Biosensors for the Detection of Bacterial and Viral Clinical Pathogens and COVID-19 Diagnosis

Biosensors are measurement devices that can sense several biomolecules, and are widely used for the detection of relevant clinical pathogens such as bacteria and viruses, showing outstanding results. Because of the latent existing risk of facing another pandemic like the one we are living due to COVID-19, researchers are constantly looking forward to developing new technologies for diagnosis and treatment of infections caused by different bacteria and viruses. Regarding that, nanotechnology has improved biosensors design and performance through the development of materials and nanoparticles that enhance their affinity, selectivity, and efficacy in detecting these pathogens, such as employing nanoparticles, graphene quantum dots, and electrospun nanofibers. Therefore, this work aims to present a comprehensive review that exposes how biosensors work in terms of bacterial and viral detection, and the nanotechnological features that are contributing to achieving a faster yet still efficient COVID-19 diagnosis at the point-of-care.

The second element is the transductor or detector, which works by sensing a signal related to a physicochemical change caused by the interaction between the bioreceptor and the analyte. It transforms the signal into another one that can be evaluated and quantified [57][58][59][60][61]. The last part of a biosensor is the reader device. It usually involves a display that depends on software and hardware to generate the results [62].
Some important attributes define the performance of a biosensor. In the first place, selectivity is the capacity of a bioreceptor to detect a specific bio-entity when analyzing a sample composed of other components. This is probably the main feature and determines the needed bioreceptor. Second, reproducibility is the ability to produce the same response for a certain experimental set-up that is performed multiple times. Reproducible signals provide high reliability and robustness. Third, stability is the capacity to endure ambient disturbances around the system that can affect the precision and accuracy of the device. Fourth, sensitivity also known as the limit of detection (LOD) is the minimum amount of the analyte that can be detected by a biosensor. For clinical applications, it is required to detect the analyte in samples of low concentrations (ng/ml or fg/ml). Finally, linearity examines how accurate are the measurements within the analyte range of concentrations (i.e. linear range), and in response to the smallest variation in terms of concentration that can cause a change in the output (i.e. resolution) [63]. microorganisms [69]. Regarding viral analysis, Ahmadi et al. evaluated single virus detection through an optical biosensor, where viral particles attached to a microsphere optical resonator's surface caused a shift of resonance to longer wavelengths [70].
The second type, electrochemical biosensor, has been extensively applied to pathogen detection. These devices sense the analyte through electrodes by measuring electrical signals resulting from catalytic reactions or specific unions. The previous is derived from the capture of electrons as a result of redox reactions between the analyte and the bio-element [71]. In addition to that, the analysis of the desired element is determined by different readouts like potentiometry, amperometry, and conductometry [72]. This type of biosensor has been subjected to improvements due to bio-and nanomaterials development [72,73]. Finally, yet importantly, there are piezoelectric biosensors. Piezoelectricity refers to the ability of a material to generate a voltage under mechanical stress [76]. These biosensors possess crystals that vibrate under the influence of an electric field. Besides, certain materials vibrate at characteristic resonant frequencies in response to interaction with other molecules. The relationship between the resonant frequency changes and the mass from the molecules adsorbed or desorbed from the crystal's surface is conceived as the working principle of transduction in this type of biosensor. Therefore, vibration provides information on the phenomenon that is being measured [77,78]. Fu  Moreover, nano-biosensor materials' high surface area is considered a major advantage compared to conventional devices and plays an important role in the sensitivity, and fast response of the devices [94,95]. Therefore, these are conceived as excellent tools used for the detection, function, and interaction of proteins and nucleic acids, which improve the quality and performance of diagnosis for bacterial and viral diseases [96]. The following sections present an overview of some promising nanotechnological features in biosensors.

NPs
NPs are a wide range of materials with dimensions below 100 nm that have been used in various areas such as medical, pharmaceutical, manufacturing and materials, environmental, electronics, and mechanical industries due to their multiple properties [97][98][99][100]. Among the mostly employed are metal NPs such as AuNPs and silver NPs (AgNPs), which can be produced in different sizes and shapes (e.g., nanospheres, nanocylinders, nanowires, and nanocages). These NPs exhibit low toxicity, as well as multiple interesting chemical, biological, and physical properties, such as photo-thermal, optical, electrochemical, and biocompatibility based on their inert nature in biological fluids [101][102][103]. Additionally, these NPs can be synthesized with ease fulfilling relevant roles for diagnostic probes, and functionalized due to the presence of functional groups for achieving ligand-binding functions with a wide range of molecules, such as antibodies or genetic material [104,105]. virus H1N1. However, due to its versatility, it was also possible to detect clinically isolated influenza virus H3N2 and norovirus-like particles [108].

