1. Introduction

Figure 1. Classification of Biopsies: Understanding the Different Categories.

Figure 1. Classification of Biopsies: Understanding the Different Categories.

Figure 2. Overview of the Electrochemical Biosensors Operation.

Figure 2. Overview of the Electrochemical Biosensors Operation.

1.1. Essential Biomarkers for Liquid Biopsy Detection and Monitoring

Liquid biopsy is increasingly used for the detection, analysis and monitoring of circulating tumor cells (CTCs), circulating tumor DNA and circulating extracellular nucleic acids [15], in blood or other body fluids such as urine, with the main advantage of diagnosing cancer at an early stage. In contrast to traditional invasive biopsy, which includes cells or tissues from the lesion, liquid biopsy is a non-invasive procedure that can also be used during the treatment planning, as well as in the follow-up of the disease. In addition to blood, other body fluids are under consideration for liquid biopsy: urine [16] is already used as a source of biomarkers (PCA3 in prostate and pancreatic cancer detection) and several example of state of art detection from saliva or seminal fluid [17] or stool have been described [18].

The liquid biopsy is a non-invasive method for detecting, analyzing and monitoring cancer cells, DNA and other nucleic acids in body fluids such as blood and urine. It offers several advantages over traditional biopsy methods, including the ability to diagnose cancer at an early stage without the need for invasive procedures. This technique can be used not only for the initial diagnosis of cancer but also during treatment planning and in follow-up procedures. Other body fluids such as urine, saliva, seminal fluid, and stool are also being studied as potential sources of biomarkers for liquid biopsy. Urine is already being used to detect PCA3 in prostate and pancreatic cancer, and several state-of-the-art detection methods have been developed for other body fluids.

Biomarkers are recognized to have a critical role in the early detection of cancer, the creation of personalized treatments, and the identification of disease-related underlying processes.

The detection targets of liquid biopsy primarily encompass cells and nucleic acids found in body fluids, including circulating tumor cells (CTCs) in blood, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), exosomes, micro-RNA (miRNA), and proteins. An innovative way of using liquid biopsy, going in the direction of point-of-care medicine is the integration of these kind of assays into portable devices, which need a small amount of sample, low power to work and minimal equipment [19]. Electrochemical methods are particularly suitable for integration into near-the-bed platform, since the most of them are label-free methods and all the components can be easily miniaturized into lab-on-chip platforms.

Table 1. Some common biomarkers used for Liquid Biopsy and electrochemical method used.

Table 1. Some common biomarkers used for Liquid Biopsy and electrochemical method used.

Biomarker	Biofluid/Sample	Electrochemical method	LOD	Ref.
Exosomes	Plasma	Potentiometric	20pM 106 mL−1 43 particles μL−1 <105 vesicles/ 10 μL	[20] [21] [22] [23]
Circulating Nucleic acids Circulating tumor DNA (ctDNA) Circulating microRNA (miRNA)	Human serum Serum	DPV DPV and EIS	3.9 x10-22 g/ml 0.45 fM	[24] [25] [26] [27]
Circulating tumor cells (CTCs)	Blood Blood Peripheral blood	Amperometry DPV DPV	5 cells/ml 27 cells/ml 3 cells/ml	[28] [29] [30]
Proteins	Human serum, saliva	CV and EIS	3.3 fg m/L	[31]

2. Miniaturization Strategies for Biosensing

2.2. Nanomaterials in Electrochemical Sensors Integrated in LOC Device: From 2D to 3D Electrodes

Nanomaterials have emerged as a promising class of materials for sensing applications due to their unique physicochemical properties [41,42,43]. The exceptional properties exhibited by nanomaterials, including high surface area, excellent electrical and thermal conductivity, as well as unique optical characteristics, make them highly advantageous for seamless integration into lab-on-a-chip (LOC) devices as electrochemical sensors. This integration enables the detection of molecules in body fluids with significantly improved sensitivity and accuracy [44].

