Highlights
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The materials, fabrication methods, implantation technologies, and applications of implantable electrochemical microsensors for animals were summarized.
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The implantable electrochemical microsensors for monitoring diseases and exploring disease mechanisms were discussed.
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The current status, ongoing challenges, and future development prospects of implantable electrochemical microsensors in monitoring animal physiological information were highlighted.
Abstract
In vivo monitoring of animal physiological information plays a crucial role in promptly alerting humans to potential diseases in animals and aiding in the exploration of mechanisms underlying human diseases. Currently, implantable electrochemical microsensors have emerged as a prominent area of research. These microsensors not only fulfill the technical requirements for monitoring animal physiological information but also offer an ideal platform for integration. They have been extensively studied for their ability to monitor animal physiological information in a minimally invasive manner, characterized by their bloodless, painless features, and exceptional performance. The development of implantable electrochemical microsensors for in vivo monitoring of animal physiological information has witnessed significant scientific and technological advancements through dedicated efforts. This review commenced with a comprehensive discussion of the construction of microsensors, including the materials utilized and the methods employed for fabrication. Following this, we proceeded to explore the various implantation technologies employed for electrochemical microsensors. In addition, a comprehensive overview was provided of the various applications of implantable electrochemical microsensors, specifically in the monitoring of diseases and the investigation of disease mechanisms. Lastly, a concise conclusion was conducted on the recent advancements and significant obstacles pertaining to the practical implementation of implantable electrochemical microsensors.
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1 Introduction
Physiological information serves as a valuable indicator of animals' health status and biological characteristics. Through the continuous monitoring of various indicators, including metabolites, body temperature, and respiratory rate, it becomes possible to detect a wide range of health concerns such as infections, organ impairment, and metabolic disorders. In the field of animal husbandry, the technology for monitoring animal physiology assists farmers and veterinarians in understanding and monitoring the health, behavior, and production performance of livestock. Consequently, it enhances production efficacy, guarantees the exportation of products, and upholds public health and safety [1]. For pet owners, monitoring physiological information about pets can help owners monitor their pets’ health issues more quickly, enhance diagnosis and treatment effectiveness [2]. In the realm of animal research, researchers are capable of gras** information about animal metabolism, and the neurological system, further providing crucial references for researching human diseases. Overall, the advancement of animal physiological monitoring technology will benefit animal health and welfare, encourage the development of animal husbandry and pet health, and provide critical assistance for the research of human diseases.
Animal physiological information encompasses various parameters such as body temperature, heart rate, respiratory rate, blood pressure, weight, exercise ability, and chemicals. The traditional method of monitoring physiological information is the collection of samples for in vitro biochemical, immune, genetic, and other analyses. Techniques such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and mass spectrometry are commonly employed in laboratory settings for humoral analysis. These techniques enable accurate early-stage disease diagnosis, leading to reduced medical expenses and improved health outcomes [3]. ELISA and PCR, as gold standards for immunological and molecular diagnostics, are particularly significant in the screening of diseases such as cancer, infectious diseases, genetic and hormonal abnormalities. However, these techniques are still costly and time-consuming, necessitating the use of skilled workers and specialized gear. Researchers are faced with the task of finding alternative diagnostic devices to replace the conventional diagnostic methods. Microsensors have been extensively utilized for monitoring animal physiological information and as auxiliary tools for disease diagnosis, which can be classified as wearable (skin) [4], implantable (tissue) [5], ingestible (gastrointestinal tract) [6] depending on the location of the microsensor application. Wearable sensors detect biomarkers in the biological fluid on the body surface non-invasively, but their accuracy is constrained due to the restricted volume of the biological fluid for detection. Ingestible sensors provide accurate measurement results; however, their long-term presence in the body may pose a threat to animal health. On the other hand, implantable microsensors offer a reliable method for monitoring physiological information that is bloodless, painless, and minimally invasive. Among them, electrochemical sensors have been at the forefront of clinical diagnostics owing to their high performance, portability, simplicity, and low cost [7]. Implantable electrochemical microsensors have been widely employed for monitoring animal physiological information, such as glucose, lactate, dopamine, and biomacromolecules. These sensors assist users in promptly assessing the health status of animals, detecting issues, and implementing suitable actions.
This review mainly focuses on monitoring animal physiological information, with a particular emphasis on the widespread use of implantable electrochemical microsensors in this field, owing to their high sensitivity and minimally invasive characteristics. In this review, we comprehensively discussed materials, the fabrication methods, and implantation technologies of microsensors, and innovatively summarized the application of implantable electrochemical microsensors in monitoring physiological information in animals. The development and marketing of implantable electrochemical microsensors require addressing important factors such as long-term durability, compatibility with the human body, reliable power source, and the ability to monitor multi-analytes. The conclusion summarizes the difficulties and potential advancements in the future of implantable electrochemical microsensors for monitoring animal physiological data in vivo (Fig. 1).