GQDs
GQDs are among the most fascinating carbon-based nanomaterials employed for the development of biosensors, mostly electrochemical. These materials present outstanding properties such as signal amplifying characteristics, biocompatibility, tunable size, electro-catalytic performance, and capacity to detect multiple biomolecules. Additionally, their inertness,  [111]. Furthermore, GQDs integration into a biosensor can improve its performance in terms of reproducibility, selectivity, and sensitivity [112].

Electrospun nanofibers
Electrospinning is a nanotechnological method in which an electrostatic field force applied to a polymer solution causes a charged liquid jet to moves downfield towards an oppositely charged collector, where the fine fibers are deposited [113]. Electrospun nanofibers have been the target of different applications like drug delivery systems or scaffolds for skin tissue engineering due to their structure and physicochemical properties such as a large surface area to volume ratio, small particle size, and high porosity, among others [113][114][115][116]. However, a novel application is their use for developing nano-biosensors focused on detecting viral and bacterial pathogens [117][118][119][120].
Nano-biosensors development using these nanostructures can be achieved by two approaches. On one hand, functional polymers are electrospun to obtain a nanofiber that is used directly as an inducing element of the corresponding biosensor, which will present fast response time, high sensitivity, and good biocompatibility. On the other hand, electrospun nanofibers are used as templates to which a sensitive material is deposited on their surface, and later the system is subjected to chemical modification in order to produce a composite film on an electrode, with nanostructures that have the intended sensing characteristics [121,122].
Although the manufacturing process is simpler, keeping bio-receptor functionality is considered a great challenge for the production of this type of device. The sensing element can be immobilized through different strategies according to its physicochemical characteristics, as well as the ones from the nanofiber scaffolds, and also, based on their interfacial interactions [123]. separation, and conductometric immunoassay using a silver electrode. Nanofiber antibody's surface functionalization and sensor assembly process allowed retaining the unique fiber morphology, and displaying a linear response to both pathogens with a detection time of 8 minutes [129]. Therefore, nanofiber-based biosensors present advantages over the conventional ones such as polymer diversity for its manufacture, high specific surface area with high responsiveness, as well as an outstanding sensibility [132][133][134].

Bacterial and viral pathogens detected through biosensors and nano-biosensors
Conventional clinical analyses including an antibody or nucleic acid-based, biochemical, and enzymatic methods, are very reliable but take a long time to obtain a result. Health disciplines demand the acquisition of faster outcomes to speed up the appropriate treatment [135,136]. In this sense, biosensors and nano-biosensors are useful tools that offer an accurate diagnosis in shorter times due to their ability to provide real-time and faster clinical results [137]. Currently, there is an increasing interest in their use to detect pathogens in the human body (Table 1) [136]. . Advantages such as small samples and low energy required to avoid complications in terms of transportation and processing, make them suitable for easy and fast use in the identification of bacterial and viral pathogens [137]. Needless to say, nanomaterials advances have benefited biosensor performance to achieve the task [155].

Bacterial pathogen detection
Focusing on the human body, bacterial infections caused mainly by gram-negative microorganisms represent a particular challenge in human health worldwide because of multidrug resistance variants, greatly influenced by their indiscriminate exposure to antibiotics discharged in water, addition to food or more commonly, due to improper use of these drugs from patients [156].
Since the previously mentioned is considered a major current health concern, different kinds of nanomaterials and biorecognition elements have been employed to develop biosensors for antibiotic detection, as well for bacteria [157]. tool is based on the proteolytic activity of the pathogen proteases on a specific peptide substrate placed in the middle of two magnetic nanobeads. In this case, the dissociation of magnetic nanobeads-peptide moieties results in color change [161]. In another approach, Ahari et al.
constructed a potentiometric nano-biosensor able to detect the bacteria through the identification of an exotoxin emitted by the microorganism. Particularly, the method is often used for contaminated food, but it can also be applied for clinical detection [162].
Another important bacteria, V. cholerae, is a gram-negative facultative anaerobe that causes Cholera disease. People would infect by consuming contaminated liquids or food, providing an ideal platform for the disease, which also spreads quickly due to its secretory nature. Therefore, its diagnosis plays an important role in the disease assessment because of its mortal rate rounds between 50-60% [163]. suggests considering this method as an alternative for Shigella detection [165].
The different approaches for biosensoring detection of pathogenic bacteria have been successful and are currently being considered by many health governments and research institutions, mainly because of their fast response, high-quality performance, and reliable results [167][168][169].