Microfluidic devices offer the ability to control the flow of fluids at the microscale, enabling the rapid and precise detection of biomolecules. When combined with nanomaterial-based sensors, real-time monitoring of low concentrations of biomolecules in body fluids such as blood [45], urine [46], tears [47] and saliva [48] can be achieved. The incorporation of nanomaterials in electrochemical biosensors holds the potential to bring about a revolutionary transformation in the field of clinical diagnostics. This advancement facilitates the rapid, sensitive, and highly specific detection of biomolecules in body fluids, paving the way for significant advancements in medical diagnosis and patient care. This enhancement is achieved through either promoting electronic transfers or increasing the volume/surface area ratio [49]. This has significant implications for medical diagnosis and treatment, as it can enable the early detection and monitoring of diseases such as cancer, diabetes, and cardiovascular diseases or timely identification of bacterial infections.

The integration of nanomaterials onto the electrode surface of microfluidic devices plays a crucial role in the advancement of high-performance electrochemical sensors. This is particularly important when electrodes are situated in less accessible locations, as often encountered in lab-on-chip systems. By incorporating nanomaterials, the sensitivity and overall performance of the electrochemical sensors can be significantly enhanced, enabling accurate and reliable detection in challenging sample environments. In this kind of device, nanomaterials are typically integrated onto the electrode surface using various techniques, such as in situ synthesis [50], drop-casting [51], spin-coating [52], electrochemical deposition [53], electrospinning [54] and inkjet printing [55]. Drop-casting and spin-coating are simple and cost-effective techniques that involve the deposition of nanomaterials onto the electrode surface using a dropper or a spinning device, respectively. Electrochemical deposition involves the deposition of nanomaterials onto the electrode surface by applying a voltage or a current to the electrode in the presence of the nanomaterials in solution. Inkjet printing involves the precise deposition of nanomaterials onto the electrode surface using a specialized printer. Among them, electrochemical deposition and inkjet printing are the techniques that allow for the most precise and controlled deposition of nanomaterials onto the electrode surface, which is critical for the development of high-performance electrochemical sensors.

Various types of organic and inorganic nanomaterials, including carbon nanotubes, graphene, metal and metal oxide, polymer, quantum dots, Prussian Blue [56], nanorods, and tubes, have been incorporated into electrochemical transducers to enhance electrochemical sensing.

Figure 3. An overview of the nanomaterials used for biosensing.

Figure 3. An overview of the nanomaterials used for biosensing.

Metallic nanostructures, such as gold [57], silver [58], and platinum [59], have been widely used in electrochemical sensors integrated into microfluidic devices for the detection of molecules in body fluids. Gold nanoparticles (AuNPs), for example, have been extensively studied due to their unique electronic, optical, and surface properties, which make them ideal for use in biosensing applications [60]. AuNPs have been used in a variety of electrochemical sensors for the detection of different biomolecules, such as glucose, cholesterol [61], and prostate-specific antigen (PSA) [62] .

Magnetic nanoparticles (MNPs), such as iron oxide nanoparticles, have also been used in electrochemical biosensors integrated into microfluidic devices for the detection of biomolecules in bodily fluids. MNPs have unique magnetic and surface properties, which make them ideal for use in biosensing applications [63]. These nanomaterials not only improve the limit of detection of the sensors, but also enable the separation and transportation of bioanalytes inside the microfluidic device, thereby allowing for the miniaturization of analytical methods [64]. For example, MNPs functionalized with iridium oxide nanoparticles and tyrosinase have been used for the detection and quantification of methimazole in microsystems. In the analytical measurements a permanent magnet was used to immobilize the magnetic complex on the electrode surface. The system was highly sensitive with a low limit of detection (0.004 μM) and demonstrated effectiveness in serum samples. Interestingly, the use of microfluidic device allows for improve limit of detection, reusability, automation, volume of the sample, and response time compared to batch configuration [65].

Among nonmetallic nanomaterials, carbon nanotubes (CNTs) [66], graphene [67], and quantum dots [68,69] (QDs) have shown high sensitivity towards various analytes, including glucose [70], cholesterol [71], and proteins, [72] with detection limits in the sub-nanomolar range. Moreover, carbon-based nanomaterials such as graphene and carbon nanotubes have also been combined with metallic nanoparticles or polymeric layers to produce nanocomposites with improved performance [73,74,75] in terms of electronic transfer or selectivity.

