Ion-sensitive field effect transistor biosensors for biomarker detection: current progress and challenges

Jie Zou; Hao Bai; Limei Zhang; Yan Shen; Chengli Yang; Weihua Zhuang; Jie Hu; Yongchao Yao; Wenchuang (Walter) Hu

doi:10.1039/D4TB00719K

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DOI: 10.1039/D4TB00719K (Review Article) J. Mater. Chem. B, 2024, Advance Article

Ion-sensitive field effect transistor biosensors for biomarker detection: current progress and challenges

Jie Zou† ^ab, Hao Bai†^ab, Limei Zhang^ab, Yan Shen^a, Chengli Yang^a, Weihua Zhuang^a, Jie Hu^a, Yongchao Yao *^a and Wenchuang (Walter) Hu *^ab
^aPrecision Medicine Translational Research Center (PMTRC), West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China. E-mail: huwenchuang@wchscu.cn; hatuu@wchscu.edu.cn
^bDepartment of Laboratory Medicine, Clinical Laboratory Medicine Research Center, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China

Received 4th April 2024 , Accepted 20th July 2024

First published on 23rd July 2024

Abstract

The ion-sensitive field effect transistor (ISFET) has emerged as a crucial sensor device, owing to its numerous benefits such as label-free operation, miniaturization, high sensitivity, and rapid response time. Currently, ISFET technology excels in detecting ions, nucleic acids, proteins, and cellular components, with widespread applications in early disease screening, condition monitoring, and drug analysis. Recent advancements in sensing techniques, coupled with breakthroughs in nanomaterials and microelectronics, have significantly improved sensor performance. These developments are steering ISFETs toward a promising future characterized by enhanced sensitivity, seamless integration, and multifaceted detection capabilities. This review explores the structure and operational principles of ISFETs, highlighting recent research in ISFET biosensors for biomarker detection. It also examines the limitations of these sensors, proposes potential solutions, and anticipates their future trajectory. This review aims to provide a valuable reference for advancing ISFETs in the field of biomarker measurement.

Yongchao Yao

Yongchao Yao completed his PhD in Chemistry in 2019 under the supervision of Prof. Shiyong Zhang at Sichuan University. In 2020, he joined the laboratory of Prof. Zhiyong Qian for post-doctoral research at the same University. Now his research activity primarily focuses on the rational design of functional nanostructures toward applications for medical devices, micro/nano biosensors, biochips, and electrochemical high-throughput drug synthesis etc.

Wenchuang (Walter) Hu

Wenchuang (Walter) Hu received a BSc degree in electronics from the School of Electronics Engineering and Computer Science, Peking University in 1999. He then obtained his PhD degree at University of Notre Dame in 2002. During 2004–2005, he carried out postdoctoral research at the University of Michigan. Subsequently, in 2005 he joined The University of Texas at Dallas as an Assistant Professor and was promoted to Professor in 2017. In 2021, he joined West China Hospital of Sichuan University as a full Professor. He published over 180 journal and conference papers. His research interests mainly focus on micro/nano biosensors, biochips, nanomaterials, molecular diagnostic technologies, and point-of-care testing etc.

1. Introduction

Biosensors, as defined by the International Union of Pure and Applied Chemistry, are devices that utilize specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles, or whole cells to detect chemical or biological compounds, typically via electrical, thermal, or optical signals.^1,2 The application of standard silicon planar technology to integrated circuits and field-effect transistor (FET)-based solid-state sensors has gained prominence, offering label-free, rapid, and highly sensitive responses to specific stimuli, along with the significant advantages of low cost, compact size, and easy integration.^3–9 FETs are further categorized into junction FETs and metal oxide semiconductor FETs (MOSFETs), with the latter enjoying wider usage. In 1970, Begivel pioneered the first ion-sensitive field-effect transistor (ISFET) by substituting the metal gate of a MOSFET with a hydrogen ion-sensitive film, marking the inception of ISFET research.¹⁰ Notably, pH-ISFET sensors serve as the foundation for the development of ISFET biosensors and have seen extensive use. Over more than half a century of development, researchers worldwide have harnessed sensitive thin films to create diverse biosensors, expanding ISFET applications to the detection of various ions (e.g., K⁺, Na⁺, and Ca²⁺), penicillin, nucleic acids, proteins, and cells.^11–17 Although they suffer from signal drift and limited dynamic range, ISFETs have distinguished themselves from other solid-state biosensors through several properties (Table 1). Specifically, ISFETs have played a pivotal role in fully integrated chemical sensing systems based on complementary metal oxide semiconductor (CMOS) technology, offering portability, high throughput, mass producibility, and seamless integration with other sensing systems.¹⁸ ISFETs provide a streamlined approach to instrument design by requiring only a single reference electrode for target detection, as opposed to the conventional three-electrode system. Furthermore, serving as all-solid-state sensors, ISFETs exhibit inherent resistance to the effects of acid, alkali, and physical impact.¹⁹ This robust nature positions them as promising candidates for clinical applications in point-of-care testing (POCT), which integrates isolation and detection of biomarkers with affordable costs, high sensitivity, and short operating time.²⁰

Table 1 Comparison of the features of representative solid-state biosensors

	ISFET	Graphene-FET	Electrochemical transistor
Structural features	The metal gate is replaced by an electrolyte solution and a metal-sensitive film	The source and drain are connected by a graphene channel with a thickness of tens of microns	Electrolyte materials containing movable ions replace silicon dioxide as the gate dielectric layer

Principle	When charged biomolecules are recognized on the ion-sensitive membrane, the surface charge density will change, ultimately changing the channel current between the source and drain	The graphene layer acts as a response layer for biosensing and a transistor channel for charge transfer. The voltage on the gate will adjust the charge of the channel, finally changing the source–drain current	When voltage is applied to the reaction interface, the ions in the electrolyte will transfer charge to the semiconductor surface, changing the conductivity of the semiconductor and the current in the channel

Application	Quantification of ions, proteins, nucleic acids, and neutral molecules; enzyme activity determination, high-throughput sequencing, POCT	Detection of disease biomarkers such as ions, glucose, nucleic acids, and pathogens; and important application in gas detection	Detection of ions, proteins, nucleic acids, pathogens; suitable for recording electrical signals from the brain, heart, or any other part of the human body

Limit of detection	K⁺ 10⁻⁸ M (ref. 21)	Hg²⁺ 10⁻¹¹ M (ref. 22)	Na⁺ 10⁻⁷ M (ref. 23)
	Glucose 10⁻⁸ M (ref. 24)	Glucose 10⁻⁷ M (ref. 25)	Glucose 10⁻⁸ M (ref. 26)
	Protein 10⁻¹⁴ g mL⁻¹ (ref. 27)	Protein 10⁻¹⁷ g mL⁻¹ (ref. 28)	Protein 10⁻¹² g mL⁻¹ (ref. 29)
	DNA 10⁻¹⁵ M (ref. 30)	DNA 10⁻²¹ M (ref. 31)	DNA 10⁻¹¹ M (ref. 32)

Advantages	Good specificity, miniaturization, easy to mass produce and integrate with other detection systems, and low-cost	Good electrical and thermal conductivity, low power loss, and good heat dissipation performance	Low operating voltage, high transconductance, high resolution, short response time, easy to manufacture, and low cost

Shortcomings	Signal drift, temperature sensitivity, and limited dynamic range	Complex and expensive manufacturing process, difficult to achieve large-scale, high-quality production	Poor selectivity, limited sensitivity

Biomarkers, the molecular indicators of normal physiological processes, pathological developments, and responses to various exposures or interventions, play a pivotal role in clinical diagnosis and treatment.³³ They offer invaluable insights into the state of an individual's health and are instrumental in guiding healthcare decisions.^34,35 In this context, ISFETs emerge as extraordinary tools, uniquely positioned to revolutionize the analysis of components within body fluids. By enabling precise pH monitoring and potential signal sensing, ISFETs have established themselves as indispensable platforms for POCT. This comprehensive review is dedicated to unraveling the contemporary clinical applications of ISFETs, with a specific emphasis on their significance in the detection of biomarkers. We delve into the utility of ISFETs in the diagnosis and monitoring of cardiovascular diseases, tumors, and infectious diseases. These medical domains represent some of the most pressing challenges in modern healthcare, and ISFETs have emerged as transformative tools for the measurement of biomarkers relevant to these conditions. Furthermore, this review provides a brief overview of the structure and working principles of ISFET biosensors. Understanding the fundamental workings of ISFETs is essential in grasping its true potential within clinical contexts. Through these efforts, this review actively contributes to the ongoing advancements in clinical diagnosis and patient care, establishing ISFETs as a transformative force within the healthcare landscape.