Viral pathogen detection
Viral pathogen diagnosis is important for early and effective treatment in patients in order to prevent outbreaks or pandemics. For that reason, biosensors are being widely employed for making diagnosis easier, avoiding hard proteins or DNA identification techniques in specific virus [170,171]. One of the most common and dangerous viral pathogens is the influenza virus because of its ability to spread easily and constantly mutation. Hence, detection at early stages can be difficult [172,173].

Hassanpour et al. developed a novel optical biosensor composed of pDNA bioconjugated citrate capped AgNPs towards target sequences for ultrasensitive and selective Haemophilus influenza
detection in human biofluids [174]. This pathogen has also been detected through other different biosensors, including the work reported by Jiang et al [148,[174][175][176]. This paper describes the development of a polydiacetylene sensitive biosensor using antibody detection for H5N1 (avian influenza), in which the polydiacetylenes vesicles show a dramatic change in color from blue to red upon the detection of the virus [176].
Other dangerous viruses that affect the population worldwide include ebolavirus, HIV, and Hantavirus [177][178][179]. The first one is a negative strand-RNA virus that belongs to the Filoviridae family and causes a deadly disease called Ebola. The infected people with this agent develop a series of symptoms, where hemorrhagic fever is considered as fatal [180][181][182]. Currently, there is no vaccine or specific treatment [183]. However, different studies have presented the development of biosensors for detecting this pathogen [184]. Ilkhani et al. fabricated a novel electrochemical-based-DNA biosensor through enzyme-amplified detection to improve the sensitivity and selectivity of the device for the pathogen [185]. In addition to that, Baca et al.
developed a biosensor that can detect the virus within 10 minutes at the POC by using surface acoustic waves, showing potential to detect it before symptoms onset [186].
On the other hand, HIV is a retrovirus that attacks a patient's immune system, causing an inability to resist many diseases, and culminating in death when the person is not under drug control. Clinical treatments for HIV are crucial for reducing mortality, but early diagnosis saves many lives as well and can decrease spread rates [187][188][189]. Shafiee et al. worked on a photonic crystal biosensor to detect multiple HIV-1 subtypes (A, B, and D) upon binding of the biological analyte with the biosensor [190]. In addition to that, Gong  polyaniline/graphene (PAN/GN) using reverse-phase polymerization for the development of an electrical DNA-biosensor that showed great selectivity, and sensitivity for the detection of HIV-1 gene fragment [191].
Hantavirus is a cluster of viruses that are part of the Bunyaviridae family. The spread begins through contact with liquids, food, or particles contaminated with rodent excreta. It causes hemorrhagic fever, respiratory insufficiency, and heart failure within 2-7 days after getting infected [192,193]. Regarding its detection, Gogola et al. have performed important research for the development of biosensors [194,195]. In a first approach, they prepared an electrochemical immunosensor based on chemical modification of the gold surface with the virus antigen/protein [194]. In a second study, the research group designed a quick electrochemical biosensor based on biochar (BC) as a carbonaceous platform for immunoassay applications due to its highly functionalized surface for covalent binding with biomolecules [195]. Both studies developed devices as promising and suitable tools for hantavirus clinical detection [194,195].
Furthermore, several bio-elements can be incorporated into a biosensor for virus detection including markers, RNA, structural proteins, and enzymes from the viral pathogens [196].