One of the challenges associated with the use of nanomaterials in electrochemical sensors integrated into microfluidic devices is the reproducibility and stability of the sensors. Due to the small size of nanomaterials, synthesizing and functionalizing these materials can be challenging, leading to variations in sensor performance. Additionally, the stability of nanomaterial-based sensors can be affected by factors such as temperature, pH, and humidity, resulting in reduced sensor performance over time. Efforts are being made to address these challenges, by developing reproducible synthesis and functionalization methods and optimizing sensor design to enhance stability.

Another challenge related to the use of nanomaterials in electrochemical sensors integrated into microfluidic devices is the integration of these sensors into practical clinical applications. While many studies have demonstrated the feasibility of using nanomaterial-based sensors for detecting biomolecules in body fluids, further development and optimization are required before these sensors can be widely adopted in clinical settings. This includes optimizing sensitivity, selectivity, and stability of the sensors, as well as developing user-friendly and cost-effective instrumentation for their use. In fact, despite the enhanced sensor performance offered by nanomaterials implemented on electrode surfaces, the two-dimensional planar electrodes can still limit component and signal transmission when used in vivo, thereby affecting sensor accuracy and sensitivity [76,77]. This limitation is particularly true for complex samples such as blood or plasma used in point-of-care-devices [78,79]. Furthermore, the planar structure of two-dimensional electrodes poses challenges in achieving adequate immobilization of active components and efficient signal transmission, leading to potential issues in sensing accuracy. To address these limitations, the integration of macroscale three-dimensional (3D) porous materials, comprising nanomaterials combined with polymers [80,81,82,83] can be employed as electrodes. This approach facilitates expanded microfluidic transport and enables the incorporation of multianalyte detection capabilities, thereby enhancing the overall performance of the sensor system.

The incorporation of porous channels in biosensing systems offers several advantages, including increased surface area, enhanced ion/mass transport pathways, and improved immobilization and stability of active components. In this context, graphene has emerged as a promising avenue for the development of three-dimensional (3D) electrodes. Graphene can be fabricated in the form of aerogel or combined with polymers, providing an excellent opportunity to create highly efficient and versatile 3D electrode structures. Furthermore, the surface of graphene can be easily engineered with other nanomaterials and biorecognition elements. For instance, Xu et al. [84] demonstrated the use of a graphene foam (GF) modified with carbon-doped titanium dioxide nanofibers (nTiO2) as an electrochemical working electrode. The three-dimensional and porous structure of the GF facilitated the penetration and attachment of nTiO2 onto its surface, resulting in enhanced charge transfer resistance, increased surface area, and improved access of the analyte to the sensing surface. The GF-nTiO2 composite was further functionalized with the ErB2 antibody for specific detection of the target ErbB2 antigen, a biomarker for breast cancer. The sensor was employed for quantification of the ErbB2 antigen using differential pulse voltammetry and electrochemical impedance spectroscopy techniques. Remarkably, both methods exhibited high sensitivity across a wide concentration range of the target antigen, demonstrating excellent specificity even in the presence of other members of the EGFR family.

In another study, Zhang et al. [85] conducted a study where they developed an enzymatic electrochemical microfluidic biosensor for glucose detection. The biosensor incorporated a three-dimensional porous graphene aerogel and glucose oxidase (GOx), taking advantage of the aerogel's high electrical conductivity and specific surface area to enhance the immobilization of GOx. The microfluidic system implemented in the biosensor reduced sample consumption during testing. The biosensor exhibited excellent selectivity and stability and successfully monitored glucose levels in serum samples. This innovative biosensor shows promise for clinical applications in diabetes diagnosis, and the method employed for preparing the graphene aerogel modified electrode holds potential for broader use in diverse electrochemical sensors.

