Label-free Electrochemical Detection Research Articles

Label-free and label-based electrochemical detection of disease biomarker proteins

Introduction: Biosensors, analytical devices integrating biological sensing elements with physicochemical transducers, have gained prominence as rapid and convenient tools for monitoring human health status using biochemical analytes. Due to its cost-effectiveness, simplicity, portability, and user-friendliness, electrochemical detection has emerged as a widely adopted method in biosensor applications. Crucially, biosensors enable early disease diagnosis by detecting protein biomarkers associated with various conditions. These biomarkers offer an objective indication of medical conditions that can be accurately observed from outside the patient. Method: This review comprehensively documents both label-free and labelled detection methods in electrochemical biosensor techniques. Label-free detection mechanisms elicit response signals upon analyte molecule binding to the sensor surface, while labelled detection employs molecular labels such as enzymes, nanoparticles, and fluorescent tags. Conclusion: The selection between label-free and labelled detection methods depends on various factors, including the biomolecular compound used, analyte type and biological binding site, biosensor design, sample volume, operational costs, analysis time, and desired detection limit. Focusing on the past six years, this review highlights the application of label-free and labelled electrochemical biosensors for detecting protein biomarkers of diseases.

Biolayer-Interferometry-Guided Functionalization of Screen-Printed Graphene for Label-Free Electrochemical Virus Detection.

Additive manufacturing holds promise for rapid prototyping and low-cost production of biosensors for diverse pathogens. Among additive manufacturing methods, screen printing is particularly desirable for high-throughput production of sensing platforms. However, this technique needs to be combined with carefully formulated inks, rapid postprocessing, and selective functionalization to meet all requirements for high-performance biosensing applications. Here, we present screen-printed graphene electrodes that are processed with thermal annealing to achieve high surface area and electrical conductivity for sensitive biodetection via electrochemical impedance spectroscopy. As a proof-of-concept, this biosensing platform is utilized for electrochemical detection of SARS-CoV-2. To ensure reliable specificity in the presence of multiple variants, biolayer interferometry (BLI) is used as a label-free and dynamic screening method to identify optimal antibodies for concurrent affinity to the Spike S1 proteins of Delta, Omicron, and Wild Type SARS-CoV-2 variants while maintaining low affinity to competing pathogens such as Influenza H1N1. The BLI-identified antibodies are robustly bound to the graphene electrode surface via oxygen moieties that are introduced during the thermal annealing process. The resulting electrochemical immunosensors achieve superior metrics including rapid detection (55 s readout following 15 min of incubation), low limits of detection (approaching 500 ag/mL for the Omicron variant), and high selectivity toward multiple variants. Importantly, the sensors perform well on clinical saliva samples detecting as few as 103 copies/mL of SARS-CoV-2 Omicron, following CDC protocols. The combination of the screen-printed graphene sensing platform and effective antibody selection using BLI can be generalized to a wide range of point-of-care immunosensors.

Emerging trends in functional molecularly imprinted polymers for electrochemical detection of biomarkers

Molecularly imprinted polymers (MIPs), functioning as artificial bioreceptors, hold significant promise for biomarker detection in healthcare, disease diagnosis, and addressing drug abuse. In contrast to natural bioreceptors, MIP-based sensors offer numerous advantages, such as high stability, cost-effectiveness, high selectivity, sensitivity, and notably straightforward preparation with customizable binding sites for diverse targets. Conventional MIP sensors often necessitate external redox reagents in analytes to transduce binding events into electrochemical signals for indirect detection, presenting challenges for practical applications in wearables or point-of-care (POC) testing. Redox-active MIP sensors have emerged as a viable alternative, enabling direct and label-free electrochemical detection, with two types developed. The first type utilizes electrocatalytic materials to expedite electron transfer and facilitate a redox reaction between the captured electroactive target and the electrode. The second type incorporates an embedded redox reactive component that allows selective binding of a target to modulate its electron transfer, leading to a change in the electrical signal. This review covers emerging trends and challenges in redox-active MIP sensors for direct electrochemical detection of biomarkers, focusing on sensing mechanisms, synthesis methods, and applications. Additionally, recent progress in wearable and POC redox-active MIP sensors is highlighted. A comprehensive outlook of challenges is further provided, aiming to advance direct biomarker detection for diverse healthcare applications.

