Carbon nanomaterials are the main building blocks in nanotechnology, which have attracted much attention because of their large specific surface area, high mechanical strength, inherent structural defects, good electrical conductivity, and excellent chemical and thermal stability (Park et al., 2013). Carbon nanotubes (CNTs), graphene (Grp) and reduced graphene oxide (rGO) are the most commonly used carbon nanomaterials in electrochemical biosensors. It appears to be a fantastic material, with a tensile strength of a hundred times that of steel, more excellent thermal conductivity than diamond, and electrical conductivity equivalent to copper but with the ability to carry a large amoiunt of current. In this section, analytical applications of carbon nanomaterials such as CNT, graphene and reduced graphene oxide have been demonstrated for wearable electrochemical biosensors.

As electrochemical sensing materials, metal and metal-oxide based nanoparticles have attracted a lot of attention because of their small size, outstanding mechanical, electrical, chemical properties and high catalytic efficiency, as well as their versatility in creating new and better sensing systems (Bhide et al., 2019; Shaikh et al., 2019; Li et al., 2020). they can be categorized into noble and non-noble metal-based nanomaterials. Rh, Ir, Pt, Ru, Au, Os, and Ag are good examples of noble metal nanoparticles (Imamura et al., 2020). As it has been described earlier, metal-based nanomaterials have excellent and promising electro-catalytic properties, especially in wearable glucose non-enzymatic sensors. Like platinum Pt, palladium Pd, gold Au, metallic and oxides such as CuO, NiO, which can directly catalyse glucose (Abellán-Llobregat et al., 2017; Toi et al., 2019; Li and Wen, 2020), some metals based electrochemical biosensors are highlighted in Table 3.

Typically, glucose’s sensing mechanism consists of non-noble metal-based nanomaterials by the redox reaction of the hydroxyl (−OH) group (Archana et al., 2019). However, metal-based nanomaterials can be used as nanowires or nanosheets to achieve maximum sensitivity; that’s why some researchers use metal nanosheets and nanowires to achieve the maximum sensitivity instead of making film electrodes as shown in (Figures 4A,B) (Wen et al., 2014;Oh et al., 2018;Bae et al., 2019). In the last few years, gold has become a common active sensing material because of its excellent biocompatibility, and electrochemical property. Amanda et al. made a thin-film gold electrode-based glucose sensor (Imamura et al., 2020). The stretchable electrodes stretchability is almost 210% of its original length and accurately detect the glucose level without enzymes, which is one of the lowest documented for flexible, enzyme-free sensor (Figure 4C). Nowadays, metallic aerogels, a new category of 3-D metal-based nanomaterials anticipated by researchers, have recently become a new field of concern, offering enormous glucose bio-electrocatalysis and promise in wearable glucose sensors (Wen et al., 2016). Its porous form and gel state are ideal for immobilization of enzymes and maintaining their activity, which is highly beneficial in extending the life span of wearable glucose sensors (Zhang et al., 2012). Wen et al. developed a glucose sensor by assembling three-dimensional gold aerogels nanostructure modified glucose oxides. The sensor can detect glucose qualitatively and quantitatively in 0.1 mol/L phosphate buffer solution (pH 7.4) (Wen et al., 2016). In addition, compared with a single metal system, a reasonably designed multi-metal nanomaterial can make extensive use of the properties of two or more metal elements. thus enhancing the efficiency of the wearable glucose sensor, which will also become the metal sensor’s production path. Wang et al. and his colleagues recently created elastomer-bonded gold nanowire coating technology. Using the same technology, they create lactate-sensing working electrodes, reference electrodes, and counter electrodes for lactate monitoring in human sweat (Figure 4D).