In addition to high sensitivity, nanomaterial-based sensors are highly selective, enabling the detection of specific molecules in complex biological samples. This selectivity is achieved through the functionalization and modification of nanomaterials, by attaching specific ligands (e.g. antibodies, DNA, RNA, aptamers, and enzymes) [86] through covalent or non-covalent interactions to enhance specificity and electronic transfer in sensors. For example, Fan et al. (2022) developed a smartphone-based electrochemical system composed of CNTs functionalized with gold nanoparticles, thionine, and antibodies for the detection of CA125, a biomarker for prostate cancer [87]. The biosensor exhibited high selectivity towards CA125, with no interference from other biomolecules present in human serum.

Another approach to obtaining sensors with high specificity is the creation of molecular imprinted polymers (MIPs). MIPs are polymer-based artificial receptors with the ability to recognize different types of target molecules such as aminoacids, peptides [88], pesticides [89] and drugs [90] but even larger molecules such as proteins [91] or whole cells [92]. The target molecules act as template and interact with functional monomers to form a complex during the polymerization, then the template can be removed and leaving cavities able to rebind template molecules thank to its geometry ant chemical moieties. MIPs have been largerly used as recognition elements in electrochemical sensors offering great advantages such as improved stability, cost-effectiveness and rapid fabrication procedure overcoming limitations of natural receptors (antibodies, nucleic acids, peptides) such as sensitivity to enzymatic digestion, low preservation temperature, etc.

So, combining the advantages of MIPs and electrochemical transducers several sensing platform have been realized joining sensitivity and ease to use of electrochemical sensors with high selectivity and stability of MIPs [93]. To realize a high-performance sensing platform based on MIP as artificial receptor it is necessary to consider at least two key aspect: (i) the choice of polymer and (ii) imprinting processes. Electrochemical sensors are compatible with different imprinting approaches such as in situ bulk polymerization, surface imprinting and electrosynthesis.

The most commonly employed approach for imprinting is bulk imprinting, wherein the transducer surface is coated with a mixture of template and pre-polymer that exhibit mutual interaction. Then after polymerization template molecules are entrapped inside the polymer matrix and can be removed by a washing step creating cavities able to recognize the analyte in the subsequent analytic steps. To apply this technique to larger molecules is necessary to realize a very thin layer of polymer so the imprinted binding sites are near the interface making template removal and rebinding easier. Another possibility for the recognition of large molecules such as proteins is epitope imprinting that consists of imprinting only a portion of the target molecules [94].

An alternative approach is based on electrosynthesis in which polymerization is induced by applying a suitable potential range to a solution containing the monomers with the template molecules without any initiator. The characteristics of resulting films can be tuned by modulating electrochemical parameters. Conductive polymers (CP) and insulators/non-conductive polymers (NCP) can be used with different advantages and disadvantages. Non-conducting MIP films self-limits their growth, so allow a fine control on their thickness, while CPs are more flexible and offer the possibility to tune not only the thickness but even the conductive properties by changing the deposition conditions. The selection of polymers is strictly related to the detection methods: for example capacitive [95] or impedimetric [96] sensors require nonconductive polymers, while for amperometric detection is better to use conductive ones [97].

Surface imprinting is one of the most used technique for the development of MIPs for large molecules, cells and microorganisms: it consists in the template imprinting only on the MIP surface. Several techniques such as soft lithography, micro-contact imprinting, and sacrificial template support methods have been exploited to confine the imprinted sites on the MIP interface [98].

The analytical performances of electrochemical MIP-based sensors can be furtherly improved by combining MIP technology with nanomaterials and realizing imprinted nanocomposite. Different nanomaterials have been used to this purpose ranging from carbon based materials (e.g. nanotubes [99,100], graphene [101]) to metallic nanoparticles [102]: this transition towards nanoMIP significantly improved the analytical performances of MIP based sensors in terms of detection limits and sensitivity and the nanostructuration of the material allows for better diffusion of the analyte on the transducer surface, resulting in a faster response time for the sensor. Nevertheless further efforts are still necessary to have standardized procedures for industrial applications and medical diagnostics.