2 Fabrication of Implantable Electrochemical Microsensors
In recent years, significant progress has been made in the field of implantable electrochemical microsensors. Figure 2 illustrates advances in implantable electrochemical microsensors. With the development of materials science, energy and microfabrication, implantable electrochemical microsensors for in vivo monitoring are undergoing a transformation toward miniaturization, multi-analyte sensing, self-powered, and integration of diagnosis and treatment. The choice of materials and fabrication methods plays a crucial role in determining the size, stability, and sensitivity of microsensors [8].
![figure 2](http://media.springernature.com/lw685/springer-static/image/art%3A10.1007%2Fs40820-023-01274-4/MediaObjects/40820_2023_1274_Fig2_HTML.png)
Copyright 2013 Springer Nature; First commercial implantable continuous glucose monitor (CGM) [10]. Copyright 2020 Royal of American Society; A self-powered implantable skin-like glucometer [11]. Copyright 2018 Springer; An implantable system for sensing of cell-free DNA [12]. Copyright 2020 Springer Nature; Flexible microneedle (MN) sensors for monitoring pH distribution in rats [13]. Copyright 2021 The American Association for the Advancement of Science; A closed-loop system for managing diabetes [14]. Copyright 2022 John Wiley and Sons; A tissue-like neurotransmitter sensor [15]. Copyright 2022 Springer Nature; A miniaturized, fully integrated, wirelessly sensor for monitoring of multi-analytes [16]. Copyright 2022 Springer Nature
Overview of important developments of implantable electrochemical microsensors in the past decade which include First implantable glucose biofuel cell [9].
2.1 Materials
The choice of materials is a critical factor that significantly impacts the performance of sensor components. Given the requirement for long-term implantation in vivo monitoring, implantable electrochemical microsensors necessitate substrate and electrode materials with enhanced biocompatibility, stability, conductivity, and mechanical strength. These characteristics are essential to ensure the biocompatibility and reliability of long-term monitoring.
2.1.1 Substrate Materials
Substrate material serves as the physical supporting structure of the sensor, facilitating the reaction and monitoring processes. In sensor applications, substrate materials are typically required to possess high stability and mechanical strength, while also exhibiting electrical characteristics to ensure optimal sensor performance and accuracy. Rigid substrate materials such as some metals, semiconductors often possess desirable properties such as processability and good mechanical properties [17]. Silicon, for instance, is a type of important substrate material for microsensors due to its excellent semiconductor properties, mechanical strength, and easy processability. Dervisevic et al. designed a high-density silicon-based MN electrochemical sensor for glucose monitoring which shows good performance [18]. Silicon-based MN has good permeability and is easy to penetrate the skin. However, silicon is prone to breakage when implanted under the skin, raising significant biological concerns [19]. Stainless steel substrate is another commonly used metal substrate material, known for its high mechanical strength and machinability, which is easy to be manufactured into a variety of shapes and structures of the sensor. Using stainless steel needles as substrates, Zhang's team fabricated electrochemical microsensors that can be used to wonderfully monitor neurochemicals such as dopamine, norepinephrine and nitric oxide in the rat brain [20,21,22,13]. MNA with Au electrodes and polyaniline (PANI) deposited on the surface was used to monitor the pH distribution in the skin layer of a rat model of peripheral vascular disease. Considering better biocompatibility, Odinotski et al. prepared an HMN-based electrode (Fig. 7b) consisting of DA-HA hydrogel combined with PEDOT: PSS, with the intrinsic catechol quinone chemistry of DA used to measure the pH of ISF. HMN pH sensor is capable of in vivo measurements with 93% accuracy, providing a new direction for wearable sensors [103]. Li et al. designed a simple electrochemical microbial sensor (ECMB) based on the in situ self-assembly of AgNPs coated on MXene Titanium Carbide (Ti3C2Tx) [13].