In artificial sweat, the sensor has a high sensitivity of 14.6 μA/mM.cm². This sensor’s sensitivity is maintained even when subjected to high tensile strains of up to 100% without the use of any external structural layout (Wang et al., 2020). Their group has also suggested a gold fibre-based wearable electrochemical biosensor for sweat PH tracking. The manufactured fibre-based pH sensor shows superior sensitivity (60 mV/pH), high selectivity against cationic interference, and high stretchability (up to 100% strain). One benefit of fibre-based sensors is their ability to be incorporated in textiles, which can be integrate into daily garments to detect “unrecognizable” personal health (Wang et al., 2020). Recently, Francesca et al. designed a wearable multifunctional sweat sensing system based on platinum. The device is highly flexible and comprises four electrodes for continuous measurement of analytes such as Li⁺, Pb⁺², K⁺ and Na⁺ in different health applications and sports activities (Figure 4E). The sensors show linear responses in artificial sweat. Because of the good biocompatibility, flexibility and accurate sample handling this wearable framework is a significant step forward in the advancement of non-invasive tracking technologies for health, opening the way for better understanding of physiological parameters and clinical needs of individuals (Criscuolo et al., 2021). Metals containing oxides, such as indium oxide and lead oxide, are more sensitive and can be used to make sensing electrodes for wearable electrochemical biosensors. Huajun et al. suggested a pH sensor based on quasi-two-dimensional metal oxide semiconductors for detecting glucose and ph in sweat. The sensor is made of In₂O₃ thin films and has a detection limit of 0.0005 for pH and high accuracy in detecting glucose content (Figure 4F) (Chen et al., 2017).

Polymer-based novel sensing capabilities represent a significant advancement in electrochemical sensing. Since the electrochemical sensors are integrated into textile structures through weaving, knitting, and embroidery, or need to be directly embedded into garments and coupled at the human skin’s surface to detect the target analyte (Allison et al., 2017). It is highly desirable that the sensors should be reliable and flexible enough to reduce motion-induced signal interference. While carbon and metal-based nanomaterials are highly sensitive but they are not flexible enough as the conductive polymer-based nanomaterials. It is well recognized that CPs have advantages such as chemical diversity, low density, durability, corrosion resistance, easy-to-handle shape, terminology and adaptable conductivity. Moreover, the outstanding properties of flexible conductive polymer nanomaterials are as follows: 1) they aided in increasing the selectivity and stability of electrochemical biosensors' sensing properties. 2) They usually have a 3-D structure. They can be manufacture in various sizes, including nanometre size and high conductivity, such as 120–130 S cm⁻¹ at room temperature, which offers a more precise surface area. 3) The modification of conductive polymers at the sensing electrodes is relatively very easy and simple because it has no special requirements for the evenness of electrodes (Matsumura et al., 2018) (Gerard et al., 2002;Gerard and Malhotra, 2005;Zeng et al., 2014). Researchers discovered and produced conductive polymer nanomaterials such as (PEDOT- PSS) (Abouraddy et al., 2007;Zhao et al., 2018), polypyrrole (ppy) (Gregory et al., 1989;Tzou and Gregory, 1992)), polythiophene (PTh) and polyaniline (PANI) (Huang and Kaner, 2004;Teli et al., 2014), to fabricate the sensing electrodes. Usually, conductive polymers are deposite at working electrodes as a compact film. Some conductive polymer nanomaterial-based wearable electrochemical biosensors are summarized in Table 4. Xuesong et al. developed PANI nanoparticle-based sensitive pH sensor. The sensor was fabricated by coaxial electrospinning of PANI nanoparticles and polyurethane (PU) into the core-shell fibres (Figure 5A). A Screen-printing method was used to create a sensing electrode assembly on a polyethylene terephthalate (PET) substrate, which consisting of gold as a counter electrode, (Ag/AgCl) reference electrode, and (PANI-PU) working electrode. Here, PU provides mechanical stability to the sensor. The feasibility of detecting sweat pH on the skin was demonstrated by attaching the chip to the arm and electrochemical workstation was used to measure the reaction. The sensor operated linearly in the pH range of 2–7 with a sensitivity of 60 mV/pH and can detect pH changes of less than 0.2 (Hou et al., 2020). Salzitsa et al. developed a fully wearable and flexible patch with completely integrated sensing system for on body human sweat testing (Figure 5B).