3. Electrochemical Biosensors

3.1. Amperometric Method

In amperometric biosensors the electrode current is measured, and most amperometric electrochemical biosensors are based on an enzyme's redox activity (most often horseradish peroxidase, (HRP)). Enzymes enhance biosensing systems by catalyzing chemical reactions, improving sensitivity [103]. The performance of enzyme-based biosensors relies on factors such as electrode surface, enzyme type, substrate, and mediator usage [104]. HRP is commonly employed as a secondary detection reagent, with the TMB/H2O2 substrate proving most effective. Amperometric biosensors measure current at a constant potential to detect the analyte. This method provides selectivity as the potential used is characteristic of the analyte. The current is measured after directly setting the desired potential, enhancing the accuracy of the analysis [105].

Zhang et al. reported the development of a nonenzymatic immunosensor for the detection of SCC-Ag, utilizing rGO-TEPA and AuAg NCs. [106]. In another study, a competitive RNA/RNA hybridization assay-based biosensor was developed using Streptavidin-Horseradish Peroxidase (SA-HRP) and biotinylated capture probes. The biosensor employed H2O2 as an enzyme-substrate and hydroquinone (HQ) as a RedOx mediator. Two separate platforms, a screen-printed electrode (SPE) with Au NPs and a GCE with tungsten diselenide and Au NPs, were used in the biosensor [107]. Additionally, a microfluidic amperometric immunosensor was developed for the detection of the cancer biomarker CLD7 in circulating extracellular vesicles (EVs). The immunosensor was validated in colorectal cancer (CC) patients [108].

Figure 4. Direct competitive hybridization assay developed for miRNA determination shown schematically (adapted from the paper 98).

Figure 4. Direct competitive hybridization assay developed for miRNA determination shown schematically (adapted from the paper 98).

Chronoamperometry is a variation of amperometric techniques that involves monitoring the current generated by the faradaic process at the electrode over time. It involves applying a sufficiently large potential step to the working electrode to initiate a chemical reaction, and then observing the current as a function of time.

3.2. Potentiometric Method

Potentiometric biosensors use a tiny amount of current to monitor the potential of an electrochemical cell [109]. Potentiometric sensors, employing the controlled-current method, utilize an electrochemical cell containing two reference electrodes to measure the potential across an ion-selective membrane. Enzymes are commonly used to facilitate ion production, which is then detected by the supporting electrode. Controlled-current methods offer the advantage of using more affordable measurement instrumentation compared to controlled-potential methods. The Nernst equation relates concentration and potential in potentiometric measurements, offering a low limit of detection (LOD) for early-stage cancer diagnosis [110]. Jia et al. developed a Light Addressable Potentiometric Sensor (LAPS) for the detection of the liver cancer biomarker hPRL-3 [111]. Another study [112] utilized surface molecular imprinted self-assembled monolayers (SAM) for a potentiometric biosensor with a linear range of 2.5-250 ng/ml [113]. Label-free potentiometric detection targeted the HAPLN1 protein biomarker in MPM, achieving pM range LOD. Goda et al. created a hybridization-based potentiometric microarray for exosomal miRNA identification [114].

Potentiometric methods have been employed to target malignant cells, investigating their electrochemistry in microenvironments affected by lactate release and pH fluctuations. Shaibani et al. achieved a LOD of 103 cells per ml, revealing pH flux alterations around cancer cells and their connection to altered cell metabolism [115]. A selective detection of circulating tumor cells (CTCs) in prostate cancer was achieved by utilizing an anti-EpCAM functionalized graphene oxide potentiometric biosensor based on the Light Addressable Potentiometric Sensor (LAPS) approach [116].

Based on pH monitoring, a sensor-integrated microfluidic technique is employed to find cancer cells. In this instance, the CTC's metabolic alteration resulted in a decrease in the pH of the surrounding environment. Potentiometric methods with Ag/AgCl and ZnO electrodes were employed to assess pH variations and cell modifications in vitro, utilizing three cell lines (A549, A7r5, and MDCK) in a microfluidic setup [117].

Figure 5. A microfluidics-based pH sensor was successfully created with the use of rf sputtered ZnO thin films and Ag/AgCl ink (Adapted from paper 108).

Figure 5. A microfluidics-based pH sensor was successfully created with the use of rf sputtered ZnO thin films and Ag/AgCl ink (Adapted from paper 108).