Label-free electrochemical detection of glycated hemoglobin (HbA1c) and C-reactive protein (CRP) to predict the maturation of coronary heart disease due to diabetes

The pathophysiological link between diabetes and heightened propensity for the development of coronary heart disease (CHD) is well-established. Prevailing evidence confirms that small increases in low concentrations of high-sensitivity C reactive protein (hs-CRP) in the human body can determine the tendency of developing CHD. Additionally, glycated hemoglobin (HbA1c) is a well-recognized biomarker to evaluate diabetes progression. Given the positive correlation between diabetes and CHD, this research presents a notably unprecedented label-free electrochemical approach for the dual detection of %HbA1c regarding Total Hb and hs-CRP, facilitating early CHD prediction and cost-effective point-of-care diagnostics. Furthermore, a novel redox probe O-(4-Nitrophenylphosphoryl)choline (C11H17N2O6P) was used for the electrochemical detection of CRP, a method not documented in scientific literature before. The calibration curves demonstrate a limit of detection (LOD) of 5 mg/mL in PBS (pH 8) and 6 mg/mL in simulated blood (SB) for a linear range of 0–30 mg/mL of HbA1c. Conjointly, a LOD of 0.007 mg/mL and 0.008 mg/mL for measurement in PBS (pH 7.4) and SB are reported for a linear range of 0–0.05 mg/mL of CRP. The electrochemical systems presented could accurately quantify HbA1c and CRP in mixed samples, demonstrating reasonable specificity and practical applicability for complex biological samples.

High-density aptamer synthesis based on primer exchange reaction for label-free electrochemical biosensing

We report an electrochemical aptamer-based (E-AB) sensor for label-free quantitative detection of thrombin. In-situ synthesis of high-density aptamer replicas on the electrode is accomplished based on primer exchange reaction (PER), by which a DNA primer is continuously extended with the presence of a catalytic hairpin template and DNA polymerase. The PER enables the synthesis of long single-stranded DNA with replicated aptamer sequence for thrombin binding. The PER products on the surface of a gold electrode increase the density of aptamers on a limited electrode area, which can provide more effective binding sites for thrombin and therefore improve the performance of the sensor based on electrochemical impedance spectroscopy. Compared with the conventional E-AB sensor based on a 15-mer aptamer immobilized on the electrode, our method has a lower detection limit of 0.91 pM, and a wider linear range of 1.0 pM to 50 nM. Therefore, we believe our method is promising for label-free electrochemical detection of biomarkers.

Simultaneous Electrochemical Detection of Dopamine and Uric Acid via Au@Cu-Metal Organic Framework.

The incorporation of noble metals with metal-organic frameworks (MOFs) are conducive to the simultaneous electrochemical detection of analytes owing to multiple accessible reaction sites. Herein, Au@Cu-metal organic framework (Au@Cu-MOF) is successfully synthesized and modified as a screen-printed carbon electrode (SPCE), which serves as an excellent electrocatalyst for the oxidation of dopamine (DA) and uric acid (UA). The sensor shows a linear range from 10 μM to 1000 μM, with sensitivity and detection limit of 0.231 μA μM-1 cm-2 and 3.40 μM for DA, and 0.275 μA μM-1 cm-2 and 10.36 μM for UA. Au@Cu-MOF could realize the individual and simultaneous electrochemical sensing of DA and UA, with distinguishable oxidation peak potentials. Moreover, it exhibits reproducibility, repeatability, and stability. Ultimately, the sensor provides an avenue for an ultrasensitive label-free electrochemical detection of DA and UA.

Carbon-stabilized porous silicon biosensor for the ultrasensitive label-free electrochemical detection of bacterial RNA gene fragments

Herein, we report a carbon-stabilized porous silicon (pSi)-based electrochemical biosensing platform for the label- and amplification-free detection of bacterial 16S rRNA gene fragments that facilitates pan-bacterial detection. The sensing approach combines thermally carbonized pSi (THCpSi) structures as novel porous electrochemical transducers, and a highly sensitive sensing mechanism based on partial blockage of the pores caused by hybridization of 16S rRNA gene fragment to the DNA capture probe immobilized within the pores. Pore blockage upon RNA hybridization was quantified via differential pulse voltammetry as a decrease in the oxidation current of the redox pair ([Fe(CN)6]3/4−) added to the measuring solution. The use of carbon-stabilized pSi to build the biosensor has additional benefits: it favors high density of the immobilized bioreceptors and a large electroactive surface area, both further enhancing the overall sensitivity of the biosensor. The easily adjustable pSi morphology is key to design diagnostic tools fit-for-purpose. By tailoring the pore diameter, pore blockage upon analyte hybridization can be maximized, thus enhancing sensitivity. By tailoring film thickness, the surface area can be adjusted to optimize the amount of immobilized bioreceptors and the electroactive surface area. An excellent sensing performance was achieved by building the biosensor on THCpSi structures featuring a 27 nm pore diameter and a 1.6 μm film thickness, whose external surface was coated with a thin layer of silicon nitride (Si3N4), the latter contributing to maximize the pore blockage. The biosensor achieved a limit of detection of 2.3 pM when tested in 5% fetal bovine serum.

