should be noted that one of the major disadvantages of MNPs is their water stability, which has been the subject of recent efforts, especially through   Figure 6. Nanobiosensor using magnetic nanoparticles (MNPs) and titanium oxide nanocrystals (NTs) attached to antibodies to detect Salmonella sp. in milk. First, an immunomagnetic separation is carried out to obtain the MNPs bound to the bacteria. Titanium oxide nanocrystals (NTs) are then added, and a second immunoseparation is carried out to obtain MNPs-NTs-Salmonella complexes and free NTs with differentiated spectra (adapted from Joo et al. 77 ).
functionalization strategies, such as the binding to dextran and polyacrylic acid. 90

Biosensors with quantum dots (QDs)
QDs are inorganic fluorophores that have replaced, to some extent, conventional fluorescent dyes, exhibiting unique light emission properties according to their size and composition. Another characteristic of QDs is their narrow emission spectrum that differs according to the size and type of material, allowing the emission of sharp colors with high resolution and improving assays' sensitivity. 11,29,40 One of the most critical advantages of QDs, in addition to the fact that they show minimal interference with natural autofluorescent particles, 75,113 is their ability to simultaneously detect several biomarkers, due to the phenomenon known as synchronous emission, which allows different QDs to show excitation at the same wavelength. 10,75 They also have disadvantages, such as unstable fluorescence generation, cytotoxicity, 101 and their "lipid-like" surface, which restricts the compatibility of QDs in aqueous environments and biological fluids. It is both the advantages and disadvantages that allow QDs to be used in cell and virus marking, immunofluorescence assays, and flow cytometry. The latter application has great potential since they replace traditionally used fluorescent organic dyes (Table 9), 11 making screening tests more efficient by reducing analysis time.
Additionally, QDs have been reported to detect biomarkers such as antigens and pathogens, 12 to act as probes to detect genomic DNA, and to be employed for fluorescent Amino-modified aptamers Salmonella typhimurium the fluorescence detection of Salmonella typhimurium followed the incubation time and the addition of CD-aptamers; a linear relationship between the concentration of Salmonella typhimurium and the intensity of fluorescence was achieved 50 CFU mL -1 ND 117 Antibody

Staphylococcus aureus
QDs coated with streptavidin and conjugated with biotin-labeled anti-S. aureus antibodies were developed as fluorescence markers; the fluorescence intensity of the bead-cell-QD complexes in the presence of S. aureus at 620 nm allows detection 10 3 CFU mL -1 2 79 in situ hybridization assays (FISH). 10 Recognition of pathogens with this type of nanosensors is based on the functionalization of the nanoparticles with specific antibodies, which upon contact with the pathogen, and due to the affinity given by the functionalization, create an antigen-antibody species that emits a fluorescence signal. 34,58 Zhu et al. 89 studied two different bioconjugates QDs as biosensors, using biotinylated antibodies generating a dualcolor image for two protozoa: Cryptosporidium parvum and Giardia lamblia. Tully et al. 29 developed a rapid immunoassay based on QDs fluorescence for the detection of two surface proteins of Listeria monocytogenes, poly(amino acids) involved in the invasion of human cells, called internalin A and internalin B. Another strategy reported to recognize pathogens, specifically Escherichia coli, uses QDs coated with mannose, nanostructures that identify the mannose-specific lectin FimH from the surface of Escherichia coli, allowing the detection of bacteria in cell suspensions with less than 10 4 cells mL -1 . 58 There is also the work with magnetic QDs conjugated with anti-Salmonella antibodies by Yang et al. 113 where the intensity of the fluorescence emitted by the QDs is used for the detection of the pathogen (Figure 7).