3.3. Impedimetric Method

Electrochemical impedance spectroscopy (EIS) is a technique that examines the resistive and capacitive characteristics of a system by applying an AC excitation signal with varying frequencies. By analyzing the impedance spectra, it is possible to determine the resistive and capacitive components of the system based on the in-phase and out-of-phase current responses. At higher frequencies, the migration rate of redox species can become rate-limiting, resulting in a frequency-dependent phase lag when analytes impede access to the electrode surface.

Electrochemical impedance spectroscopy (EIS) evaluates interfacial characteristics, ion passage, and biomolecule interactions with electrode surfaces. It involves applying AC potential to an electrochemical cell and measuring the resulting current signal. The resulting frequency-dependent impedance is represented on a Nyquist plot using a Randles circuit, showing semicircles at higher frequencies for electron transfer restrictions and a linear line at lower frequencies indicating diffusion-limited electron transfer.

Figure 6. The Nyquist plot exhibits a depressed arc, indicating polarization caused by a combination of kinetic and diffusion processes.

Figure 6. The Nyquist plot exhibits a depressed arc, indicating polarization caused by a combination of kinetic and diffusion processes.

R_ct value reflects electron-transfer kinetics, while RS represents bulk electrolyte characteristics and ZW represents diffusion. The Nyquist plot helps calculate ZW, represented by a 45-degree sloped straight line intercept. EIS has been utilized for the label-free detection of cancer cells.

Elshafey et al. developed a label-free impedimetric biosensor for detecting the cancer biomarker EGFR. The biosensor utilized protein G and gold nanoparticles on a modified gold electrode for efficient immobilization. The calibration curve exhibited a wide dynamic range of 1 pg/mL to 1 g/mL, with a low detection limit of 0.34 pg/mL in PBS and 0.88 pg/mL in human plasma. Interference from various substances in human plasma led to slight variations in the electrochemical signal during real-world experiments [118]. Han et al. developed a label-free cytosensor for cancer cell identification using phage display technology and EIS, demonstrating rising Rct values with increasing cell concentration, indicating reduced electron transfer efficiency. The approach employed a specific phage immobilized on a gold electrode, with [Fe(CN)6] as the redox probe indicator, offering high specificity, repeatability, and eliminating the need for complex recognition element purification [119].

Hu et al. utilized EIS for the detection of liver cancer cells. They immobilized a mannose-specific lectin (con A) on a gold electrode, leading to changes in charge transfer resistance that correlated with the concentration of cancer cells (Bel-7404). This label-free approach directly targeted cancer cells, providing a direct, selective, and sensitive method with a detection limit of 234 cells/mL, eliminating the requirement for probe labeling [120]. Azzouzi et al. developed an impedimetric electrochemical biosensor using biotinylated DNA/LNA molecular beacon (MB) probe linked with gold nanoparticles (AuNPs) for miRNA-21 detection in blood samples. The biosensor demonstrated high selectivity, good repeatability, and a wide linear detection range of 1-1000 pM, with a low detection limit of 0.3 pM. The use of neutravidin as a recognition element on the electrode surface enhanced biosensing properties, including sensitivity [121]. Kilic et al. introduced a label-free electrochemical sensor to compare the secretion levels of extracellular vesicles (EVs) in hypoxia and normoxic MCF-7 cells. The sensor utilized functionalized gold electrodes and employed Differential Pulse Voltammetry (DPV) and Electrochemical Impedance Spectroscopy (EIS) for EV detection. The sensor exhibited a linear operating range of 102-109 EVs/ml, with a limit of detection (LOD) of 77 EVs/ml. Selectivity was assessed using RhD protein, and the results were compared with ELISA and Nanoparticle Tracking Analysis (NTA) [122].

Figure 7. Differential pulse voltammograms recorded for various EVs concentrations (10–1010 EVs/ml) (left) and EIS measurements in the concentration range of 102–5×106 EVs/ml (right) – (adapted from paper 113).

Figure 7. Differential pulse voltammograms recorded for various EVs concentrations (10–1010 EVs/ml) (left) and EIS measurements in the concentration range of 102–5×106 EVs/ml (right) – (adapted from paper 113).