2. Structures, principles, and features of ISFET biosensors

2.1. Basic structures and sensing mechanisms

ISFET biosensors are ingeniously designed, featuring a silicon semiconductor gate, an ion-selective membrane, a reference electrode, and an insulating layer (Fig. 1A). The gate region, a fundamental part of an ISFET biosensor, is covered with an ion-selective membrane tailored to the specific ion being measured.³⁶ When exposed to changes in ion concentration in the surrounding solution, the surface potential at the gate region is influenced, thereby modifying the conductivity of the silicon channel. This change effectively transforms ISFET biosensors into transducers capable of converting chemical information into an electrical signal. The fundamental working principle of ISFETs hinges on the modulation of the threshold voltage (V_th) of the transistor. When an ISFET is exposed to a solution with varying ion concentrations, the surface potential of the gate region is altered, affecting the gate-source voltage (V_gs). This, in turn, changes the V_th of the transistor. The V_th shift is directly proportional to the ion concentration in the solution. As a highly versatile sensor, there have been a lot of attempts to use ISFET-based electrochemical sensors in the detection of various biomarkers. In this section, we emphasize the sensing mechanisms for ions, nucleic acids, proteins, and cells.


	Fig. 1 (A) The schematic diagram of the ISFET. (B) The chip is composed of ISFET arrays compatible with CMOS technology. Reproduced with permission.³⁷ Copyright 2021, Springer Nature.

2.1.1. Ions. Specific sensing of certain ions (K⁺, Ca²⁺, etc.) can be achieved by additional surface modification of the gate insulator or by depositing a specific film on top of the gate insulator. Taking a pH-sensitive ISFET as an example, the surface of the gate insulator (usually Si₃N₄, Al₂O₃, or Ta₂O₅) is hydrolyzed into ionizable sites (such as –OH groups) in aqueous solution, which can bind or release H⁺ during dynamic exchange, so their protonation state can change with the pH value of the surrounding solution. The interface potential is generated on the surface of the insulator in response to variations in the H⁺ concentration, resulting in the activation of the drain current through the conduction channel. Therefore, the pH of the solution can be accurately quantified according to the change in the drain current.

2.1.2. Nucleic acid. The gate insulator is usually an oxide film or a nitride film, which has hydroxyl groups on its surface in solution. Silane coupling reagents with active groups can covalently bind to the hydroxyl groups and then modify the oligonucleotide probe at the end. The phosphate backbone of the nucleic acid molecule carries negative charges. The specific recognition and hybridization of nucleic acids forms a double strand, thus increasing the negative charge density on the gate surface and causing an electrical signal change.³⁸

2.1.3. Protein. The principles of common protein detection can be divided into two types: direct method and indirect method. The base of the direct method is most proteins such as antigens and antibodies are charged molecules. The formation of an antigen–antibody complex at the gate oxide/solution interface can cause changes in charge distribution, affect the threshold voltage of the immuno-field effect transistor, and directly adjust the drain current of the ISFET.³⁹ However, only charge changes within the Debye length range can be detected. However, the sizes of antibody macromolecules (usually 10 nm) are much longer than the Debye length in physiological solutions (1 nm). The distance between the protein charge and the surface is greater than the Debye length and will be shielded by counterions. Therefore, immuno-field effect transistors can only be used in low ionic strength solutions. The indirect method is to connect labeled molecules (such as enzymes) to the antigen/antibody, and then detect the enzyme-catalyzed product or reduced substrate based on the specific binding of antigen and antibody.

2.1.4. Cell. Carbon dioxide, a byproduct of cellular respiration, dissolves in the solution and causes pH changes. The pH change of the solution depends on the cell activity, so ISFET can monitor cell activity in real-time. On the basis of cellular respiration, embryonic activity, allergic reactions, chondrocyte organization, autophagy, and carcinogenesis can also cause changes in the interface pH or specific ion concentration at the nanogap between cells and gates, which can be captured and output by ISFET.

2.2. Modification of surface functional membranes

The effective coupling of recognition elements and signal sensors in electrochemical biosensors is the key to information transmission. Functional membrane modification on the electrode surface can significantly increase the binding constant (K_a) between the biological sample and the recognition element, play an important role in the specific recognition of targets, and effectively improve the signal-to-noise ratio and detection limit. Therefore, we should also pay great attention to the ways of functional membrane modification on the ISFET, which can be mainly divided into chemically synthesized, physically or chemically structured, and biologically induced, according to the diversity of materials.^40,41

2.2.1. Chemical substances. A polymer membrane with functional monomers can be synthesized on the surface of the sensor. And the chemical substances on the chemical polymer membrane can interact with charged ions and biomolecules. For example, the hydrophobic polymer layer can capture ion carriers with poor water solubility, while water-soluble biomolecules such as glucose can easily bind to the hydrophilic polymer membrane. The above polymer membrane helps to change the charge density of ions and molecules and convert them into electrical signals. For example, some researchers have used the negatively charged deprotonated boric acid diol ester formed by phenylboronic acid to interact with low molecular weight oligosaccharides such as paromycin and kanamycin to achieve quantitative analysis.⁴²

2.2.2. Nanostructures. Adding nanostructures such as nanoshells and nanopores to the electrode surface can increase the surface area of the electrode. Polymer porous nanofilters reduce the nonspecific electrical signals of small molecules and biomacromolecules, effectively improving the signal-to-noise ratio of electrode interface signal detection. Sakata et al.⁴³ found that polyaniline-based nanofilters can capture interfering substances and reduce the effect of nonspecific adsorption of biological molecules such as proteins on potential, but it will not prevent the penetration of small molecules. The introduction of AuNPs significantly enhanced the potential signal. In addition, surface modification such as electrografting of aromatic diazonium salts on the electrode surface is also an excellent choice for preparing the filter layer. The gaps between the grafted aromatic molecules allow small molecules to reach the electrode surface while inhibiting the adhesion of large biological molecules. Moreover, the preparation process of aromatic diazonium salt membranes is controllable, which helps to control the thickness of the anchoring layer to be consistent with the Debye length.^44–46 Besides, due to aptamers’ specific recognition ability, they have important potential in capturing target molecules, preventing the adhesion of interfering substances, and improving the detection signal-to-noise ratio.^47,48

2.2.3. Biological materials. Cells cultured on the electrode surface themselves can serve as biological materials to detect biomarkers, such as antibodies, through specific membrane antigens. Other biologically derived materials including antibodies and enzymes are also useful in providing the specificity and activity, thus triggering immunological reactions and generating electrical signals. Some researchers have proposed that basophils release histamine in immune responses based on IgE antigen reactions, and the potential of antigen-specific IgE to activate patient basophils can be evaluated by detecting the pH changes caused by histamine.⁴⁹ Besides, when cells are cultured on the electrode surface, some proteins in the culture medium are non-specifically adsorbed on the gate during the pre-culture process, which not only helps the cell adhesion but also prevents the approach of charged molecules. But much smaller H⁺ can easily pass through and bind to the hydroxyl group on the gate surface, achieving a specific pH response.

2.3. Integration of ISFETs with CMOS technology

The integration of ISFETs with CMOS technology further enhances these sensors, enabling miniaturization, power efficiency, and integrated signal processing, and this powerful synergy has transformed the landscape of sensing technology (Fig. 1B). This miniaturization not only reduces the space required for sensor installations but also makes it possible to incorporate these sensors into smaller, more portable devices. The reduction in power consumption is another key advantage of this integration. ISFET biosensors integrated with CMOS technology consume significantly less power compared to traditional ISFETs. This improvement is essential for ensuring the longevity of battery-powered devices and reducing the energy demand of sensor networks. It opens up opportunities for long-term, unobtrusive monitoring applications in remote or resource-constrained environments.

One of the most exciting developments in the wake of this integration is the potential for POCT applications. Portable diagnostic devices that leverage ISFET biosensors integrated with CMOS technology can provide rapid and highly accurate results. These devices are particularly valuable in healthcare settings, where timely clinical decisions can be a matter of life and death. From clinics to remote field locations, these portable diagnostic tools are becoming indispensable for healthcare professionals. In clinical settings, POCT devices equipped with integrated ISFET biosensors and CMOS technology can offer rapid and reliable diagnostic capabilities. They are especially useful for conducting on-the-spot assessments, monitoring disease progression, and providing real-time feedback to healthcare providers. For example, a handheld device could measure a patient's pH levels, detecting abnormalities or trends that might require immediate attention. Beyond clinical environments, portable diagnostic devices equipped with ISFET biosensors integrated with CMOS technology have the potential to transform healthcare in remote or resource-limited regions. They can be used for the rapid diagnosis of infectious diseases, enabling early detection and timely intervention. This can help prevent the spread of diseases and improve overall public health outcomes.

In conclusion, ISFET biosensors, especially integrated with CMOS technology have ushered in a new era of sensing technology. The combination of miniaturization, power efficiency, and integrated signal processing has not only enhanced the capabilities of these sensors but has also had far-reaching implications for a variety of applications, especially in the field of POCT. The development of portable diagnostic devices that utilize these sensors has the potential to revolutionize healthcare, making rapid and accurate diagnostics accessible in a wide range of settings, from clinical environments to remote field locations. This technology has not only transformed the landscape of sensing technology but also has promise for addressing some of the most pressing challenges in healthcare and environmental monitoring.