Au nanoparticle decorated reduced graphene oxide and its electroanalytical characterization for label free dopamine detection

Au nanoparticle decorated RGO as effective nanocomposite for label-free electrochemical detection of dopamine.

Pseudouridine-modified RNA probe for label-free electrochemical detection of nucleic acids on 2D MoS2 nanosheets.

RNA modification, particularly pseudouridine (Ψ), has played an important role in the development of the mRNA-based COVID-19 vaccine. This is because Ψ enhances RNA stability against nuclease activity and decreases the anti-RNA immune response. Ψ also provides structural flexibility to RNA by enhancing base stacking compared with canonical nucleobases. In this report, we demonstrate the first application of pseudouridine-modified RNA as a probe (Ψ-RNA) for label-free nucleic acid biosensing. It is known that MoS2 has a differential affinity for nucleic acids, which may be translated into a unique electronic signal. Herein, the Ψ-RNA probe interacts with the pristine MoS2 surface and causes a change in interfacial electrochemical charge transfer in the MoS2 nanosheets. Compared with an unmodified RNA probe, Ψ-RNA exhibited faster adsorption and higher affinity for MoS2. Moreover, Ψ-RNA could bind to complementary RNA and DNA targets with almost equal affinity when engaged with the MoS2 surface. Ψ-RNA maintained robust interactions with the MoS2 surface following the hybridization event, perhaps through its extra amino group. The detection sensitivity of the Ψ-RNA/MoS2 platform was as low as 500 attomoles, while the results also indicate that the probe can distinguish between complementary targets, single mismatches, and non-complementary nucleic acid sequences with statistical significance. This proof-of-concept study shows that the Ψ-RNA probe may solve numerous problems of adsorption-based biosensing platforms due to its stability and structural flexibility.

Advances in Droplet-Based Microfluidic High-Throughput Screening of Engineered Strains and Enzymes Based on Ultraviolet, Visible, and Fluorescent Spectroscopy

Genetic engineering and directed evolution are effective methods for addressing the low yield and poor industrialization level of microbial target products. The current research focus is on how to efficiently and rapidly screen beneficial mutants from constructed large-scale mutation libraries. Traditional screening methods such as plate screening and well-plate screening are severely limited in their development and application due to their low efficiency and high costs. In the past decade, microfluidic technology has become an important high-throughput screening technology due to its fast speed, low cost, high automation, and high screening throughput, and it has developed rapidly. Droplet-based microfluidic high-throughput screening has been widely used in various fields, such as strain/enzyme activity screening, pathogen detection, single-cell analysis, drug discovery, and chemical synthesis, and has been widely applied in industries such as those involving materials, food, chemicals, textiles, and biomedicine. In particular, in the field of enzyme research, droplet-based microfluidic high-throughput screening has shown excellent performance in discovering enzymes with new functions as well as improved catalytic efficiency or stability, acid-base tolerance, etc. Currently, droplet-based microfluidic high-throughput screening technology has achieved the high-throughput screening of enzymes such as glycosidase, lipase, peroxidase, protease, amylase, oxidase, and transaminase as well as the high-throughput detection of products such as riboflavin, coumarin, 3-dehydroquinate, lactic acid, and ethanol. This article reviews the application of droplet-based microfluidics in high-throughput screening, with a focus on high-throughput screening strategies based on UV, visible, and fluorescence spectroscopy, including labeled optical signal detection screening, as well as label-free electrochemical detection, mass spectrometry, Raman spectroscopy, nuclear magnetic resonance, etc. Furthermore, the research progress and development trends of droplet-based microfluidic technology in enzyme modification and strain screening are also introduced.

