The fundamental principle of an electrochemical biosensor is to convert biological events, such as enzyme-substrate interactions and antigen-antibody interactions, into electrical signals, such as current, voltage, or impedance (Cho et al., 2020[26]). To obtain valuable information regarding flaviviral infections (such as DENV, ZIKV, and JEV), various electrochemical detection methods have been developed and utilized. These methods include potentiometry (measuring the potential of an indicator electrode), conductometry (measuring conductivity or resistance) and amperometry/voltammetry (measuring current as a function of imposed potential) (Khristunova et al., 2020[76]). The combination of amino-reduced graphene oxide (NH₂-rGO) and β-cyclodextrin (-CD) was utilized to modify the surface of a glassy carbon electrode (GCE) to detect the HIV gene using differential pulse voltammetry (DPV). The electrochemical biosensor exhibited excellent sensitivity and selectivity, achieving a limit of detection (LOD) of 8.7 fM. The electrochemical biosensor was developed by Li (2020[89]) for detecting the HIV segment in human serum samples, demonstrating successful detection of the target using electrochemical methods (Li, 2020[89]). To detect the Listeria hlyA gene, an impedimetric biosensor was created. It worked by immobilizing a DNA-detecting probe on a poly-5-carboxy indole (5C Pin) polymer. When it came to target DNA concentration, this label-free biosensor showed a detectable linear range of 1 × 10^-4 to 1 × 10^-12 M (moles per liter). After being covalently immobilized on the 5C Pin polymer, the target DNA sequence underwent further hybridization with the probe. Charge transfer resistance, expressed in ohms (Ω), was used to assess the biosensor's performance and provide information on changes in electrical impedance at the electrode interface. With potential uses in clinical diagnostics and food safety, this novel method shows promise for the extremely sensitive and specific identification of Listeria hlyA gene sequences (Kashish et al., 2015[72]). Electrochemical biosensors provide quick, precise, and sensitive results at an affordable cost. Nanomaterials and nanocomposites enhance sensitivity. Microfluidic systems integrate with biosensors for miniaturized platforms. Challenges include stability, repeatability, low LOD values, and sample sensitivity (Singh et al., 2021[138]).

Optical biosensors, including colorimetric, fluorescence, SPR, optic fibers, and biological luminescence, are compact diagnostic devices used for pathogen detection, generating electrical signals (Damborský et al., 2016[32]; Eksin and Erdem, 2023[46]). These provide an alternative technique for viral detection, offering reliability, user-friendliness, and cost-effectiveness. They also reduce the reliance on nucleic acid amplification. These biosensors have been used to identify pathogens such as HIV, Ebola, norovirus, and influenza virus, utilizing techniques like fluorescence, surface plasmons, and colorimetry. Nano-biosensors enable targeted and single-virus scanning for infection detection (Maddali et al., 2020[99]). Colorimetric biosensors utilize ligand-target interactions that result in a visible color change, enabling simple and portable optical quantification (Piriya VS et al., 2017[120]). To quantify fluorescence, a fluorophore substance must be activated at one wavelength to produce light emission at another wavelength. Fluorescent dye-labeled reporter molecules provide for sensitive analyte detection (Li et al., 2013[87]). A fluorescent biosensor for HBV DNA sequence recognition based on gold nanorods (AuNRs) was created by Lu et al. (2013[96]). By mixing fluorescein-tagged single-stranded DNA (FAM-ssDNA) with the AuNRs solution, they produced a ternary complex known as FAM-ssDNA-CTABAuNRs. According to Lu et al. (2013[96]), this led to fluorescence resonance energy transfer (FRET) from FAM to AuNRs, which decreased the fluorescence intensity of FAM. One effective method for identifying molecular species at the single-molecule level is surface-enhanced Raman scattering (SERS). The sensitivity and chemical specificity of current optical detection techniques can be substantially exceeded by attaching molecules to nanostructured substrates, hence increasing the Raman signal (Lin et al., 2023[91]). An essential aspect of surface-enhanced Raman scattering (SERS) is surface plasmon resonance (SPR), which takes place at the interface between a metal and a dielectric. SPR occurs when longitudinal electron waves, induced by light that is polarized parallel to the surface, cause a concentration of oscillating electrons (Lazcka et al., 2007[83]). Liu and Huang (2012[94]) used DNA-silver nanoparticle (AgNP) conjugates in a range of 0.30 to 2.0 nmol/L to quantitatively detect HIV DNA using a sandwich approach based on SPR (Liu and Huang, 2012[94]). To detect changes in mass on the probe/transducer surface, piezoelectric sensors that employ quartz crystal microbalances (QCM) measure resonance frequency fluctuations (Lazcka et al., 2007[83]).