3. Application of ISFET biosensors in biomarker detection

Early diagnosis and personalized precision medicine are pivotal strategies for improving patient prognosis and extending survival. In recent years, ISFET sensors have demonstrated their effectiveness in both qualitative and quantitative analysis of ions, nucleic acids, disease-related molecular markers, and molecular interactions. In the subsequent sections, we present a comprehensive discussion, replete with specific examples, of their applications in the detection of major diseases and related methodologies, with a specific emphasis on the utility of ISFET biosensors.

3.1. Cardiovascular diseases

Cardiovascular diseases represent a broad spectrum of conditions that impact the heart and blood vessels, presenting a significant global health challenge. This category encompasses various ailments, including ischemic heart disease, coronary artery disease, heart failure, and other related disorders. Early detection and ongoing monitoring of cardiovascular diseases are essential for enhancing patients' outcomes. In this context, ISFETs offer valuable capabilities. Of these, immuno-ISFET, which utilizes antigen–antibody specific binding, is the most widely used. By leveraging the foundational principles of an enzyme-linked immunosorbent assay (ELISA) detector, specific antibodies immobilized on the surface of the ISFET sensor capture target biomolecules in the patient's blood sample (Fig. 2A). Through the application of enzymatic reactions and subsequent signal amplification, ISFET biosensors can quantitatively detect biomarkers such as cardiac troponins I (cTnI), brain natriuretic peptide (BNP), N-terminal pro-BNP (NT-proBNP), tumor necrosis factor-alpha (TNF-α), and interleukin-10 (IL-10). These biomarkers furnish crucial insights into heart health, facilitating early diagnosis and continuous monitoring of cardiovascular diseases, thereby enhancing patient care and management.


	Fig. 2 (A) The detection principle on a handheld test device for cTnI measurement and the physical picture of it. Reproduced with permission.⁵⁰ Copyright 2021, Elsevier. (B) Schematic diagram of the rGO-ISFET biosensor interface for the detection of NT-proBNP.⁵¹ Copyright 2019, Elsevier. (C) Sensitivity and selectivity of TNF-α detection using an ISFET immunosensor.⁵² Copyright 2021, Elsevier.

According to data released by the World Health Organization (WHO), ischemic heart disease remains the leading global cause of death, accounting for 16% of all worldwide fatalities.⁵³ Acute myocardial infarction (AMI) is typically a condition of ischemic heart disease. The American College of Cardiology guidelines designate cTnI as the gold standard biomarker for diagnosing AMI, with a sharp increase in peripheral blood concentration indicating a possible heart attack.^54,55 Given the rapid onset of AMI, early and sensitive monitoring of changes in peripheral blood cTnI concentration is crucial for ensuring timely and accurate resuscitation, a critical factor in saving the patient's life and improving their prognosis. To guarantee the effectiveness of resuscitative measures, the total duration of cTnI testing should not exceed 60 minutes.⁵⁶ ISFET biosensors, renowned for their compact size, swift response, and easy integration, have become the core detection components for constructing POCT devices for clinical analytics of cTnI concentration.^57–59

Wang and his research team addressed a pressing need by developing a rapid, accurate, and portable diagnostic device aimed at the early detection of AMI (Fig. 2A).⁵⁰ Their approach harnessed In₂O₃ nanobelts and circuit modules to successfully integrate these components into a compact, portable platform, enhancing accessibility for POCT. Central to this device was the incorporation of a dual-gate ISFET, specifically designed to enable early AMI detection through the identification of cTnI. Their innovative methodology featured a one-step ELISA strategy, facilitating the conversion of trace amounts of cTnI in serum to a drain–source current, with the ultrasensitive ISFET assuming a pivotal role in this process. The portable device showcased a wide linear detection range spanning from 1 to 1000 pg mL⁻¹, and an impressively low detection limit of 0.3 pg mL⁻¹, significantly surpassing the current diagnostic cutoff of 40 pg mL⁻¹ for AMI. Furthermore, the device exhibited exceptional selectivity and consistently delivered reliable results within a mere 20-minute timeframe when analyzing actual serum samples. This groundbreaking technology holds significant promise for the early screening and diagnosis of AMI, particularly in non-hospital settings, as it substantially reduces the reporting time. Moreover, it offers highly specific and accurate cTnI recognition, potentially expediting the process of early diagnosis and emergency care for non-hospitalized patients.

Heart failure is also a complex clinical syndrome arising from various cardiovascular diseases, encompassing structural and functional abnormalities of the heart. This condition leads to compromised heart pumping or filling capacity, ultimately progressing to heart failure, necessitating lifelong treatment and monitoring, which is often cost-intensive.⁶⁰ Globally, an estimated 640 [thin space (1/6-em)] 000 individuals are afflicted by heart failure, emphasizing the importance of early detection and intervention. Timely interventions can significantly enhance recovery prospects, reduce complications, and improve patients’ quality of life.⁶¹ One valuable biomarker for assessing heart failure is BNP, a protein released by heart ventricle cardiomyocytes in response to stimuli such as tension and strain.⁶² A growing body of research confirms the correlation between peripheral blood levels of NT-proBNP and BNP and the severity of heart failure. Elevated concentrations of these biomarkers indicate poor cardiac function, while the absence of a substantial decrease in concentration after anti-failure therapy suggests limited treatment effectiveness and a gloomier prognosis for patients.^63–65 Real-time monitoring of NT-proBNP and BNP at home offers a significant opportunity for early diagnosis, intervention, and treatment monitoring. On-site quantitative testing at outpatient and emergency clinics provides crucial guidance for timely treatment adjustments. Walid and his team selected reduced graphene oxide (rGO) as a highly sensitive transducer to achieve lower detection limits.⁵¹ The rGO-ISFET enables rapid response and the generation of electrostatic charge signals from biomolecules (Fig. 2B), contributing significantly to rapid, accurate, and reproducible detection. This innovative technology has given rise to a highly sensitive immunobiosensor platform that allows label-free detection of NT-proBNP antigens in a reusable manner. Notably, this platform boasts an impressive detection limit as low as 1–10 pg mL⁻¹, considerably below the NT-proBNP concentration threshold for Stage 1 heart failure as specified by the New York Heart Association. The assay's linear range aligns well with the clinically relevant range of NT-proBNP, offering substantial clinical utility.

The two-dimensional electron gas structure established by the AlGaN/GaN heterojunction exhibits exceptional carrier mobility and electron density potential. Zhang et al. innovatively developed an AlGaN/GaN-based immunosensor platform to effectively mitigate background signal interference. This technological advancement enabled the achievement of ultra-sensitive BNP detection with an astonishingly low detection limit of 0.097 pg mL⁻¹.⁶⁶ Furthermore, the utilization of magnetic bead-immobilized antibodies contributed to heightened device reproducibility. TNF-α is a pivotal pro-inflammatory cytokine closely associated with the severity of cardiac diseases.⁶⁷ Remarkably, the serum concentration of TNF-α exhibits a robust linear correlation with salivary levels. Saliva, as a non-invasive sampling medium, offers the advantage of minimal technical complexity and reduced psychological burden on the examinee. This characteristic significantly enhances patient compliance, rendering it particularly suitable for long-term monitoring of target biomarkers.^68,69 Albert and colleagues introduced an innovative label-free ISFET immunosensor, featuring a biofunctionalized silicon nitride sensing membrane. This cutting-edge technology is specifically tailored for the detection of TNF-α in saliva. Moreover, it has been seamlessly integrated into an e-health system, facilitating efficient health data management. This integrated approach has the potential to revolutionize cost-effective patient health monitoring.⁷⁰ To enhance sensitivity and enable automated result transmission, Hamdi and colleagues ingeniously combined an ISFET immunosensor with electrochemical impedance spectroscopy for the first time. This resulted in a highly selective platform with a wide linear range for analyzing salivary samples, achieving a remarkable detection limit as low as 1 pg mL⁻¹ for TNF-α (Fig. 2C).⁵²

Salivary cortisol, a steroid hormone, plays a role in numerous metabolic pathways and serves as an effective predictor of heart failure. Monitoring cortisol concentrations serves as a useful tool for the early diagnosis of heart failure and the assessment of disease progression.⁷¹ This research team further explored the value of the immuno-ISFET biosensing platform in salivary cortisol testing, providing a comprehensive analytical tool for the diagnosis and long-term health management of heart failure patients.⁷² Recognizing the limitations of single biomarker assays in disease diagnosis and progression monitoring, the research team in Evanthia's group introduced the KardiaTool platform for multi-target analysis of heart failure biomarkers. This platform can simultaneously quantify NT-proBNP, TNF-α, IL-10, and cortisol in saliva samples.⁷³ It comprises two components: KardiaPOC and KardiaSoft. KardiaPOC integrates microfluidic channels, sensors, actuators, microelectromechanical systems, microelectronics, biochemical components, and functionalized magnetic nanoparticles into a disposable lab-on-a-chip. Saliva samples undergo pre-concentration and purification with functionalized magnetic beads. Following thorough washing, the samples enter the detection chamber constructed by ISFET immunosensor arrays to determine marker concentrations. The results of the tests and patient clinical data are automatically transmitted to KardiaSoft for analysis and diagnosis, as well as recommendations for monitoring treatment efficacy. This POCT device efficiently follows a “sample in, result out” approach, characterized by low sampling technical requirements, multi-target detection capabilities, and robust analytical functions. The KardiaTool platform is poised for productization and has the potential to become an essential tool for heart failure patients, facilitating self-testing at home and aiding medical professionals in on-site assessments.