The sensing device is designed so that a steady stream of sweat can flow through an array of compact microneedles with a diameter of 50 μm. The sensors are embedded in a microfluidic channel, which can simultaneously track metabolites (lactate) and electrolytes, for example, pH and sodium ions. The promise of the multi-sensing platform for monitoring the metabolite and electrolyte (sodium, lactate, and cortisol) from saliva is demonstrated in detail. For that, a potentiometric sodium ion sensor made of poly (3, 4-ethylenedioxythiophene) (PEDOT) and an amperometry lactate sensor were proposed. This sensing device can transmit data wirelessly for easy processing and storage, with the potential for real-time data analytics (Anastasova et al., 2017). As it has been seen, various groups have achieved device dimensional stability through lithographic or coating processes that are either costly or complicated on a large scale. So, for the first time, AJ Bandodkar et al. filled this technological gap by using the screen-printing method to create a low-cost and highly stretchable (PEDOT: PSS) based wearable electrochemical biosensor (Figure 5C). The stretchable device exhibits 2-D serpentine interconnects with 180° turns between electrode areas and touchpads (Bandodkar et al., 2015c). This electrochemical device has high sensitivity, can undergo high tensile stress, and meets the stretchability criteria of many applications; it was a good sign for potential and next-generation wearable systems. Tomas Guinevere et al. also developed a screen printed Potentiometric pH sensor based on conductive polymer (PANI) to measure the pH level of wounds in the body (Guinovart et al., 2014). The device works by carefully incorporating a pH sensor into bandages (pH range 5.5–8). These new pH-sensitive bandages opened a new possibility for the realization of telemedicine. In 2018, Xu et al. introduced a new and innovative technique to develop a highly sensitive PEDOT-based electrochemical biosensor for the detection of dopamine (DA) (Figure 5D) (Xu et al., 2018). The sensor was fabricated using a PEDOT-modified laser scribed graphene (LSG) method, which shows higher sensitivity and selectivity for detecting dopamine in a complex mixture. These PEDOT-LSG electrodes have a lot of potential for infield or point-of-care biosensing and some other incorporated bioelectronics products. Recently, Zehad et al. used the same technique to develop a PEDOT-PSS modified 3-D stable porous, porous, laser-induced graphene (LIG) to detect glucose and pH in human sweat (Zahed et al., 2020). Where, PEDOT-PSS is used to increase the tensile stability and uniform conductivity of the electrode. The fabricated electrochemical biosensors display a good current response to glucose in a wide linear range of 10 mmol/L to 9.2 mmol/L, with high sensitivity of 247.3 mA L mmol cm⁻² and a low detection limit of 3 mmol/L. As a new kind of multifunctional sensor, this versatile substrate was further improved with Pt/Pd nanoparticles for glucose detection (Figure 5E). Recently Mariani et al. proposed a PEDOT: PSS film based electrochemical potentiometric sensor through a new and different approach. A pH-dependent modification of the current flowing through the PEDOT: PSS film was obtained through the random electrochemical gating caused by the potentiometric transducer (PEDOT: BTB), PEDOT: Bromothymol Blue [27]. The feasibility of this technique was demonstrated by creating a screen-printed pH sensor on a bio-ceramic fabric (Figure 5F). Compared to the rigid analogous fabricated on a glass substrate, this textile pH sensor demonstrated a standardized sensitivity of (7.5) x10⁻³ pH⁻¹ in the range of 2–7, with no penalty of sensing efficiency. By using the same content PEDOT:PSS and PEDOT:BTB (Mariani et al., 2020). The same technique was used to develop a thread-based pH sensor, as shown in (Figure 5G). In conjunction with another thread-based sensor for multi-sensing network and chloride ion (Cl⁻) detection, the textile sensor could detect pH selectively during continuous recordings (Possanzini et al., 2020).

In accordance with the sensing nanomaterials described above, various novel nanomaterials with good electrical conductivity, large specific surface area, and good biocompatibility are developing in the field of wearable electrochemical biosensors. For example, Polymer nanomaterials flexibility, conductivity, durability and long-term stability would be enhanced further after compound with Carbon and metal-based nanomaterials (Zhou and Shi, 2016; Jia et al., 2019) as some of them have been listed in Table 5.

