**3.5 On-skin biofluid extraction: electrical-based approaches**

Each person has 2.03 million sweat glands; sweat gland densities vary broadly across the skin surface and subjects, ranging from 16 to 530 glands cm<sup>−</sup><sup>2</sup> [11, 13, 69]. Normally, during exercise, sweat can be secreted around 20 nL gland<sup>−</sup><sup>1</sup> min<sup>−</sup><sup>1</sup> [11]. For example, the forehead or arm can generate sweat around 3 μL cm<sup>−</sup><sup>2</sup> or even lower. The fluctuation of sweat rate is also related to numerous factors, such as activity intensity and hydration level. Therefore, the limited volume of sweat causes a challenge in sweat analysis and operations. This leads to the development of miniaturized skin-worn electrochemical devices that can be practical in such small dead volume. For instance, the textile-based energy-harvesting BFC requires sweat volume per area of 40 μL cm<sup>−</sup><sup>2</sup> to deliver steady outputs [31]. Designing a capillary chamber is a possible route for low-volume electroanalytical systems [70].

In addition to a passive way to collect sweat, one strategy is an active electricalbased approach, called "iontophoresis" [71, 72]. This active strategy offers

#### **Figure 5.**

*Electrical-based strategies using iontophoretic electrodes to extract biofluids, including (A) pilocarpine iontophoresis and (B) reversed iontophoresis.*

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interferents, such as ascorbate [53].

and polyphenol oxidase.

provides the apparent *k*s, of 1.5 s<sup>−</sup><sup>1</sup>

values of −0.35 and +0.6 V, respectively.

In addition, extra membranes can be a biocompatible barrier to address challenges from biofouling and interferents, especially when electrochemical operations are made in real matrices, samples, such as sweat. A perfluorinated sulfonated membrane (Nafion®) is an example membrane, which is also easy to drop-cast. This coating membrane can protect the enzymatic layer and also prevent anionic

Shelf life and operational stabilities of enzymatic electrodes are among the most critical challenges. The enzyme and active materials, such as mediators, can also leach during operations. Extensive studies have been made to improve enzyme bioelectrodes, such as by crosslinking hydrogels in the presence of the enzyme [54, 55]. Such crosslinking can entrap the enzyme to be more stable; moreover, this way enhances the loading of the enzyme, while the three-dimensional structure can facilitate the transport of analytes or biofuels, improving bioelectrode functions. Nevertheless, crosslinking enzyme or covalent binding of the enzyme can change the conformation of the enzyme and thus affect the activity [56]. Furthermore, one alternative to stabilize the enzyme electrode is the addition of stabilizers, such as polyelectrolytes, dextrans, glycerol, polyethyleneimine, and hydrophobic oils [57–59]. For instance, hydrophobic mineral oil or silicone grease can be used to minimize enzyme denaturation [58, 59]. The pasting liquid helps to lower protein mobility, maintain conformational rigidity of enzymes, and barrier to hydronium ions from acid environments. This strategy can stabilize many enzymes, such as GOx, LOx, AOx, horseradish peroxidase, amino acid oxidase,

Increasing enzyme loading can also improve the performance of biocatalytic devices. Employing high surface nanomaterials is useful to enhance the surface loading of the target catalyst. A graphene-based electrode is a good example

have high conductivity and specific surface, represent an outstanding candidate nanomaterial for electrochemical wiring [38, 61]. The thin nanoscale structure can intimately incorporate with the active enzyme. Adsorption of GOx on CNTs

can be as high as 11.2 s<sup>−</sup> 1 [63]. Therefore, mediatorless bioelectrodes with excellent electron transfer could be demonstrated. Their high three-dimensional architecture also offers an enhanced loading of enzyme and/or redox mediator immobilizations. As a result, this can enlarge the current output from the biosensor or BFCs. Importantly, for BFCs, the maximized OCV and current density could be observed [43]. This BFC consists of a GOx/catalase/CNT bioanode and laccase/CNT biocathode without additional mediators. The CNT/enzyme matrix was compressed together under high hydraulic pressure (10 kN). The resulting output in an airsaturated electrolyte (200 mM glucose in 0.2 M phosphate buffer solution, pH 7 at room temperature) after 3 days displayed a high maximum OCV of 1 V. Note that the GOx/catalase/CNT bioanode and the laccase/CNT biocathode showed OCV

Importantly, biofluids from the skin (such as sweat and extracted interstitial fluids) contain a variety of chemicals that can inhibit enzyme activity, reflecting challenges in biosensing and BFC functions in real-time on-body applications. For instance, heavy metals can be found in sweat as the body expels chemicals or balances the charges. One example is Cu2+ which has been reported as an inhibitor to deactivate the enzyme. The Cu2+ in sweat can be in a range of 1.6–16 μM [11]. 0.1 μM Cu2+ could decrease the OCV value of the glucose BFC [64]. However, this enzyme-inhibitor electrochemical behavior is analytically attractive toward the development of self-powered biosensors, such as for direct heavy metal screening

); in addition, it offers a fast

[60]. Moreover, CNTs, which

[62]. The *k*s of GOx at the hybrid biocomposite

platform to offer a high enzyme loading (1.1 nmol cm<sup>−</sup><sup>2</sup>

heterogeneous electron transfer rate (*k*s) of 2.8 s<sup>−</sup><sup>1</sup>

**42**

on-demand sweat generation as the device can be placed to a local skin target. There are two main approaches to extract sweat: (1) iontophoresis with pilocarpine drug and (2) reversed iontophoresis without the drug. These are attractive routes for continuous sweat analysis.

First, pilocarpine iontophoresis can be used to stimulate the sweat. In principle, a small electrical current is applied to enable the pilocarpine administration across the epidermis as illustrated in **Figure 5A**. For example, the tattoo-based enzymatic alcohol sensor consists of a pair of electrodes located in contact with the skin surface. Small constant current (0.2 mA cm<sup>−</sup><sup>2</sup> ) was applied through the cryogel material containing pilocarpine at the anode (positive) iontophoretic side [18]. The applied electrical force will push the pilocarpine drug, which possesses a large positive charge, to eventually enter into the skin. Such transdermal drug delivery of pilocarpine can induce the local sweat, sufficient for the subsequent electrochemical detection. In addition, interstitial fluid (ISF) located under the skin can be extracted. Without this iontophoretic strategy, it is challenging to access ISF through wearable technology.

Second, the reversed iontophoresis without pilocarpine drug can be used to extract relevant analytes, such as glucose [17]. For instance, as presented in **Figure 5B**, a current (0.2 mA cm<sup>−</sup><sup>2</sup> ) is applied to extract glucose in ISF. During the reverse iontophoresis process, glucose is pulled out at the negative iontophoretic compartment. Even though glucose holds no charge, the inherent permiselective characteristic of the skin prefers to transport positive species, allowing such glucose extraction. Applying electric field on mobile electric charge can cause Coulombic force, leading to a net convective flow in the skin from the anode to cathode direction. Accordingly, dissolved analytes (e.g., glucose) are also moved toward the cathode where they can be extracted and monitored. Therefore, the glucose amperometric working electrode, adjacent to the cathodic iontophoretic side, can detect the glucose level from the extracted sample.