3.2. Infectious disease

Infectious diseases, caused by living pathogens like bacteria, viruses, parasites, and others, are capable of rapid transmission among human or animal vectors through various means.⁷⁴ Effectively managing the source of infection is pivotal for limiting pathogen spread. Therefore, precise diagnosis not only provides a scientific foundation for accurate and efficient treatment but also plays a crucial role in infectious disease prevention.⁷⁵ Traditionally, the gold standard for most microbial testing involves the isolation and culture of pathogens, a time-consuming process, typically taking 2–4 days. Meanwhile, commonly used molecular techniques like polymerase chain reaction (PCR) testing have their limitations, including complex procedures, reliance on specialized equipment, and high costs. Immunological tests struggle to achieve early infection diagnosis and are susceptible to false negatives. In contrast, POCT enables a “sample in, result out” approach without requiring separate sample handling and extraction. It offers advantages such as safety, speed, user-friendliness, and reduced chances of errors and contamination in intermediate steps.⁷⁶

Respiratory tract infections stand as a leading cause of morbidity and mortality in both children and adults worldwide, representing one of the deadliest infectious diseases globally.⁷⁷ The emergence of SARS-CoV-2 in December 2019 has further emphasized the urgent need for rapid and precise diagnostic tools to enable early detection of viral infections and effectively manage outbreaks.⁷⁸ In response to this critical challenge, SEO et al. reported an immuno-FET biosensor for the SARS-CoV-2 spiking protein, with high sensitivity and without the need for sample pre-treatment or labelling (Fig. 3A).⁷⁹ Nicolas and his research team have developed a handheld POCT device for the detection of SARS-CoV-2, aptly named “Lacewing technology” (Fig. 3B).⁸⁰ This innovative device can efficiently extract RNA from viral swabs within 5 minutes and subsequently detects the H⁺ ions generated during the reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) process through a 78 × 56 ISFET array. Within a mere 15 minutes, the test results can be relayed to mobile applications and synchronized with electronic health records, further offering geotagging for real-time pandemic monitoring. This technology proves invaluable in shaping effective pandemic management strategies. Furthermore, Mohsen and colleagues have introduced an electrical, label-free detection technique based on CMOS-compatible silicon nanowire ISFETs. This method allows for the direct detection of COVID-19-specific T cells in the patient's blood, as well as the assessment of the activation status of antigen-specific T cells. This breakthrough facilitates real-time monitoring of human immunity, enabling the effective differentiation between early infections and asymptomatic cases and mitigating the misuse of medications.⁸¹ In the context of respiratory infections, influenza viruses are among the most prevalent culprits causing acute respiratory illnesses.⁸² In this regard, Bong and collaborators have constructed an immunosensor featuring an antibody targeting the hemagglutinin protein as the recognition element (Fig. 3C).⁸³ The presence of the influenza virus triggers the formation of a “capture bead-virus-urease” complex in the reaction system, resulting in no pH change upon the addition of urea. Conversely, in the absence of the influenza virus, substantial production of hydroxide ions in the filtrate is observed, leading to a noticeable change in the potential signal output from the ISFET biosensor (Fig. 3D). This immunosensor facilitates a wash-free detection process completed within 20 minutes, offering a swift and efficient means of influenza virus detection.


	Fig. 3 (A) A highly sensitive immune-FET-based detection process for COVID-19 virus in nasopharyngeal swabs. Reproduced with permission.⁷⁹ Copyright 2020, American Chemical Society. (B) Diagnostic workflow of Lacewing from sample to result based on the CMOS microchip integrating over 4000 ISFET sensors. Reproduced with permission.⁸⁰ Copyright 2021, Institute of Electrical and Electronics Engineers. (C) Principles of a one-step immunoassay for influenza A virus based on two magnetic beads and filters. Reproduced with permission.⁸³ Copyright 2020, Elsevier. (D) The pH changes according to the concentration of influenza virus colorimetrically determined using the indicator phenol red and with ISFET. Reproduced with permission.⁸³ Copyright 2020, Elsevier. (E) The specific quantitative test for Plasmodium falciparum based on a novel CMOS lab-on-a-chip assay. Reproduced with permission.⁸⁴ Copyright 2019, Elsevier.

Preoperative and pre-transfusion testing for four infectious markers, including human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and syphilis spirochetes, serve a vital role in identifying infections in patients prior to hospitalization. This not only reduces the risk of exposure to medical staff but also lowers the incidence of medical disputes. In cases of emergency surgery, rapid acquisition of a patient's infection information is essential for implementing effective preventive measures. Currently, laboratory-based antigen, antibody, and nucleic acid detection remain the primary methods for detecting these pathogens. However, their significant drawbacks, such as time consumption and dependence on bulky equipment, limit their clinical utility. There is an urgent need to expedite the development of POCT products for these infectious markers. To address this challenge, researchers have made several attempts. For instance, Lee and colleagues leveraged the strong negative surface charge of hepatitis B surface antigen (HbsAg) to create an immunosensor with an anti-HbsAg immobilized sensing film, achieving a remarkable detection limit of 22.5 fg mL⁻¹.²⁷ Chang and his team enhanced the design of extended electrodes and the size of the ISFET sensor, enabling the detection of DNA hybridization signals for HBV at fM levels.¹⁸ Another group harnessed integrated CMOS electronic devices to overcome detection obstacles arising from the screening effect of high ionic strength solutions. They achieved sensitive capture of HBV DNA strand hybridization signals down to fM levels in a 1 × PBS solution environment.⁸⁵ Furthermore, Naqeebullah and his team introduced a handheld device that combines ISFET and RT-LAMP technologies for the rapid detection of hepatitis viruses. This device, by monitoring real-time pH changes in the system, can detect positive samples within 30 minutes and automatically transmit results, offering the potential for short-term and accurate bedside testing.⁸⁶ Similarly, Cooke et al. developed a novel chip-based CMOS assay for the 8-minute detection of HIV RNA, providing an effective means for timely virus detection and ensuring effective treatment to prevent drug resistance emergence.⁸⁷ Significantly, the main methods for syphilis spirochete detection mainly involve serologic detection, and efficient molecular detection methods remain limited. The potential of using ISFET in this context requires further exploration.^88,89

Malaria stands as one of the most menacing infectious diseases in low-income regions. According to the WHO, a staggering 247 million individuals were infected with malaria in 2021, with Africa bearing the brunt of 95% of these cases. In light of these alarming statistics, the development of low-cost testing devices that do not depend on specialized laboratories or technicians has immense value in screening for malaria infections and ensuring timely treatment of the disease.⁹⁰ To address this critical need, Malpartida-Cardenas and their team have introduced an innovative molecular detection platform for Plasmodium falciparum, the parasite responsible for a significant portion of malaria cases.⁸⁴ This platform combines ISFET with highly efficient RT-LAMP technology. This unique synergy allows for the quantification of plasmodium falciparum DNA and the detection of single-nucleotide polymorphisms in the artemisinin-resistant malaria-associated C580Y gene within 20 minutes (Fig. 3E). The clinical utility of this platform shows immense promise as it empowers healthcare professionals to provide patients with rapid diagnosis and conduct pathogen resistance analysis. This, in turn, equips doctors with a crucial foundation for formulating effective treatment plans, ultimately reducing the risk of delayed treatment and the ineffective use of medications. This advancement is a significant step toward the efficient and timely management of malaria infections, particularly in resource-constrained settings.

In conclusion, the integration of advanced diagnostic tools like ISFET-based devices with artificial intelligence and data analytics has great promise for real-time monitoring and response to major epidemic diseases. Furthermore, the development of more compact utility in resource-limited regions, where the burden of infectious diseases is often the greatest, is important. Despite the persistent challenges posed by infectious diseases, the progress made in POCT and related technologies offers hope for more efficient and effective disease control. Looking ahead, scientific research and technological innovation will continue to lead the way in our ongoing battle against infectious diseases, providing better tools for global communities to achieve rapid diagnosis and response. These advances underscore the vital role of diagnostics in maintaining public health and controlling the spread of infectious diseases. In this context, ISFET technology stands as an integral component with the potential to offer even more possibilities for infectious disease monitoring and control in the future.