Electrochemical immunosensors have been extensively used in medical diagnostic and therapeutic applications, doping or impurities, and the food industry to track biological components and biological molecules of environmental origin and influence. where antibodies are probes that form ion complexes with the same antigen pairs with specific targets. Nanomaterials are the best candidates for developing electrochemical immunosensors with good sensitivity and signal transduction capability. A significant number of electrochemical label-free immune strategies have recently been developed and used to identify multiple target biomarkers associated with many kinds of diseases (Tuteja et al., 2018), including viruses (Kaushik et al., 2018), cardiac markers, and other proteins (Dutta and Lillehoj, 2018; Haji-Hashemi et al., 2019). Focused on this label-free method, a non-faradaic impedimetric portable/wearable cortisol sensor was designed using semiconductive MoS2 nanosheets and vertically aligned metal electrodes to calculate cortisol concentration in artificial sweat samples (Kinnamon et al., 2017). As a new immunosensing platform, Stretchable and disposable electrochemical biosensors are eager to identify biomarkers from body fluids such as saliva, interstitial fluids, sweat, or wound fluids, as well as various biomechanical organisms (Kim et al., 2011; Cao et al., 2014; Gao et al., 2016c; Liu et al., 2016; Liu et al., 2017). However, the realization of such stretchable electrochemical biosensors has been narrowed by the challenges of obtaining electrochemical electrodes with high sensitivity, stretchability, and stability during deformation. With that in mind, Bo-Yeong et al. proposed a simple, durable and stretchable disposable point of care electrochemical immunosensor based on metallic nanomaterials. A three-dimensional, micro-patterned stretchable substrate was used to form thermally evaporated gold as working and counter electrodes (Figure 8A). The sensor shows high sensitivity and stability when stretching up to 30%. The sensor can detect low concentrations of target molecules, down to 100 fM of TNF- α protein, an inflammation biomarker. After all, it was still challenging to develop a soft, skin-interfaced biosensor patch entirely operative lab-on-patch technology, with particular significance for non-invasive detection and differentiation of biomarkers from body fluids (Heikenfeld et al., 2018, 80; Zhao et al., 2019; Lee et al., 2020). It will also be more user-friendly than lab-based immunoassays or in-hospital (POCT) point-of-care testing, which involves numerous in vitro sample processing steps and user expertise in sample analysis (Daniels and Pourmand, 2007). Many current immunoassay procedures use labelling to produce a detection signal, in which an antibody, fluorescent markers, or redox pair is added to the detection probe. These methods are time-consuming, need several steps, and cause discomfort to the wearer (Joung et al., 2019). To meet these challenges, Lee et al. developed a stretchable, wearable lab-on-a-patch (LOP) device made up of a label-free impedimetric biosensor and a stretchable microfluidic system for on-body measurement of the hormone, biomarker, and cortisol. A three-dimensional nanostructured gold was used as a sensing electrode to achieve the high sensitivity required to measure the pM-levels of cortisol in sweat (Figure 8B). Using an antibody as a probe biomolecule, this biosensor measured sweating cortisol accurately during exercise, ranging from 1 pg/ml to 1 μg/ml, under a 30% strain (Lee et al., 2020). This LOP platform may be enhanced to track other biomarkers in sweat such as cytokines, neuropeptides, therapeutic drugs in sweat and a broader range of biomarkers in other biofluids such as interstitial fluids or wound exudate. Further, we see a great roll of nanomaterials in molecular imprinted polymer-based sensors (MIP’s). Huang et al. recently proposed a flexible electrochemical urea sensor (Figure 8C). In their first work, they developed the MIP by imprinting urea with electropolymerized (PEDOT) on a network of carbon nanotubes and gold nanotubes (AuNTs) (Liu et al., 2018). The developed flexible sensor demonstrated a strong linear response to physiologically significant urea levels while showing negligible cross reactivity. Their second work fabricated an ECL sensor by coating a specific MIP layer on highly luminescent nanospheres immobilized AuNTs networks (Parlak et al., 2018). The sensor can detect lactate and urea from sweat accurately with high stability. Parlak and his colleagues created a wearable organic electrochemical system focused on a molecularly selective nanoporous membrane for non-invasive cortisol sensing (Figure 8D) (Parlak et al., 2018). The cortisol biorecognition is based on a MIP membrane with a laser-patterned microcapillary channel array for sample acquisition and organic electrochemical transistors (OECTs) based on PEDOT:PSS. The wearable sensor’s stability and stretchability are offered by the styrene-ethylene-butylene-styrene (SEBS) elastomer substrate. Lately, laser-burned graphene (LBG) has been developed as an excellent electrode matrix for wearable electrochemical sensing applications because of its one-shot fabrication and excellent electrochemical performance (Ugur et al., 2014;Hamblin, 2016). Jong et al. recently proposed a Ti₃C₂T_x (MXene/LBG) based wearable electrochemical impedimetric immunosensor with a 3-D electrode network for noninvasive cortisol biomarker identification in human sweat at the point of treatment (POC). Laser-induced graphene (LBG) is the basic material used in electrode construction since it is stable and has strong electrical properties. Ti₃C₂T_x MXene, which has excellent electrochemical properties and outstanding enzyme loading capabilities, was deposited on the electrode (Figure 8E). The cortisol sensor had a very low concentration limit of 3.88 pM and excellent selectivity. This MXene LBG-based flexible noninvasive patch can be used to identify other biomarkers or pathogens. The developed path can be coupled with a wearable electrochemical front-end for impedance signal monitoring and wireless data transmission for smartphone-based biomarkers or pathogen diagnosis properties (San Nah et al., 2021).