Biosensors with carbon nanotubes
Several zero-dimensional (0D) to three-dimensional (3D) carbon nanomaterials have been developed as electrochemical biosensors, showing that the analysis of bacterial pathogens in food through carbon nanotubes (CNTs), graphene, and their derivatives, improves the detection sensitivity, mainly when electrochemistry is used as an analysis method. For example, carbon nanotubes with a large surface-to-volume ratio make it possible to acquire and distinguish electrical signals before and after biological elements hybridization. 68  Carbon nanotubes are graphene tubules made up of monoatomic layers of hexagonal bonded carbon atoms wound on their axis. They are found as simple nanotubes (rolled graphene sheets) or multiple nanotubes (several nested concentric sheets) that absorb infrared light, giving them properties such as biorecognition-signal amplifiers and transducers. 33 Due to their attractive electronic, optical, and thermal properties, they can be used in nanomedicine (Table 10).
It is known that CNTs can be bio-persistent, maintaining their physicochemical characteristics unaltered in the environment without degrading and having the capacity to generate adverse effects in the exposed organisms. For that reason, strategies have been developed that improve their biocompatibility through surface modification with proteins, antibodies, and nucleic acids. On the other hand, it has recently been reported that these structures may be susceptible to biodegradation,  which increases the possibility of application in living systems. 10,33 DNA probes have been shown to degrade when exposed to cellular enzymes or nucleases present in samples during the development of molecular techniques. However, the use of CNTs protects these biomolecules since the interaction between single-stranded DNA nucleotides and simple nanotubes allows the formation of a stable complex, making its degradation more difficult. 10  when compared to the same hybridized probe, but without nanotubes. These results have important implications in molecular techniques employed in food samples where DNA probes are easily degraded by cellular enzymes or nucleases present in food. 38 In another contribution to this area, Karimi et al. 121 reported in 2019 an aptamer immobilized on multi-walled carbon nanotube (MWCNTs) to detect Bacillus anthracis (Figure 8). Upon the adsorption of the labeled aptamer on MWCNTs, fluorescence emission was quenched. In contrast, by adding the recombinant protective antigen domain 4 (rPAD4) to the hybridization reaction and Table 10. Carbon nanotubes used for pathogenic agent detection (cont.) incubation for 10 min, the fluorescence emission was significantly recovered to 85% as compared to the control.

Biosensors with metal-organic frameworks (MOF)
In recent years, MOF applications, especially nanosized (NMOF), have generated significant interest in biomedicine. Unlike traditional MOFs, NMOFs exhibit highly ordered porosity and inherent pore size but with higher surface areas, which gives them improved biological activity, chemical/colloidal stability, and surface modification facilities. When compared to other nanomaterials, NMOFs demonstrate the following advantages for biomedical applications: (i) The chemical diversity of MOFs provides a library of versatile porous materials, making them highly available for various applications related to biology.
(ii) The balance between stability and degradability allows reuse and degradation as needed.
(iii) Controllable porosity and surface area allow efficient encapsulation/loading of both small molecules and biomacromolecules, providing dispersibility, biocompatibility and biosafety for in vivo applications. 122 More recently, works such as that of Bhardwaj et al. 67 using the metallorganic fluorescence framework IRMOF-368 have shown that these MOFs can be used in various biological applications ( Figure 9) such as molecular detection, recognition of adenosine triphosphate (ATP) and cancer biomarkers, and in various types of samples, including liquids and gases. 67,123 The MOF applicability is extensive due to its fascinating framework architecture properties. Several studies have shown that MOFs' chemical nature allows unlimited structural variations during and post-synthesis, which can control the stability of the network, porosity, internal and external surface area, thermal and chemical stability, selectivity, crystal structure, toxicity, and luminescent properties, among others (Table 11). 48 Figure 8. A labeled fluorescent ssDNA aptamer binds to a multi-walled carbon nanotube, which causes its deactivation. When this complex interacts with the rPAd 4 protein, which is a Bacillus anthracis antigen, fluorescence is restored, evidencing the presence of the bacteria (adapted from Karimi et al. 121 ).

Figure 9.
A bacteriophage specific to S. arlettae bioconjugated with a fluorescence metal-organic framework (IRMOF-3) changes its photoluminescence intensity with different bacterial densities (adapted from Bhardwaj et al. 67 ).