3.3. Cancer

Cancer is a major global health concern, responsible for nearly 10 million deaths in 2020.^53,91 Moreover, cancer incidence continues to rise each year, emphasizing the need for early detection and prompt intervention.⁹² Liquid biopsy, as an alternative to invasive tissue biopsy, offers several advantages, including minimal patient discomfort, reduced sampling errors, and lower psychological burden. Tumor marker testing serves a pivotal role not only in early cancer screening and classification but also in tumor staging, progression, and chemotherapy resistance.⁹³ In healthy individuals, tumor markers typically exist at low concentrations. However, when a tumor develops, these markers can increase in the bloodstream, with the degree of elevation often corresponding to the stage and progression of the cancer. Given the complexity of body fluids and the need to detect subtle changes in marker concentrations, there is an urgent demand for rapid, highly specific, and sensitive assays for clinical applications. Notably, the field of tumor-associated marker detection has witnessed significant advancements, with ISFET biosensors emerging as a label-free technique.

Lung cancer remains one of the deadliest malignancies, with an approximate daily toll of 350 lives.⁹¹ Human breath contains a plethora of metabolites and volatile organic compounds (VOCs) arising from various sources within the body, including the respiratory tract, internal organs, and the microbiome. The composition and quantity of these compounds can vary according to an individual's health status, rendering them potential biomarkers with various clinical applications, including disease screening and diagnosis.⁹⁴ An innovative approach to detecting VOCs involves the utilization of drosophila LUSH proteins, known odor-binding proteins that interact with various alcohols.⁹⁵ Lin and his team used LUSH proteins as recognition elements for ethanol and amplified the signals generated by molecular interactions through a dual-gate ISFET sensing system (Fig. 4A).⁹⁶ This method proved highly successful in detecting ethanol concentrations ranging from 0.001–1%. Furthermore, it led to the development of a novel olfactory sensing platform with broad applications, including non-invasive diagnostics, detection of toxic substances, and freshness testing of food. Research has shown that elevated levels of 1-propanol and hexanal are associated with the development of lung tumors, underlining the potential of this platform for lung cancer screening through breath analysis. The heavy metal cadmium has been linked to the risk of lung cancer. Monitoring cadmium levels in water and blood is crucial for safeguarding human health.^97,98 Wang and his team addressed this challenge by developing a cadmium ISFET. This innovative technology involved modifications to the FET extended gate structure, ensuring that detection performance remains unaffected by the complex components present in serum samples. The cadmium ISFET device demonstrated a wide linear detection range from 10⁻¹¹ to 10⁻⁷ M, making it a highly valuable tool for clinical applications.⁹⁹ Moreover, ISFET biosensors have emerged as indispensable tools for monitoring treatment efficacy and investigating drug mechanisms. The approval of cisplatin as an anticancer drug has not only paved the way for a new generation of platinum-based therapies but also heralded an era of transition metals as potential anticancer agents. Researchers, such as Zhang and his team, have explored the cytotoxicity and ligand-controlled reactivity of cyclometalated rhodium(III) complexes using ISFET-based mini-pH-meters.¹⁰⁰ These complexes demonstrated a remarkable 5-fold higher antiproliferative effect on A549 lung cancer cells in comparison to cisplatin, hinting at their potential as novel transition metal-based chemotherapeutic agents.


	Fig. 4 (A) The representation of ISFET sensors equipped with special sensitive membranes used to detect ethanol, and several detection results under different conditions. Reproduced with permission.⁹⁶ Copyright 2017, American Chemical Society. (B) The ISFET enabled the detection of EGFR in breast cancer cells by combining enzymatic reactions, as well as the trend of detection signal with pH change. Reproduced with permission.¹⁰¹ Copyright 2022, American Chemical Society. (C) The expected workflow for prostate cancer patients screening from liquid biopsy extraction to deriving clinically viable responses by mRNA detection using the ISFET biosensor and optimized RT-LAMP assay. Reproduced with permission.¹⁰² Copyright 2022, American Chemical Society.

Breast cancer stands as the most prevalent malignant tumor among women, comprising 31% of female cancers.⁹¹ Notably, a subset of breast cancer patients, approximately 3–20%, harbor mutations in the PIK3CA p.E545K gene, a critical therapeutic target.¹⁰³ Promising outcomes have been achieved with the small molecule inhibitor Alpelisib for patients bearing the PIK3CA p.E545K mutation. To distinguish between the wild type and mutant type, Alexandrou and colleagues combined a 78 × 56 ISFET sensor array with LAMP technology.¹⁰⁴ The presence of a mutation in the examined sample triggered the LAMP reaction, while the ISFET captured the resulting pH change in the system, converting it into a voltage signal. This innovative combination not only shortened the reaction time and simplified experimental procedures but also substantially improved detection sensitivity through multi-stage signal amplification. Remarkably, this technology enabled the differentiation between wild-type and mutant samples in just 15 minutes, with a detection limit of 100 copies per reaction. Furthermore, the team developed a portable real-time nucleic acid testing platform that facilitates automated data transmission to the cloud. This platform boasts evident advantages, including high sensitivity, high specificity, and cost-effectiveness, making it a valuable asset in healthcare, especially in resource-constrained regions. Another approach proposed by Tabata and colleagues centered around the encapsulation of the ISFET gate with an extracellular matrix gel, which could capture breast cancer cells with varying levels of epidermal growth factor receptor (EGFR) expression.¹⁰¹ Subsequently, they introduced an anti-EGFR antibody linked to glucose oxidase in the system. When glucose oxidase interacts with its substrate, it generates gluconic acid, resulting in a decrease in system pH. These pH changes were effectively captured and output by the ISFET, with the output signal varying based on the level of EGFR (Fig. 4B). This device represents a novel biomarker detection technology for living cells, combining cellular FETs and enzyme-based signal amplification. This innovative approach holds great promise for the capture and detection of circulating tumor cells. Additionally, it was proposed that apoptosis, a key cellular process, leads to changes in pH.¹⁰⁵ To explore this phenomenon, Shah and his team suggested the use of staurosporine to treat breast cancer cells and monitor the resulting pH changes in the cell culture medium using ISFET arrays.¹⁰⁶ This approach allows for the determination of whether breast cancer cells are undergoing apoptosis. The use of a precisely calibrated and programmable floating gate channel ISFET effectively controls drift and widens the detection range, showing promise for monitoring drug efficacy.

Prostate cancer has emerged as the most prevalent malignancy in men as of 2023, accounting for 21% of all cases.⁹¹ Elevated serum levels of prostate-specific antigen (PSA) often serve as a precursor to prostate cancer, underscoring the importance of highly sensitive and specific PSA detection for early diagnosis and prolonged patient survival.¹⁰⁷ In pursuit of this goal, Ma and a team of researchers made significant advancements by modifying a nanoribbon-based ISFET (NR-ISFET) with polyethylene glycol on its surface.¹⁰⁸ This modification effectively mitigated the shielding effect imposed by the Debye length on molecular detection, enhancing the sensor's affinity for protein interactions. The improved sensor exhibited the capability to detect PSA in high ionic strength solutions and human plasma, achieving detection limits as low as 10 pM. Furthermore, the simultaneous connection of the NR-ISFET chip to multiple microchannels allowed for the concurrent detection of various functionalized NR-ISFETs, opening the door to multiple cancer biomarker detection through this platform. In light of the remarkable progress in silicon nanotechnology, Rani highlighted the exceptional advantages, including a high surface area ratio and precise fabrication control, which make silicon nanowire ISFETs (SiNW ISFETs) an enticing platform for biomolecular detection.¹⁰⁹ By covalently bonding PSA-specific aptamers to the SiNW surface and amplifying the response signals using nano-sized transistors, Rani significantly improved protein detection sensitivity. The detection limit reached an impressive low of 300 fM, well below the clinically relevant range. This assay platform has the potential to significantly enhance prostate cancer detection rates. Nonetheless, some studies have proposed that the sensitivity and specificity of PSA screening require enhancement to mitigate the risk of underdiagnosis and overtreatment.^110,111 Therefore, there is a pressing need to explore novel markers for early prostate cancer screening. Researchers led by Schostak discovered that urinary membrane-bound protein A3 (ANXA3) is a highly specific and novel non-invasive prostate cancer biomarker. Zheng and colleagues made substantial improvements in the detection sensitivity of ANXA3 in unprocessed samples by enhancing the capacitive coupling between the gate dielectrics at the top and bottom of the thin-film transistor. They further proposed measuring the reference signal of each patient's urine sample for self-normalized detection, minimizing the influence of variations between different urine samples.^112,113 Combining these enhancements, the team introduced an ISFET-based urine immunoassay with a remarkable detection limit below 1 fg mL⁻¹. In addition, Broomfield and colleagues introduced a non-invasive lab-on-a-chip platform for the immediate detection of circulating mRNA in prostate cancer (Fig. 4C).¹⁰² Distinguishing it from other methods, they established a molecular detection platform that fuses RT-LAMP with ISFET sensors to enable the multiple amplification of signals. This platform can complete the detection of target RNA in prostate cancer cells within 22 minutes.