Fluid and electrolyte imbalance is a significant clinical issue that is directly associated with morbidity and mortality [105]. Electrolyte imbalance often involves multiple ions. Zhao et al. proposed a bifunctional electrochemical biosensor for simultaneous monitoring of glutamate and Ca2+ [106]. The bifunctional ME consisted of a Pt ME co-modified by glutamate oxidase (GluOx) and PtNPs for glutamate monitoring, along with a fully solid-state Ca2+-selective microelectrode (Ca-ISME) as a potential recording channel for Ca2+. The sensor exhibited excellent selectivity and limit of detection (LOD), and successfully recorded the dynamic changes in glutamate and Ca2+ in ischemic and normal mice. Zhu et al. created an MN sensor that is integrated with screen-printed electrodes and can monitor pH, Na+, K+, and Ca2+ (Fig. 7c) [107]. When MN penetrates the skin, ISF is quickly attracted to the electrode modified with the ion-selective membrane (ISM), causing the open circuit potential to change in a concentration-dependent manner. The sensor successfully monitored dietary-induced electrolyte imbalance in mice, with the potential for application in pet electrolyte monitoring. Molinero-Fernande et al. proposed an MN electrochemical sensing system for multi-analyte monitoring of pH, Na+, K+, Ca2+, Li+, and Cl− (Fig. 7d) [60]. Selective detection of ions is achieved by modifying f-MWCNT and ion-selective membrane by dropwise addition on the MN surface of stainless steel substrate. In vivo experiments demonstrated good sensitivity, selectivity, and stability, although some differences in ion concentrations between ISF and blood require further investigation.
Implantable electrochemical sensors have been extensively used in animal health monitoring research. These sensors can detect various biological indicators, such as glucose, lactate, DNA, and proteins, and can monitor the changes in these indicators in real-time in the body, which is of great significance for the dynamic monitoring of animal health. In recent years, the development directions of implantable electrochemical microsensors for monitoring chemical substances in animal body fluids include multi-analyte monitoring, wireless remote monitoring, long-term stability, miniaturization, and biocompatibility. At the same time, there are issues with sensors such as stability, biocompatibility, data processing, energy supply, and cost to be addressed.
4.4 Neurotransmitters
Neurotransmitters are essential chemicals that amplify, transmit, and transform signals in the brain [109]. Changes in neurotransmitter concentrations may lead to several brain diseases such as Alzheimer's disease (AD) [110] and Parkinson's disease (PD) [111]. Therefore, monitoring neurotransmitters has significant implications for understanding pathological mechanisms and improving prevention strategies. The basic structure and function of the animal nervous system and the human nervous system are similar, and many neural substances and pathways are highly conserved between animals and humans. Therefore, by studying animals’ neurotransmitters, we can gain a better understanding of the function and disease mechanisms of the human nervous system.
4.4.1 Dopamine
Neurotransmitters can be classified chemically into biogenic amines, amino acids, and choline [112]. In vivo electrochemical studies using MEs have long been used to investigate neurotransmitters, providing real-time insights into their function in the brain and body [113]. Dopamine (DA), an important neurotransmitter in the central nervous system, is mainly found in the hypothalamus and pituitary gland. Abnormal DA concentrations may contribute to diseases such as Alzheimer [114], PD [115], and schizophrenia [116]. In vivo electrochemistry of carbon-fiber electrode (CFE) is one of the most useful methods for tracking neurochemicals in specific brain regions. Tang et al. first used CFE to quantify single DA. Intrasynaptic DA release and were surprised to find that harpagide (a natural product) could both enhance synaptic DA release and restore normal levels of DA release from damaged neurons, adding hope for Parkinson's treatment and prevention [117]. Since DA itself or the reactant can easily form a film on the CFE surface, unmodified CFE will inevitably suffer from surface biofouling and lead to a significant decrease in sensitivity [118]. To address this issue, Feng et al. prepared CFE modified by a nano-conducting PANI mixed with poly tannic acid (pTA) (Fig. 8a) and experimentally showed that the electrode has good antifouling properties, higher sensitivity than the unmodified electrode as well as excellent enrichment ability for DA electrochemical measurements [119]. Carbon-based nanomaterials are often used to modify the electrode surface of DA electrochemical sensors, which typically have high electrical conductivity, are biocompatible, and have a large specific surface area [120]. Taylor et al. studied CFE coated with a highly conductive PEDOT/CNT coating that exhibited extremely high sensitivity and selectivity for DA [121]. Metal ME is also used for in vivo monitoring of DA due to its high electrochemical catalytic activity and excellent conductivity. Chen et al. used gold nanoparticles (AuNPs) and reduced graphene oxide (rGO) modified Pt wires for in vivo electrochemical monitoring of DA (Fig. 8b). The modified electrodes exhibited high sensitivity, selectivity, and resistance to interference for DA, which was validated by implantation into the rat striatum. This experimental electrode has promising future applications for monitoring various neurotransmitters in vivo [122]. In addition, acupuncture needles' ability to be used as sensing devices for in vivo electrochemical monitoring of various biomolecules has been well shown. Zhou et al. fabricated a unique needle field-effect transistor (FET) microsensor based on an acupuncture needle (Fig. 8c), and it is shown to be capable of monitoring DA as well as neuropeptide Y in vivo [20]. The above work is excellent which gives a new insight into the FET domain and broadens the range of real-time in vivo detection species.