3.4. Conductometric Method

Conductometric biosensors offer exciting possibilities for advanced bioanalytical detection. They measure changes in electrical conductivity during chemical reactions, using enzymes to modify the ionic strength of the sample solution. These biosensors are advantageous due to their miniaturization potential, low voltage requirement, and lack of a reference electrode. They are promising for applications in healthcare, environmental monitoring, and food safety [123]. Capacitive biosensors have the benefit of capacitance measurements being more informative of the biosensor's insulating qualities [124]. Even slight sensor layer desorption generally results in a rise in the capacitance baseline. Furthermore, nonspecific binding is less likely with capacitive sensors.

Liang et al. established a conductometric immunoassay for the detection of alpha-fetoprotein (AFP) in the serum of liver cancer patients. The assay demonstrated robust conductometric responses, achieving a low detection limit of 4.8 pg/mL across a dynamic linear range of 0.01-100 ng/mL [125]. For the quantification of prostate cancer antigen, Bhardwaj et al. [126] introduced a conductometric immunosensing platform utilizing tetracyanoquinodimethane (TCNQ)-doped thin films of copper-MOF, Cu3(BTC)2. The platform exhibited a dynamic linear range of 0.1-100 ng/mL for PSA detection, with a limit of detection as low as 0.06 ng/mL. Lin and co-authors devised conductometric sensors based on silicon nanowires for the detection of apolipoprotein A1, a biomarker associated with bladder cancer. The sensors demonstrated a wide dynamic range spanning from 0.2 ng/mL to 10 µg/mL, with a detection limit of approximately 1 ng/mL [127].

Figure 8. A general presentation illustrating diverse electrochemical techniques employed in biosensing.

Figure 8. A general presentation illustrating diverse electrochemical techniques employed in biosensing.

4. Sensors Integration

4.1. Lab-on-Chip Platforms: Wearable and Portable Devices for POC

Lab-on-a-chip (LOC) devices notably multitasking devices, show the most attractive advantages of performing several lab procedures on a single chip with little volumes of chemicals and great efficiency. One of the most challenging feature of LOCs, also known as a micro total analysis system (μTAS) is to fully integrate microelectromechanical system (MEMS) with automated microfluidic tools to allow their widespread use in medical applications (liquid biopsy, therapeutic follow-ups, health and diseases monitoring). These user-friendly, sensitive and portable LOC sensors for real-time analysis has various benefits over traditional analytical techniques. In this review papers we have listed several examples of sensors suitable for integration into LOC devices. Nevertheless, the step over technological gap to overcome limitations in the widespread diffusion of such devices is still linked to scalability of systems and poor affordability of highly integrated platforms. Only few examples indeed are the commercially available platforms including electrochemical systems and their use for monitoring health parameter is going in the direction of self-diagnostics and fitness applications but not toward becoming a gold standard in clinical practices.

Recently, Atkcakoca and coworkers reported on the realization of an electrochemical biosensor with integrated microheater improving the performance of the nucleic acid hybridisation assay based on Electrochemical Impedance Spectroscopy, paving the way for the development of highly sensitive and specific integrated label-free biosensors [156]. Kasturi and collaborators developed a microfluidic channel with integrated valves and electrochemical biosensor for the detection of beta-amyloid, a biomarker for Alzheimer's disease. Gold microelectrodes are transducer holding the antibody-antigen complexes inside the fluidic chambers governed by pneumatic valves. A linear response of the sensor through DPV measurements was reported for beta-amyloid antigen concentrations from 2.2 pM to 22 uM [157]. One of the most promising applications of integrated LOC for diagnostic purposes is the realization of smartphone-based and wearable devices, which can really realize all the features of portability, low-costs and rapid response needed to fully embody the Point-Of-Care concept.

Flexible electronic devices, making use of specific methods of fabrication and detection, have properties such as flexibility, wearability, conductivity, stretchability, mechanical resistance, and, biocompatibility. In this scenario, a plethora of material suitable as substrates for electrochemical transducers have been considered [158]: cellulose-based, polyaniline (PANI), polyimide (PI) and specific fabrication methods have been demonstrated. Most of them are able to electrochemically detect ions concentrations from skin and sweat as well as monitoring fitness parameters like heart bit, blood pressure, body temperature and oxygen concentration [159,160].