In summary, the field of ISFET biosensors has made significant strides in cancer detection, offering highly sensitive and specific assays for various cancer biomarkers. From lung cancer to breast cancer and prostate cancer, ISFET-based technologies have showcased their potential to revolutionize cancer screening, diagnosis, and treatment monitoring. These innovative approaches not only enhance early detection but also provide valuable insights into treatment efficacy and the development of novel therapeutic agents. The continued advancement of ISFET technology, coupled with ongoing research into new biomarkers and assay techniques, has great promise for improving cancer patient outcomes and reducing the global burden of this disease. As we move forward, we can anticipate that ISFET-based biosensors will play an increasingly vital role in the fight against cancer, offering faster, more accurate, and non-invasive diagnostic solutions.

3.4. Diabetes

Diabetes, a metabolic disease characterized by chronically higher than standard blood glucose levels, has become one of the global public health threats. Approximately 194 million people worldwide had diabetes (5.1%) and 314 million had impaired glucose tolerance (8.2%) in 2003, which are projected to increase to 7.3% and 8.0% respectively, by 2025.¹¹⁴ Diabetes and its complications (such as diabetic retinopathy, diabetic nephropathy, diabetic retinopathy, etc.) have brought heavy health and economic burden to individuals, families and the public health system.^115,116 The main way to diagnose and control diabetes is real-time monitoring of blood glucose and glycosylated hemoglobin levels. Following the advances in electrochemistry, the measurement of glucose concentration has become one of the classical applications of ISFET biosensors, and there has been extensive literature on the composition and innovations of the relevant platforms, which are briefly described below. Nilsson et al. proposed the use of an enzyme-pH-electrode to determine glucose concentration at an early stage of research.¹¹⁷ The enzyme is immobilized in a polyacrylamide gel around the electrode, and the pH change of the solution is detected after the addition of the glucose substrate to achieve indirect quantification of glucose concentration. The detection capability of the ISFET sensor with immobilized enzyme for serum glucose was further explored by Saito and colleagues.¹¹⁸ In this device, a highly cross-linked albumin outer membrane of glutaraldehyde was added to the sensor surface, aiming at limiting the diffusion of glucose in solution and improving the sensitivity of the detection. This device is less disturbed by the complex components of serum and realizes the testing of non-diluted serum glucose, yet with a sensitivity that needs to be improved. Zhao et al. attached dextran-capped silver nanoparticles probes to the ISFET gate (Fig. 5A).¹¹⁹ Subsequently, the high affinity between ConA and carbohydrates was utilized to achieve highly sensitive quantitative detection of large ConA proteins, electrically neutral glucose molecules by introducing a biocompetitive mechanism (Fig. 5B and C). The detection limit of glucose was as low as 10 nM in the final results. Meanwhile, a matching cell phone application was created by the researchers to visualize and wirelessly control the sensing system through data acquisition, saving, analysis, and sharing functions, which reduces the impact of human operational errors on results.


	Fig. 5 (A) The sensing mechanism of ConA. (B) A diagram of glucose detection using the ISFET/Dex-AgNPs/ConA unit. (C) The results of ConA using the ISFET/Dex-AgNPs unit and the linear correlation results of Vout on the glucose concentration. Reproduced with permission.¹¹⁹ Copyright 2020, Elsevier.

More than just invasive, the results of commonly used blood collection are affected by the specification of the needle, the depth of puncture, and the volume of collection. The levels of small molecules such as glucose and creatinine in interstitial skin fluids including suction effusion fluid are quite comparable to those of serum, according to a study.¹²⁰ In this way, a sensing device for transcutaneous effusion fluid collection and real-time monitoring of glucose concentration was proposed by Kimura and other researchers.¹²¹ This innovation represents a good technical basis for a wearable blood glucose monitoring device. Wearable biosensors are attracting attention for their ability to continuously measure and screen markers in disease treatment, diagnosis, and health management.^122,123 Needles used in wearable miniature sensing devices are typically less than 1 mm in length and width and can penetrate the epidermis while avoiding blood vessels and nerve endings, which allows the microneedle arrays in the patch to enable real-time, painless, non-invasive, or minimally invasive detection of a wide range of biomarkers.¹²⁴ Zimmermann and other researchers have proposed a disposable minimally invasive self-calibrating continuous glucose monitor, which is composed of a hollow microneedle that collects epidermal mesenchymal fluid, an integrated porous poly-Si dialysis membrane, and an ISFET sensor that integrates glucose enzymes.¹²⁵ This monitor addresses the compatibility challenges of high-temperature wafer bonding with temperature-sensitive enzymes.

In conclusion, ISFET biosensors have made notable contributions to the effective management of diabetes by offering sensitive, specific, and invasive real-time monitoring of glucose levels. As we look ahead, the evolution of ISFET technology continues to have great promise. With further advancements, we anticipate even more precise, minimally invasive, and user-friendly monitoring solutions for diabetes and various other applications, ensuring better health outcomes and improved quality of life for individuals worldwide. The interdisciplinary collaboration between materials science, biotechnology, and nanotechnology is propelling the development of ISFETs, and their integration into wearable devices is on the horizon, bringing us closer to the next generation of cutting-edge biosensors.

3.5. Application in other situations

In addition to its role in the above applications, ISFET-based wearable biosensors have demonstrated their value in the analysis of sweat composition for human health monitoring. Garcia-Cordeo and colleagues introduced a fully on-chip integrated wearable sweat sensing system designed to track biochemical information conveyed by the skin's surface in real-time.¹²⁶ This system incorporates n-type nanoribbon ISFET sensors on an ultrathin silicon body insulator with a thin layer of embedded oxide in the substrate, effectively controlling electrostatic interference by eliminating parasitic capacitance. Furthermore, the device's microfluidic system passively collects small amounts of sweat (∼20 nL) from the human skin, allowing real-time compositional analysis triggered by ultra-small amounts of sweat in the resting state. This low-energy sensing system enables simultaneous monitoring of multiple markers (H⁺, K⁺, Na⁺) with stable and reproducible results. Bellando and their team integrated double-layer SU-8 micro-nano-channels into silicon-on-insulator FETs and incorporated micro-silver/silver chloride electrodes in the first layer to achieve accurate monitoring of biomarkers in sweat, resulting in the development of a technology platform known as “skin lab” successfully.¹²⁷ This platform leverages the remarkable advantage of zero-energy sweat pumping by gathering sweat through capillary force and delivering it to FET sensors. It has great potential for wearable devices used in precision medicine and healthcare. Moreover, various surface modification strategies for ISFETs have significantly expanded their application possibilities. Liu and other researchers innovatively immobilized the taste receptor (Gr5a) of Drosophila cells on the surface of an extended gate ISFET (EG-ISFET). They harnessed Gr5a's specific binding ability with alginate to achieve precise quantification of salivary alginate, serving the purpose of rapid screening for Alzheimer's disease.¹²⁸ Satake and their team reported a measurement platform for the accurate monitoring of cellular metabolic differences based on ISFETs.¹²⁹ By adding growth factors to chondrocytes, FETs monitored the electrical signals generated by pH changes resulting from cellular glycolysis or oxidative phosphorylation. This approach allowed for the indirect assessment of the proliferation ability of chondrocytes and elucidation of the mechanism of extracellular matrix synthesis by chondrocytes based on the signals. ISFET also stands out in the monitoring of anaphylactic reactions. Yang and their team skillfully induced rat basophilic leukemia cells to undergo an anaphylactic reaction and release histamine on the sensor's surface, enhancing the alkaline strength of the solution. This achievement realizes real-time, non-optical monitoring of anaphylactic reactions in living cells.⁴⁹

The versatility and growing range of applications for ISFET technology are driving innovation across various fields. From medical diagnostics to environmental monitoring, and wearable biosensors to real-time health tracking, ISFETs have established themselves as indispensable tools for researchers and healthcare professionals alike. The ability to measure ions, pH, and biomolecular interactions in a label-free, rapid, and highly sensitive manner offers unparalleled advantages. With ongoing research, we can expect even more sensitive and specific sensors, miniaturized devices for POCT, and new avenues for monitoring complex biological and environmental processes. ISFETs are poised to play a pivotal role in the evolution of sensing technologies, enhancing our ability to understand and interact with the world around us. Their potential is limited only by our imagination and commitment to exploring the frontiers of science and technology.

4. Performance optimization strategies

Although there has been substantial literature underscoring the importance of ISFETs in biomarker testing, most of these platforms remain uncommercialized. This can be attributed primarily to the inherent shortcomings of ISFET sensors, including signal drift, temperature sensitivity, and a constrained linear range. To enhance their sensing capabilities, extensive efforts have been dedicated to investigating strategies for optimizing ISFET performance. These strategies are primarily centered around bolstering the sensitivity of biosensing platforms and transcending the limitations imposed by the Debye length. The following paragraphs provide a concise overview of these endeavors, focusing on these two critical aspects.