Label-free electrochemical cancer cell detection leveraging hemoglobin-encapsulated silver nanoclusters and Cu-MOF nanohybrids on a graphene-assisted dual-modal probe

Breast cancer detection at an early stage significantly increases the chances of successful treatment and survival. This study presents an electrochemical biosensor for detecting breast cancer cells, utilizing silver nanoclusters encapsulated by hemoglobin and Cu (II)-porphyrin-metal organic framework (BioMOF) in a graphene-incorporated nanohybrid probe. This Hb-AgNCs@MOF-G probe demonstrates high electrochemical activity, superior dispersity, porosity, and a large surface area for effective functionalization. Using a green ultrasonic-assisted stirring method, we fabricate ultra-small 5 nm particles that readily immobilize on a glassy carbon electrode, generating a detection signal when interacting with ferricyanide/ferrocyanide redox probes. The resulting immunosensor detects as few as 2 cells/mL using Electrochemical Impedance Spectroscopy (EIS) “signal on” and 16 cells/mL via Square Wave Voltammetry (SWV) “signal off”, within a broad range of cell concentrations (102–5 × 104 cells/mL). Our designed sensor shows improved selectivity (5- to 16-fold) and robust detection in human blood with a recovery efficiency between 94.8–106% (EIS method) and 95.4–111% (SWV method). This sensor could streamline early cancer diagnosis and monitor patient treatment without requiring labelling or signal amplification. As a pioneering endeavor, we've utilized integrated porous MOFs with Hb-encapsulated silver nanoclusters in cancer detection, where these components collectively enhance the overall functionality.

Label-free and ultrasensitive electrochemical transferrin detection biosensor based on a glassy carbon electrode and gold nanoparticles

In this study, we developed a label-free and ultrasensitive electrochemical biosensor for the detection of transferrin (Tf), an important serum biomarker of atransferrinemia. The biosensor was fabricated by using glassy carbon electrode (GCE) and modified with gold nanoparticles (AuNPs) via electroless deposition. The electrochemical characteristics of the GCE-AuNPs biosensors were characterized using cyclic voltammetry and electrochemical impedance spectroscopy analysis. Differential pulse voltammetry was used for quantitative evaluation of the Tf-antigen by recording the increase in the anodic peak current of GCE-AuNPs biosensor. The GCE-AuNPs biosensor demonstrates superior sensing performance for Tf-antigen fortified in buffer, with a wide linear range of 0.1 to 5000 μg/mL and a limit of detection of 0.18 μg/mL. The studied GCE-AuNPs biosensor showed excellent sensitivity, selectivity, long-term storage stability and simple sensing steps without pretreatment of clinical samples. This GCE-AuNPs biosensor indicates great potential for developing a Tf detection platform, which would be helpful in the early diagnosis of atransferrinemia. The developed GCE-AuNPs biosensor holds great potential in biomedical research related to point of care for the early diagnosis and monitoring of diseases associated with aberrant serum transferrin levels. These findings suggest that the GCE-AuNPs biosensor has great potential for detecting other serum biomarkers.

Metallo-base pair-mediated DNA probe for label-free, amplified and catalytic electrochemical detection of metallothionein

Metallothionein (MT) is an important biomarker for the early diagnosis of heavy metal toxicity and malignancy and the detection and quantification of MT therefore has important biomedical significances. In this work, we present a new metallo-base pair-mediated DNA probes for label-free and sensitive electrochemical assay of MT via hybridization chain reaction (HCR) and catalytic redox recycling signal amplifications. The DNA probe is immobilized on the gold electrode to prepare the sensor. Due to the strong binding capability between MT and the metal ions in the DNA probes, the presence of the MT target molecules leads to both disruption of the duplex structure of the DNA probes and the release of ssDNA strands, which trigger subsequent HCR formation of long dsDNA polymers on the electrode from two hairpins. Large amounts of [Ru(NH3)6]Cl3 can then be captured onto the dsDNA polymers to show substantially enhanced catalytic currents with co-existence of K3[Fe(CN)6] for highly sensitive assay of MT down to 0.15 nM in the range of 0.5–500 nM. The sensor also shows the capability for monitoring MT in diluted human serum samples, indicating its great potential for detecting various metal ion binding proteins and small molecules for different application purposes.

Bibliometric analysis of aptamer-conjugated nanoparticles for diagnosis in the last two decades

Objective. Aptamer-conjugated nanoparticles for diagnosis have recently gained increasing attention. Here, we performed a bibliometric analysis to provide an overview of this field over the past two decades. Methods. The terms ‘aptamer, nanoparticles and diagnosis’ were used to search for relevant original articles published in English from 2003 to 2022 in the Web of Science database. VOSviewer and CiteSpace software were employed to analyze the development process, knowledge structure, research hotspots, and potential trends in the field of aptamer-conjugated nanoparticles for diagnosis. Results. A total of 1076 original articles were retrieved, with a rapid increase in the annual output and citation. The journal ‘Biosensors and Bioelectronics’ has contributed the most in this field, and the most influential researcher, institution and country were Weihong Tan, the Chinese Academy of Sciences, China, respectively. Gold nanoparticles and quantum dots were the most used, but in the past three years, research hotspots focused on carbon dots and graphene quantum dots. Diagnostic directions primarily focused on cancer. The most used strategy was label-free electrochemical detection, but in the past two years, colorimetric analysis and fluorescence imaging emerged as hot topics. Conclusion. The bibliometric analysis reveals a rapid increase in the research on aptamer-conjugated nanoparticles for diagnosis, major contributors at the levels of journals, authors, institutions, and countries, and research preferences in diagnostic objects, nanoparticle types, and detection methods, as well as the evolution of research hotspots and future trends.