Sempionatto and collaborators, recently developed a non-invasive device for the simultaneous monitoring blood pressure and heart rate via ultrasonic transducers and multiple biomarkers via electrochemical sensors. They optimized the integrated device conformal to curved skin thus ensuring mechanical resistance and reliable sensing of several compounds: glucose in interstitial fluid; lactate, caffeine and alcohol in sweat [161].

Integration of small microfluidic channels into wearable devices is also a challenging topic for the development of such devices, since biological fluids, easily exploitable for liquid biopsy (sweat, interstitial fluid) could be conveyed toward the sensing areas of the devices.

Electrochemical sensors measure the reaction caused by the interaction between the sensing surface and the analytes and the corresponding response is then converted into electric signals that can be monitored by potentiometry, amperometry, and conductometry measurements [162]. The integration of electrochemical biosensors into point-of-care (PoC) platforms, especially when combined with smartphones, has emerging as a powerful tool for personalized health monitoring, thus enhancing the practicality of the diagnosis compared to the traditional laboratory-based diagnostic methods [163]. In the last years, smartphone-based biosensors have been widely employed for human health PoC testing to improve the diagnosis and treatment of several diseases, thanks to their cost-effectiveness, ease of use and portability. Figure 9 illustrates the diagnosis steps in smartphone-based electrochemical biosensors.

Figure 9. Smartphone-based electrochemical biosensors for health monitoring.

Figure 9. Smartphone-based electrochemical biosensors for health monitoring.

One of the first application of a smartphone-based electrochemical biosensor system was described as amperometric sensing [164]. Amperometry is a type of voltametric technique in which a constant voltage is applied to the working electrode, and the current provided by the oxidation/reduction of an electroactive analyte is then measured as a function of time. Liu et al. reported an amperometric aptasensor integrated with a smartphone assisted portable wireless biochip, for the simultaneous real-time monitoring of insulin and glucose in saliva with the lowest detectable concentration of 0.85 nM and 0.8 nM, respectively. The sensing platform, combined with a Bluetooth transmission system to generate the digital diagnosis by smartphone signal readout, provided a PoC testing tool for the diagnosis of diabetes and other insulin-resistance-associated diseases [165]. Voltammetry is another electrochemical technique which includes different type of measurements such as differential pulse voltammetry (DPV), cyclic voltammetry (CV), square wave voltammetry (SWV), and linear sweep voltammetry (LSV), widely used in smartphone integrated electrochemical sensors [166]. In 2020 Low et al. developed a DPV-based electrochemical sensor combined with smartphone for the detection of circulating miRNA-21 biomarker in saliva. They demonstrated that the smartphone based biosensing system, equipped with a specific Android application, and displayed comparable performances with commercial workstations for the detection of miRNA-21 [167]. Combining a screen-printed immunosensor with a smartphone based electrochemical system, Fan et al. reported the detection of cancer antigen 125 (CA125) by DPV measurements. Data were transmitted to smartphone through Bluetooth, acting as interface for the communication with remote medical center via Internet. The developed system provided a sensitive detection of CA125 with a LOD of 2 mU mL-1 [168].

Electrochemical impedance spectroscopy (EIS), in which the impedance of the system is measured as a function of frequency, is also widely used in the fabrication of EIS-based electrochemical sensors combined with smartphones. For example, Talukder et al. reported a portable system for personalized monitoring of blood cell count consisting of a smartphone-based microfluidic impedance cytometer [169].

The recent advances in this field highlight the advantages of electrochemical sensing systems integrated with smartphone, thanks to their high simplicity of fabrication [170]. Moreover, the development of multiplexing smartphone-based electrochemical systems can be used for the simultaneous detection of various biomarkers, thus enabling remote control of diseases by doctors and accelerating the diagnosis and treatment of pathology.

5. Comparison of Liquid Biopsy Electrochemical Methods: Advantages and Limitations

Table 2. A comparison of various electrochemical methods employed in biosensing applications for liquid biopsy analysis.

6. Future Perspectives and Concluding Remarks

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