4.1. Improved sensitivity of biosensing platforms

Improving sensitivity in biosensing platforms involves three primary approaches: amplifying the detection signal, optimizing sensor performance, and reducing interfering signals.

Specifically, in the realm of ISFET-based nucleic acid detection, there are two key methods for amplifying the detection signal. The first method involves affinity-based nucleic acid detection, which works by increasing the charge density on the sensor's surface. This is achieved through the pairing of complementary DNA strands. The second method relies on specific catalytic reactions between enzymes and substrates, leading to a high ionic strength within the system. Unfortunately, high ionic strength solutions lead to shorter Debye lengths, significantly reducing detection capabilities. To overcome this limitation, scientists introduced peptide nucleic acids (PNAs). These artificial polymers resemble DNA but lack phosphate groups, making them electro-neutral. In low buffer solutions, sensor surfaces coated with PNAs significantly amplify electrical signals when binding to DNA molecules with negatively charged backbones.¹³⁰ By immobilizing PNAs on the sensor's surface, researchers like Hahm developed the first ultra-sensitive SiNW electronic device for detecting DNA hybridization, achieving detection limits as low as a few femtomoles (Fig. 6A).¹³¹ In a groundbreaking move, Gao's team utilized rolled circular amplification (RCA) to generate negatively charged long single-stranded DNA products (Fig. 6B). This innovation enhanced the signal response of silicon nanowire field effect tubes, further improving sensitivity and the signal-to-noise ratio, enabling the detection of incredibly low DNA molecule concentrations of 50 aM L⁻¹ (Fig. 6C).¹³² Moreover, properly preparing the sample is another crucial step in increasing sensitivity. For ultra-sensitive DNA methylation detection, Maki and colleagues used magnetic beads modified with DNA probe molecules to capture and enrich target DNA.¹³³ Subsequently, target molecules released through a competitive reaction were bound to antibodies on the sensor's surface, generating electrical signals based on their negative charges.


	Fig. 6 (A) Ultrasensitive nanowire nanosensors and PNA modified on their surface for DNA trapping. Reproduced with permission.¹³¹ Copyright 2003, American Chemical Society. (B) Enhanced response signaling by RCA generation of long single-stranded DNA products. Reproduced with permission.¹³² Copyright 2013, American Chemical Society. (C) Plot of current versus time for a range of DNA probe concentration conditions. Reproduced with permission.¹³² Copyright 2013, American Chemical Society. (D) The principle and details of InN RUM-FET biosensors’ design. Reproduced with permission.¹³⁴ Copyright 2021, Elsevier.

The performance of the sensor device itself is another significant factor affecting sensitivity. Researchers have developed techniques like body effect reduction to enhance the accuracy of ISFET interface electronics. For instance, Chung and colleagues compensated for the temperature dependence and signal drift of ISFETs,¹³⁵ while Panteli's team thinned the top passivation layer inherent in the CMOS fabrication process using reactive ion etching.¹³⁶ By fine-tuning these aspects, they achieved substantial increases in sensitivity, passivation capacitance, and reduced capacitance attenuation, leading to more reliable detection results. Furthermore, Song's research introduced a FET biosensor based on coiled indium nitride microtubules, harnessing the unique properties of these microtubules to achieve label-free detection of HIV gp41 antibodies (Fig. 6D).¹³⁴ This novel approach achieved detection limits as low as 0.1 ng mL⁻¹, representing a remarkable 20-fold improvement over standard ELISA techniques. These advancements in ISFET technology play a critical role in pushing the boundaries of sensitivity in biosensing, ultimately enabling the detection of minuscule quantities of target molecules with greater precision.

The surface of the ISFET sensor has an ion-sensitive membrane. The chemical substances on the surface of the functional membrane can interact with the ions or charged biomolecules to be detected in the electrolyte solution and convert them into electrical signals. The detection sensitivity depends not only on the density of ion groups for specific capture on the ion-sensitive membrane, but also on the biological contamination such as proteins in blood samples, which can produce obvious non-specific adsorption on the surface of the ion-sensitive membrane and generate obvious noise signals. When the biosensor interface does not have sufficient selectivity for the biomolecules to be tested, the signals generated by interferers (i.e., noise) can severely interfere with the analyte signals and reduce the signal-to-noise ratio. How to achieve the separation and identification of trace target analytes by effectively reducing or eliminating the matrix effect in complex biological samples is still a challenging problem.

Surface chemical treatments such as modification of the electrode surface by hydrophilic polydopamine can also improve its anti-bonding properties, reducing the nonspecific adsorption of proteins but not decreasing the detection sensitivity to ions, keeping it near the Nernstian response.¹³⁷ The output signal of FET can be enhanced by increasing the electrode surface area, but the noise will also be amplified at the same time. Some researchers pointed out that if the nanofilter is modified on the surface of the sensor, the biomarker to be tested can pass through the special nanostructure, while the interferents of different sizes will be intercepted outside the Debye length and cannot approach the electrode surface to produce nonspecific noise signals.⁴⁴ The nanofilter membrane designed by Sakata et al. is mainly composed of an anchoring layer and a filter layer.^43–45 The anchoring layer is coated on the electrode surface, and the filter layer has a chromatographic effect, which can intercept large interfering molecules outside the high-density nanomultilayers, and at the same time, phenylboronic acid in the nanofilter membrane can capture small interfering molecules (Fig. 7A). Considering the screening effect of the Debye length, the thickness of the anchoring layer can be controlled and the distance from the filter layer to the electrode surface can be placed outside the Debye length, so that nonspecific negative charge signals will not be captured. Fig. 7B shows a structural model of a polymer nanofilter for specific detection of I-cysteine. Adding phenylboronic acid (PBA) to the filter layer can selectively capture L-DOPA, and the added hydrophilic cross-linking agent cross-links the filter layer to form a rigid polymer structure, reducing the electrical noise caused by the conformational change of the filter caused by the binding of L-DOPA and PBA, and ultimately reducing the noise signal by 70–90%.


	Fig. 7 Schematic diagram of the structure of a polymer nanofilter. (A) Structural schematic of FET biosensor modified with nanofilters; (B) schematic representation of the interface for capturing L-DOPA and specifically detecting cysteine using nanofilters polymerised with PBA. Reproduced with permission.⁴⁴ Copyright 2019, American Chemical Society.

The combination of nanofilters and ISFET biosensors greatly improves the signal-to-noise ratio for the detection of small biomolecules, such as serotonin. Molecularly imprinted polymer (MIP) is a high molecular weight polymer that can simulate the interaction between antigen and antibody. It can extract and purify target analytes from various complex biological samples and effectively eliminate the influence of matrix effects. MIP not only has the advantages of high specificity and high affinity, but also has the advantages of high stability, low cost and simple preparation compared with macromolecules such as antibodies. Its separation and screening capabilities have important application value in the development of high-sensitivity detection technology.¹³⁸ MIP film modified on the gate electrode of an FET biosensor can be used for the specific detection of small biological molecules. PBA can form stable esters with diol biomolecules (such as sugars, catecholamines and lactic acid) through covalent bonds, and is usually used as a functional monomer for synthesizing MIP film on the surface of FET electrode.¹³⁹ At the same time, the esterification reaction of PBA–diol compounds is a reversible reaction, which is determined by pH. For example, the pK_a of glucose borate is 6.8. After the glucose molecule binds to PBA, it can be removed under acidic conditions, and it can stably bind under relatively alkaline conditions and produce changes in negative charge density for signal reading.¹⁴⁰ Meanwhile, according to the above principle, the biosensor modified with MIP of PBA monomer can not only improve the detection specificity and reduce the noise signal, but also clean the analyte by changing the pH of the electrolyte solution, so as to realize the repeated use of the sensor and reduce the cost of use.

In conclusion, the field of ISFET biosensors has evolved significantly, offering versatile applications in various domains. The pursuit of enhanced sensitivity has led to innovative approaches, such as PNA utilization, RCA-based amplification, and sensor device optimizations, collectively pushing the boundaries of what is possible in terms of biosensing. The future holds immense promise for ISFET technology, as continued research and development are expected to drive even greater sensitivity and specificity. These advancements will undoubtedly contribute to the development of more precise and efficient diagnostic tools, facilitating breakthroughs in healthcare and scientific discovery. The journey of ISFETs in biosensing is far from over, and we can anticipate exciting developments in the years to come.

4.2. Break the limits of Debye length

In order to maintain the charge neutrality requirement at the electrolyte solution–electrode interface, there are equal numbers of positively and negatively charged ions in an electrolyte solution. Any charged particle will be surrounded by particles of the opposite sex, and its electric field can only act within a certain distance. The electric field force between two charged particles will interact only if the distance is less than this. If the distance exceeds this, they will be shielded by the electric field of the surrounding opposite sex particles. This distance is called the Debye length, also known as the Debye screening distance (λ_D), which is defined in eqn (1).