COVID-19 pandemic
Currently, many viruses are being considered to have the capacity of causing future pandemics. Different factors such as fast dissemination, a high transmission rate of new variants, difficulties to develop efficient and sensible diagnostic techniques, as well as the lack of specific vaccines and safe drugs for treatment, make them one of the major threats for mankind [197,198].
The most recent case is the COVID-19 announced as pandemic on March 13 th , which is an infectious disease with rapid human-to-human transmission caused by SARS-CoV-2. This pathogen belongs to the positive-strand RNA viruses [199,200].
Like any other viral outbreak, an early diagnosis is fundamental for preventing an uncontrollable spread of the disease. However, this pandemic has the particularity that more than 30% of the confirmed cases are asymptomatic, thus making it harder to control [200][201][202]. RT-PCR is the most used suitable and reliable method for detecting SARS-CoV-2 infections until now.
Nevertheless, the technique is time-consuming, labor-intensive, and unavailable in remote settings [203,204]. Although several other methods can be employed for that purpose, such as immunological assays, thoracic imaging, portable X-rays, or amplification techniques, the pandemic spread of COVID-19 demands to develop POC devices for rapid detection (Figure 3) [205][206][207][208]. The industrial sector has developed some suitable POC biosensors for the qualitative detection of SARS-CoV-2 IgM and IgG antibodies using samples as low as 10 µl of human serum, whole blood, or finger prick, obtaining results within 10-15 minutes (Table 2) [214]. Many of these rapid serological tests are paper-based biosensors that perform a colorimetric lateral flow immunoassay. In this method, SARS-CoV-2 specific antigens are typically labeled with gold, and bind the corresponding host antibodies, which migrate across an adhesive pad. As can be seen in (C) will be shown [215]. Although the use of serological tests to detect SARS-CoV-2 is still under debate, these are foreseeing as crucial tools for the implementation or ceasing of lockdowns established worldwide [216].  MDPI [215].
Other research groups have developed Lab-on-a-Chip-based biosensors for SARS-CoV-2 detection [208,217]. This technology avoids the need for specialized personnel through the integration of microfluidic components into a biosensor, allowing increasing their production, and reducing the costs of the assay [218]. POC commercialized instruments based on this microfluidic technology are having an important role in this pandemic, like ID NOW ® , Filmarray ® , GeneXpert ® , and RTisochip ® [219].
Cell-based biosensors have also contributed to COVID-19 diagnosis. Mavrikou et al.
developed a biosensor based on membrane-engineered mammalian cells that possess the human chimeric spike S1 antibody. The device can detect SARS-CoV-2 S1 spike protein selectively, where the binding of the protein to the membrane-bound antibodies results in cellular bioelectric properties modification measured by Bioelectric Recognition Assay. The LOD is 1 fg/ml and the response time is about three minutes. In addition to that, the biosensor includes a portable read-out device that can be operated by a smartphone [220].
Moreover, nano-biosensors have shown an outstanding potential to contribute to the fight against COVID-19, providing holistic insights for developing ultrasensitive, cost-effective, and rapid detection devices for mass production [221]. Advanced materials are the basis of nano-enabled or integrated micro-and nano biosensing system technologies that can detect earlier the virus, and even show good binding properties allowing them to inactive or destroy the pathogen upon the application of an external stimulus [222].
Different research groups have developed carbon-based and graphene-based POC biosensors [208,217]. Graphene is foreseeing to have a leading role in the attempt of fighting against COVID-19. This low-cost material can be employed for virus detection since its sensitivity and selectivity can be enhanced by modifying its hybrid structure (e.g., antibody-conjugated graphene sheets) that allows tuning its optical and electrical features. Some graphene-based sensors that can be explored for SARS-CoV-2 detection are photoluminescence, colorimetric, and SPR biosensors [223,224]. Seo et al. employed the material for the development of a field-effect transistor (FET)-based biosensor for detecting SARS-CoV-2 ( Figure 5). In this case, graphene sheets from the FET were coated with a specific antibody against the virus spike protein, which was successfully detected at concentrations of 1 fg/ml in a phosphate-buffered saline medium. In addition, the device was able to detect the virus in clinical samples, exhibiting a LOD of 2.42 × 10 2 copies/ml. The fabricated biosensor is considered as a promising immunological diagnostic alternative for the disease [225].

Conclusions
Last few decades, viral and bacterial pathogens have become a real menace to human safety.
Their rapid identification must be considered as a priority task in order to prevent an outbreak that represents a high risk of disruption of the healthcare system, and a disastrous socio-economic Funding: This research received no external funding.

Conflicts of Interest:
The authors declare no conflict of interest.