Discussion
Nanobiosensors are new devices that improve traditional biosensors' effectiveness due to the use of nanomaterials in their manufacturing. The development of these devices has allowed the detection of pathogens or some of their components in a fast, efficient, and highly sensitive way in different samples. This has practical implications in the clinical field, food industry, and the environmental area. By implementing this type of technology, up to 1 cell mL -1 of the analyzed sample can be detected. 25 This technology has been widely used to detect pathogens, thanks to the use of some recognition elements present on their surface that function as markers, or the presence of genetic material that can hybridize specifically with designed probes anchored on the nanomaterial. In the development of these devices, aptamers have also been used as new functionalizing molecules. Based on nucleic acids, these have shown an affinity for some structural characteristics of the pathogen in question, facilitating their detection.
Similarly, the technology has also been used to detect toxins or other secreted substances responsible for infectious diseases through colorimetric tests using various types of nanoparticles, which decreases the cost of conventional tests to detect such substances and lowers the limit of detection. Due to the development of these devices, the processing time, the equipment required, and the trained personnel to perform tests for pathogens that compromise public health are reduced.
The most significant number of detection studies are related to Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium. However, the most used microorganism in screening tests has been E. coli, which is a bacterium of great importance because it is considered an indicator of fecal contamination in waters, and it is recognized as a cause of diseases such as gastroenteritis, urinary tract infections, and meningitis.
On the other hand, QDs have been primarily used to detect different types of pathogens, such as Cryptosporidium parvum and Giardia lamblia, two species of protozoa present in water samples and which are also recognized as indicators of fecal contamination. 89 This type of detection test could expand this technology area since it could be applied not only to bacteria but to other organisms of environmental concern. It should also be noted that it would be interesting to verify these detection devices' effectiveness in other areas such as agriculture, where very little has been done.
Each type of nanobiosensors has advantages and disadvantages. It is not easy to define which type of nanobiosensor is more useful for each specific case since there are a variety of devices with multiple principles of operation, those based on nucleic acids, those based on the detection of specific proteins, or those that detect the organism of interest as such. All of the above makes a possible comparison between them more complicated.
To define the effectiveness of this type of device, criteria such as type of bioreceptor, transduction system used, limit and time of detection, and ease of scaling must be considered. It can be seen after carrying out the review that most of the biological and non-biological interactions used as a basis for the design of nanobiosensors are based on antigen-antibody binding, electrostatic interactions, and streptavidin-biotin interaction. Nanobiosensors also use the affinity that specific proteins, such as concanavilin A and lectins, show for carbohydrates present in pathogens' cell walls, which can be used as target molecules. As for the most widely used transduction systems for nanobiosensors, the electrical and optical types stand out.
The limit of detection must also be taken into account to define the device's effectiveness. These data can usually be reported in different units depending on the type of nanobiosensor, though, in general, it is evident by this review that the best limit of detection for a nanobiosensor based on silica nanoparticles is 1 cell mL -1 of sample. 25 However, several nanobiosensors based on metallic nanoparticles, specifically on AuNPs, also show outstanding limit of detection, detecting 10 bacteria mL -1 of sample. This indicates that efforts should be directed to improve the design of devices based on metallic nanoparticles, not only because of the limit of detection they present but also because they require fewer chemical precursors for their elaboration, making them the simplest to build and the less expensive.
The above also shows the currently growing interest in developing green chemistry methodologies, where live species are used, including bacteria, fungi, or plants, to obtain nanoparticles. These approaches could replace traditional nanoparticles synthesized using toxic chemical precursors, and then, we would have nanobiosensors composed of nanoparticles obtained through biosynthesis, making the technology more feasible for commercial use, specifically in the food industry.
Additionally, it is essential to highlight a novel approach within this technology, the design of hybrid nanobiosensors, where different types of nanomaterials are used to make the detection process more efficient. In this field, the use of magnetic nanoparticles fused with titanium dioxide nanocrystals or quantum dots to detect various types of bacterial and fungal pathogens appears to be the most promising technology.

Conclusion
The application of nanotechnology to detect pathogenic microorganisms has led to significant advances, reaching limit of detection of up to 1 cell mL -1 of sample, requiring only minutes to perform the detection, and using techniques as simple as UV spectroscopy. Furthermore, nanotechnology's advantages predict that we will continue to reduce limit of detection, analysis time, and instrumental requirements. This makes it possible to foresee that we are very close to the commercialization of portable nanosensors for on-site application in agroindustry, food, and human health. However, the specificity shown by the currently reported works and the increasingly recurrent appearance of pathogens in humans and animals possibly show us the need to develop nanostructured systems with the ability to detect multiple pathogenic microorganisms at the same time, an area until now unexplored.