	(1)

where l_B is the Bjerrum length (0.7 nm), and ρ_i and z_i are the density and the valence of the i-th ionic species.³⁹ The ions in the solution will be redistributed near the channel surface of the biosensor and exert capacitance on the channel to form an electric double layer (EDL), and charge shielding occurs on the EDL, as shown in Fig. 8A. According to the EDL formation theory, charge shielding occurs when the detection distance (distance from the channel surface) of the molecule to be detected exceeds the neutral charge region.^39,141


	Fig. 8 (A) Schematic diagram of charge distribution, double layer formation and Debye length at the solid–liquid interface. Reproduced with permission.³⁹ Copyright 2019, Elsevier. (B) Molecular pendulum structure, and the timing current diagrams for antibody-MP and aptamer-MP sensors. Reproduced with permission.¹⁴² Copyright 2023, John Wiley & Sons, Ltd.

The Debye length limit has posed a significant challenge in the application of ISFET sensors. Despite notable progress in ISFET biosensor research, a charge shielding effect at the electrode–electrolyte interface has restricted their utility in various fields. Stern et al. conducted investigations into the impact of Debye length on streptavidin sensing in buffers with varying ionic strengths.¹⁴³ Their findings revealed that lower ionic strength solutions result in longer Debye lengths, enhancing molecular detection sensitivity. In commonly used buffer solutions such as 1× and 0.1× PBS solutions, the Debye length ranges from 0.7 to 2.2 nm. However, the size of most biomolecules to be tested is in the range of a few nanometers (e.g., antibodies ∼10 nm, and aptamers with 30 base pairs are up to 10 nm). The charge change after the specific binding of macromolecules to the test substance cannot cause a significant change in the surface potential of the ISFET electrode due to the screening of the Debye length, which greatly limits the application of ISFET. And low ionic strength buffers may affect the conformation of biomolecules and weaken the activity and binding affinity of the analyte (such as the secondary structure of proteins based on hydrogen bonds, and the tertiary and quaternary structures that rely on electrostatic and hydrophobic interactions). Current research mainly optimizes ISFET biosensors to break the limitation of Debye length by changing the Debye screening length, such as selecting new semiconductors with special carrier distribution, modifying special molecules on the channel, etc., or shortening the detection distance, such as using aptamers instead of antibodies, coupling specific enzyme catalytic reactions, etc. to eliminate charge shielding.^144–146

This challenge is associated with the morphology of the sensor channel material, particularly in concave regions of the channel.¹⁴⁷ To address this limitation, researchers have explored various strategies. For instance, Wang adjusted the roughness of graphene surfaces, and their results demonstrated that the pleated sensor exhibited weaker charge screening effects within the detectable range, substantially improving detection performance.¹⁴⁸ Another approach involves the application of a permeable polymer layer to increase the Debye length. Gao et al. coated the surface of SINW-FET biosensors with a bio-permeable polymer called polyethylene glycol (PEG) and immobilized specific antibodies on SiNW surfaces, enabling the detection of target molecules even in high ionic strength solutions.¹⁴⁹ PEG, with its densely distributed polymer chains and gaps between them, enhances the dielectric constant of the solution and extends the effective Debye shielding length.

Researchers have also introduced innovative sensing methods, such as using small molecule biological receptors to reduce the distance between the sensor surface and the biological molecules.¹⁵⁰ Nucleic acid aptamers can form nanostructures through intramolecular base pairing and have selectivity and affinity for specific biological molecules comparable to antibodies. After the interaction of biological molecules, the detection distance of the electrode surface modified by aptamers or antigen-binding fragments (Fab) is reduced from 10–15 nm to 3–5 nm, which is completely in line with the Debye screening range (7.4 nm) of 0.01× PBS buffer. Therefore, they can replace antibodies and become new molecular recognition elements.^19,151,152 Das and their team developed a novel approach that modifies a DNA-linked recognition element on the sensor's surface.¹⁵³ They harnessed the inverted molecular pendulum motion to induce changes in charge distribution, allowing real-time electrochemical signal tracking to determine whether the target is bound to the recognition element. The active motion of the molecular pendulum probe, coupled with strong electric field-mediated transport, overcomes the limitations of the Debye length, providing significant sensing advantages. In addition, researchers have utilized aptamers for sensor construction, resulting in aptamer-based molecular pendulum sensors for the detection of biomarkers like BNP and NT-proBNP with wide linear ranges (Fig. 8B).¹⁴² Aptamers, with their similar recognition capabilities to antibodies but smaller size and ease of chemical synthesis, offer competitive advantages, particularly in limited sensing ranges. Moreover, for the detection of electrically neutral substances, nucleic acid aptamers with negatively charged backbones show great promise. Sheibani et al. leveraged this feature to design an extended-gate field-effect transistor for cortisol detection.¹⁵⁴ The binding of the charged capture probe to cortisol alters the gate potential, inducing a change in drain current and enabling highly specific, label-free detection of electrically neutral small molecules. This innovative approach allows the conversion of signals from protein detection to nucleic acid detection, presenting an essential strategy to overcome the Debye length limitation and indirectly amplify detectable signals.

In conclusion, the development and application of ISFET sensors represent a fascinating journey of scientific progress. From their early days as pH sensors to their current role in detecting a wide range of biomolecules and analytes, ISFET sensors have come a long way. The challenges of the Debye length limit and sensitivity have been addressed through various innovative strategies, including surface modifications, aptamer-based approaches, and novel sensing methods. These advancements have significantly broadened the scope of ISFET applications, promising enhanced accuracy, specificity, and versatility.

5. Conclusion and perspectives

ISFET biosensors have demonstrated significant promise in the realm of biomarker detection, owing to their remarkable attributes, including ultra-sensitivity, label-free operation, seamless integration, and cost-effectiveness. In this comprehensive review, we elucidate the structure and operational principles of ISFETs, and delve into the application of ISFET biosensors in disease detection. This encompasses both qualitative and quantitative analysis of nucleic acid and protein markers associated with cardiovascular diseases, infectious diseases, cancer, and diabetes mellitus. While ISFET-based biosensors possess numerous strengths that render them highly competitive for marker detection, they face challenges stemming from the stringent clinical diagnostic requirements for detecting trace quantities and limitations associated with the sensor's detection performance due to the Debye length. Consequently, researchers have proposed several strategies to enhance the efficient detection of target molecules or ions. These strategies encompass adjustments to the sensor's structure, the incorporation of signal amplification and conversion techniques, and modifications to the conduction mode. Nonetheless, there remain certain shortcomings in the current application of ISFET biosensors for early diagnosis and health monitoring.

Firstly, the composition of the sensing platform is complex, and the integration needs to be improved. At present, most ISFET devices are mainly based on detection and analysis, and fail to integrate sample pretreatment, signal readout, and analysis steps, which hinders the realization of a seamless process of “sample input, result output”. At the same time, the capture and output of biosensor signals still require bulky and expensive professional instruments. In the future, the composition of the device can be simplified, and its integration can be improved, which can be widely used in the development of wearable detection equipment and POCT detection devices. Secondly, the development of multi-target integrated microarrays is an approach that has not been fully explored. This method not only speeds up multi-sample testing and minimizes the use of reagents and samples, but also enables multiplexed analysis, providing substantial support for early disease screening and accurate diagnosis. For instance, the technology of pooling four preoperative infectious indicators has significant clinical application value, as test results can be obtained from the same blood sample, thereby reducing the risk of pathogen transmission. In addition, by combining multiple ISFETs, joint testing of multiple types of patient samples (such as serum, saliva, urine, cerebrospinal fluid, ascites, etc.) can be performed to achieve an accurate and rapid diagnosis of the cause of the patient's disease, which has important application prospects in outpatient clinics, emergency departments, etc. However, the above-mentioned detection model increases the difficulty of integration, increases manufacturing costs, and limits commercialization. New technologies need to be explored to overcome the difficulties of integration and industrial production. Moreover, this approach has great value for in vivo diagnosis. In vivo diagnostic and monitoring technology can maintain the original state of the target to be tested, reduce the loss of detection sensitivity caused by changes in physical properties and biological functions of the target after leaving the body, and sample pre-treatment, and provide the test results that are closest to the health status of the body. At the same time, real-time dynamic monitoring of the target in the body can provide objective reference information for physical health status, disease treatment effects, recurrence risks, drug resistance, etc. at the earliest, which can serve as an important basis for improving lifestyles and adjusting patient treatment plans. Therefore, optimizing the biocompatibility of ISFET manufacturing materials and in-depth exploration of implantable ISFET chips are also important directions. In summary, the application of ISFETs in the field of biomedicine is gaining unstoppable momentum, riding the wave of rapid growth across various cross-disciplines. We hold a firm belief that ISFET technology is poised to become a major player in biomarker detection, as the field of micro and nanotechnology continues to innovate and evolve.

Data availability

The data and information presented in this review are based on previously published studies, which are cited throughout the article.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (32201090).

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Footnote

† Jie Zou and Hao Bai contributed equally to this work.