![figure 8](http://media.springernature.com/lw685/springer-static/image/art%3A10.1007%2Fs40820-023-01274-4/MediaObjects/40820_2023_1274_Fig8_HTML.png)
Copyright 2019 American Chemical Society. b Schematic of AuNPs/rGO-Pt for in vivo electrochemical monitoring of DA [122]. Copyright 2019 Elsevier. c Schematic of the application of needle FET [20]. Copyright 2022 John Wiley and Sons. d Schematic illustration of the soft implant for sensing neurotransmitters in the brain [15]. Copyright 2022 Springer Nature. e Schematic of glutamate sensing with the nickelate-Nafion sensor [125]. Copyright 2020 American Chemical Society. f Illustration of the measurement with the LLIM in the rat brain [127]. Copyright 2021 American Chemical Society
Schematic of implantable electrochemical microsensors for monitoring neurotransmitters. a An illustration of the structure of PTA-PANI-coated CFE [119].
4.4.2 Serotonin
Many psychological disorders, such as depression, and anxiety, are related to low serotonin levels. Therefore, model experiments conducted on animals will help to explore the mechanism of psychological disorders. Fast scanning cyclic voltammetry (FSCV) is an electrochemical technique with precise temporal resolution and sensitivity for the measurement of fast neurotransmitter kinetics in vivo [123]. In a study by Abdalla et al., a novel method for stimulating and measuring serotonin concentrations was characterized in three brain regions [124]. It was shown that stimulation of axons leads to the production of serotonin in the three measured regions of the brain. Recently, Bao's team developed a stretchable, tissue-mimicking neurochemical bio-interface, NeuroString (Fig. 8d) [15]. The NeuroString sensor allowed real-time, multichannel, and multiplexed monoamine sensing in the brains of normally behaving mice, as well as the measurement of serotonin dynamics in the gut in the absence of undesired stimuli and interference with peristaltic motility. The microsensor has the potential to be used as a biomolecular sensing for soft organs throughout the body.
4.4.3 Glutamate
Glutamate is one of the most abundant and predominant excitatory neurotransmitters in the central nervous system, accurate monitoring of excitatory neurotransmitter levels is critical for understanding the underlying neuronal processes of various illnesses. Sun et al. created and evaluated a new calcium titanate-nickelate-Nafion electrode for recording glutamate release from electrically activated brain slices in vitro and primary visual cortex (V1) in vivo in awake mice exposed to visual stimuli (Fig. 8e) [125]. The electrode was shown to have high selectivity for glutamate, a fast response time (1.2 s) and LOD (16 nM). Recently, Chu et al. developed a flexible electrochemical microsensor for simultaneous monitoring of L-glutamate and GABA levels, which are anticipated to play a vital role in the excitation: inhibition balance [126]. The flexible electrodes were electrochemically deposited with Pt to increase the electrochemically active surface area, and in vitro and in vivo experiments in mice demonstrated their accuracy for long-term monitoring.
4.4.4 Choline
Choline is another neurotransmitter that participates in the transmission of neural signals and plays an important role in motor control, memory, and other aspects. Wang et al. developed a liquid/liquid interface microsensor (LLIM) (Fig. 8f) for monitoring the redox inactive neurochemical choline (Ch) in the rat brain [127]. Ch was found at a specific ion transfer potential and a unique ion transfer current signal based on the difference in the solvation energy of choline in cerebrospinal fluid (aqueous phase) and 1,2-dichloroethane (organic phase). LLIM responded well to choline, with strong linearity and selectivity with a LOD of 0.37 µM. For the first time, it was demonstrated that LLIM can be employed in the brain.
Overall, these advancements in neurochemical sensing techniques provide valuable insights into the functioning of neurotransmitters and their role in various diseases and physiological processes. The development of these techniques not only helps us better understand the working principles of the nervous system, but also provides researchers with more tools to explore the relationship between neurotransmitters and diseases.
4.5 Oxidative Stress Metabolites
Small molecule products of oxidative metabolism have a substantial impact on physiological processes and pathological pathways. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are two examples of significant metabolites [113]. They play diverse roles in the regulation of several physiological processes at relatively low concentrations.
4.5.1 H2O2
Overproduction of ROS/RNS contributes to the development of cardiovascular disease, cancer [63, 134]. Copyright 2019 Royal Society of Chemistry. f Diagram of a three-electrode device used for in vivo electroanalysis in the brain of an anesthetized mouse [137]. Copyright 2022 John Wiley and Sons