Aptamer-based biosensors, also known as aptasensors, are biosensors that use aptamers for identification. They are versatile, can be easily changed, and have predictable secondary structures. They are effective in detecting blood infections and are known for their rapid detection sensitivity due to their integration with signal amplification techniques (Liu et al., 2021[93]). However, the absence of excellent aptamers for therapeutically significant targets is one of the field's current constraints. To enhance its accuracy and dependability, the aptamer technology must also undergo comprehensive testing in a clinical sample matrix (Zhou et al., 2014[175]). A simple method was devised to construct aptamer-based fluorescence biosensors for the quantitative measurement of Hepatitis B antigen (HBeAg). The molecular detection element in this technique is an HBeAg aptamer that has been fluorescence-labeled, and a tiny DNA molecule that is similar to the aptamer is used as a competitor. It is reported that this approach yields a limit of detection (LOD) for HBeAg of 609 nanograms per milliliter (ng/ml). Fluorescence tests were performed on blood serums that were positive and negative for HBeAg after this biosensor was established, and statistical significance was noted (p < 0.05). Notably, it just takes two minutes to finish the detection test in its whole. These recently identified aptamers have the potential to enhance the chronic hepatitis B diagnosis process (Huang et al., 2016[65]). Furthermore, a different aptamer-based biosensor was created with good specificity for the diagnosis of the Hepatitis C virus (HCV) and the identification of the virus core antigen in infected individuals. A random library of 60 RNAs, consisting of roughly 10¹⁵ sequences, was used to generate high-affinity aptamers using the systematic evolution of ligands by the exponential enrichment (SELEX) approach. Subsequent protein chip tests revealed that the chosen aptamers bound to the HCV core antigen selectively, but not to the other HCV antigen, NS5. The concentrations and the limit of detection are accurately represented in nanograms per milliliter (ng/ml), giving a clear picture of the sensitivity of the detection. These biosensors based on aptamers have a great deal of promise to improve HCV infection detection and treatment (Lee et al., 2007[85]).

Affinity solid-state-based biosensors, known as immunosensors, are utilized to monitor specific analytes, such as antigens (Ag), by establishing a stable interaction between the Ag and an antibody (Ab) that acts as the binding agent. This immune reaction generates a measurable signal through a transducer, as described by Gil Rosa et al. (2022[53]). Using highly specific antibody-antigen bond, a variety of biomolecules, including viruses, bacteria, protein markers, nucleic acids, and other tiny particles, are identified (Mahato et al., 2018[100]). According to one of the investigations, a gold film electrode from a recordable compact disc (CD-trade) was used to create an electrochemical immunosensor for the NS1 protein (Cavalcanti et al., 2012[19]). The immunosensor responded linearly to NS1 concentrations between 1 and 100 ng/ml, with a 0.33 ng/ml detection limit. With a detection limit of 200 fg/ml, a highly sensitive label-free immunosensor was created for the detection of HIV-1. It is a potential strategy for viral identification and evaluation of biological and medicinal samples (Lee et al., 2013[84]). Additionally, a completely novel amperometric immunosensor for the HIV-1 p24 antigen (p24Ag) detection was found. With a relatively low detection limit of 0.0064 ng/ml (S/N = 3), the predicted immunosensor demonstrated good electrochemical sensitivity to the presence of p24 in a concentration range of 0.01 to 60.00 ng/ml (Kheiri et al., 2011[75]). An ultrasensitive immunosensor for measuring hepatitis B surface antigen at a detection limit of 0.36 pg/ml exists, according to one of the assessments. Hepatitis B surface antigen was detectable by the immunosensor in the linear concentration range of 1.7 to 1920 pg/ml (Shourian et al., 2015[133]). Immunosensors have a significant deal of potential to develop into effective measuring devices because of their real-time estimation, constrained sample intake, relatively inexpensive price, and easy apparatus operation (Janik-Karpinska et al., 2022[69]).