Conductive nanomaterials and nanocomposites have lately gained popularity as potential materials for DNA biosensors. Electrochemical biosensors for DNA detection are being developed and used in many human applications such as food, healthcare, environment, etc. (Ramanavičius et al., 2006; Booth et al., 2011). A DNA biosensor is designed by immobilizing a DNA probe on an electrode surface and then using hybridization to label the target DNA sequence. This hybridization, known as transduction in the technological era, can be observed optically and electrochemically. A DNA biosensor for detecting the H5N1 gene sequence of the influenza virus was created using a DNA aptamer immobilized hybrid nanomaterial-modified (MWCNT/PPy) electrode. The modified electrode nanoparticles provide a porous structure with a wide effective surface area. The latest (PANI-AuNPs) nanocomposite based DNA biosensor detected micro RNA-16 using a streptavidin-AP conjugate to biotinylated recognition sites via transduction with a 0.1 nM detection limit (Saberi et al., 2013). Very recently, for the first time, Jiang and his team proposed a cloth-based DNA biosensor by using nanocomposite (CdTe-MWCNTs) to get a stronger electrochemical signal. Under ideal conditions, target DNA samples (75-bp DNA fragments produced by PCR amplification) were determined in a range of 20 fM to 5 nM, with a detection limit of 8.74 fM and relative standard deviations of 2.04 and 4.75 percent for intra- and inter-assays at 50 pM TD, respectively (Jiang et al., 2020).

Wearable electrochemical biosensors can be manufactured from a variety of nanomaterials depending on the needs of real-time applications. Nowadays, more conductive nanomaterials will be developed and used to generate new sensing electrodes for wearable electrochemical biosensors. The new development trends of sensing nanomaterials in the future will be as follows.’

Firstly, sensing nanomaterials with porous structures, high electrical conductivity, and catalytic activity will be often used in both enzymatic and non-enzymatic sensors to enhance the sensor sensitivity. Second, sensing nanomaterials should have superior mechanical properties in order to improve the sensor’s durability and flexibility throughout everyday activities. Third, because there are different interferences (such as glucose, lactate, Na+, K+) in body fluids, nanomaterials with specific identification of analytes like glucose or lactate may attract more attention to increase the selection of sensors. Finally, to produce low-cost, high-performance, and reliable wearable electrochemical biosensors and biosensors, more knowledge of nanomaterial characteristics, as well as advancements in manufacturing and processing procedures, is required. For example, replacing Ag with Cu or carbon-based components and using low-cost CNT synthesis and processing methods. Only preliminary studies on the biocompatibility of nanomaterials for wearable applications have been conducted. More methodical reports on nanomaterials' long-term biocompatibility are urgently needed to support the practical applications of nanomaterials. Graphene has been used to create a variety of wearable electrochemical biosensors, especially for healthcare applications. To ensure high efficiency, additional efforts should be made to improve large-scale advancement with reasonable uniformity and defect-free deposition onto different substrates with high-pitched consistency and yield. However, nanotechnology applications are beginning to emerge, and more research is needed to obtain novel results and uses.