Microfluidic biosensors are integrated chip devices that combine various activities, offering reduced reagent and specimen consumption, flexible liquid manipulation, and reduced detection times due to their integration with characterization techniques (Xing et al., 2022[160]). Many microfluidic LOC devices have been used in a variety of assays and biosensors, such as paper microfluidic assays, magnetic bead cell sorting assays, particle immunoassays, and microfluidic ELISAs for pathogen detection. These advancements have raised the levels of specificity, sensitivity, usability, and mobility (Fronczek, 2013[52]). There are many instances where microfluidic approaches have proven to detect pathogens well. According to Huang and Huang (2019[64]), SERS may be utilized to quickly identify E. coli strains that cause sepsis when combined with microfluidic microwell technology. Conventional detection methods don't require long culture durations since the bacteria in the microwell may be contained there, increasing the effective bacterium concentration by up to 10⁷ folds (Huang and Huang, 2019[64]). Micro- and nanotechnologies have permitted clear advances in HIV diagnosis, with the commercial availability of microfluidics-based CD4+ T cell counts utilizing a drop of blood from a finger prick (Damhorst et al., 2015[33]). In contrast to conventional methods, the employment of fluorescent microsphere-based lateral flow immunoassay strips (FM-LFIAs) for standard swine fever (CSFV) detection yielded a sensitivity of 5.28 ng/ml, covering a linear range of values from 9.77 to 625 ng/ml (Xie et al., 2020[159]). Ikeda et al. (2009[67]) showed how to use an on-chip flow cytometer and a disposable microfluidic device to detect Listeria monocytogenes (pathogenic bacteria) in milk (Ikeda et al., 2009[67]). Hsieh et al. devised a nanofluidic pre-concentrator and a nanoslit Fano resonance biosensor to identify latent membrane protein 1 (LMP1) for diagnosing Epstein-Barr virus (EBV). The cost-effective nano-slit plasmonic sensor chip can be produced on a large scale using nanoimprinting and aluminium deposition. The nonporous membrane was designed as an ion-selective channel integrated into the sensor chip to enhance the concentration of LMP1 proteins. Subsequently, a sensor chip employing anti-LMP1 IgG was developed to detect LMP1, achieving a limit of detection (LOD) of 100 pg/ml, while the sensing range spanned from 100 pg/ml to 10 g/ml. (Hsieh et al., 2022[63]). Studies show microfluidics offer accurate, convenient, and quick live pathogen detection techniques. However, they lack sensitivity and have limited capacity for high sample quantities. Obstacles like usability, cold storage, and electricity-free operations need to be addressed for low-resource applications (Spatola Rossi et al., 2023[143]).

Aptamers are molecules having properties in a fashion like antibodies with complex 3-D structures. These single-strand nucleic acids (DNA, RNA) work by forming an affinity bond with their target molecules. Due to their increased stability, aptamers, which are typically 22-100 nucleobases in length, are capable of denaturation and renaturation. The variable region edged by constant regions in the aptamers allows the amplification and identification of sequences (Devi et al., 2021[39]; Sharma and Shukla, 2014[132]). Aptamers have been employed for pathogen detection and concentration applications because of their excellent specificity and affinity in binding to their target, which is dependent on the main sequence which is described in Table 5(Tab. 5) (References in Table 5: Bachour Junior et al., 2021[10]; Figueroa-Miranda et al., 2020[49]; Mohsin et al., 2020[109]; Rahmati et al., 2021[125]; Tang et al., 2016[146]; Zhu et al., 2012[176]) (Nagarkatti et al., 2014[111]). The detection and recognition of targets rely on factors such as the length of nucleic acid molecules within aptamers, the primary sequence, and various environmental variables. In addition, the hydrogen bonds, the forces of van der Waals, hydrophobic contacts, and the combination of complementarity in form all contribute to the intermolecular interaction that occurs between the target and aptamers. As a result, the aptamers gain distinctive qualities such as in vitro creation, stable temperature, pH intervals, non-toxicity, and better immune response (Yue et al., 2021[167]).

An electronic nose (E-nose) is a device with chemical sensors that mimic human olfactory perceptions. It identifies volatile organic molecules with varying specificity and uses a signal-pre-processing unit and pattern recognition system. The gas sensor array converts molecular signals into electrical signals, generating unique odor patterns. E-nose offers rapid sensing and less bias, providing more consistent measurements between devices (Chen et al., 2013[22]; Ye et al., 2021[166]; Kumar et al., 2020[80]; Tan and Xu, 2020[145]). An illustration of the concept of utilizing an electronic nose to identify multiple categories of volatile organic compounds (VOCs) is present in the gas phase of blood samples. In this investigation, sera from 24 control cattle from para-TB non-suspect herds and sera from 43 dairy cattle with spontaneously occurring para-TB infections were obtained. Additionally, 24 sera from control cattle that were not infected and 26 sera from naturally brucellosis-infected animals were collected. At the population level, the e-nose could only discriminate sera from brucellosis and paratuberculosis-infected animals from healthy animals. The study's findings therefore demonstrate the potential of VOC analysis for the distinction of viral illnesses in animals, despite the limitations of the technology. Also, disease-specific volatiles must be found utilizing techniques like gas-chromatography. It is necessary to develop an e-nose approach so that VOC analysis can eventually be used as a cutting-edge diagnostic test (Pardo and Sberveglieri, 2010[114]).