Proximity hybridization induced bipedal DNA walker and rolling circle amplification for label-free electrochemical detection of apolipoprotein A4

Apolipoprotein A4 (Apo-A4) is considered as a prospective molecular biomarker for diagnosis of depression due to its neurosynaptic toxicity. We develop a proximity hybridization-induced DNAzyme-driven bipedal DNA walker strategy for Apo-A4 quantification based on rolling circle amplification (RCA) triggered by poly adenine binding to Ag nanoparticles (AgNPs). With the help of DNAzyme, the free-running bipedal DNA walker can quickly and sequentially shear a molecular beacon that acts as a primer to initiate the RCA process, producing a large number of long DNA strands containing numerous adenines. The long repetitive adenine strands then absorb large amounts of AgNPs on the electrode interface, which is then electrochemically stripped of the AgNPs. The method has a linear detection range of 0.001 ∼ 100 ng mL−1 and a detection limit of 0.46 pg mL−1. The presented detection strategy is label-free, which allows high sensitivity and selectivity for detection of a wide range of protein targets by corresponding DNA-based affinity probes, which have potential applications in bioanalysis.

Label-Free and Sensitive Electrochemical Detection of DNA Methylation Damage Based on DNA Glycosylase Recognition and Excision Coupled with Exonuclease III Amplification

Because the cytotoxic DNA methylated bases are chemically inert and small in size, limited biosensing strategies especially with high sensitivity were established to detect DNA methylation damage. In this work, a label-free electrochemical assay was proposed for the sensitive detection of DNA methylation damage in dsDNA film on indium tin oxide electrode. The human alkyladenine DNA glycosylase was employed to recognize and selectively remove DNA methylated bases, generating apurinic site. Subsequently, exonuclease III not only further converted the methylation sites into strand breaks, but also progressively removed nucleotides from the 3′ to 5′ end starting from the apurinic sites. These processes could maximum amplify DNA methylation damage, also confirmed by gel electrophoresis and fluorescence measurements. The damaged DNA film bound much less electrochemical indicator, Ru(bpy)2(dppz)2+, and accompanied by an anodic current drop. As a result, DNA methylation damage produced with as low as 10 μM of methyl methanesulfonate can be detected, which was 100-fold lower than the previously reported photoelectrochemical sensing strategy. Other DNA base modification products showed much less decrease of oxidation current, demonstrating its good selectivity. This strategy can be utilized to sensitively and rapidly assess the genotoxicity of environmental pollutants.

Attomole Electrochemical Detection of MicroRNAs Based on Surface-Initiated Enzymatic Polymerization Coupled with Copper Enhancement

The sensitive and effective detection of microRNAs (miRNAs) is of great significance since miRNAs have been proven to have undeniable importance in participating in many biological processes. Herein, we present a novel, sensitive, label-free electrochemical miRNA detection method. Three signal amplification techniques are incorporated in this method, including the efficient conjugate of primer-modified polystyrene spheres (PS) with magnetic beads (MBs) triggered by target miRNA, template-free surface-initiated enzymatic polymerization (SIEP) on the primers, and the use of copper ions in square wave voltammetry (SWV) for detecting acidically depurinated primers. Cooperating with the electrochemical approach, this method was able to achieve a detection limit of 120 aM. With an attomole level of sensitivity and easiness of manipulation, this novel method is suitable for miRNA routine detection in both research and clinical aspects.

Proximity hybridization induced bipedal DNA walker for label-free electrochemical detection of apolipoprotein A4 based on DNA meditated Ag nanoparticles growth

Apolipoprotein A4 (Apo-A4) is considered as a prospective molecular biomarker for diagnosis of depression due to its neurosynaptic toxicity. Here, we propose a neighboring hybridization induced catalyzed hairpin assembly (CHA) driven bipedal DNA walker that mediates hybridization of Ag nanoparticles (Ag NPs) with DNA probes for highly sensitive electrochemical quantitative detection of Apo-A4. Driven by CHA, this bipedal DNA walker can spread all over the surface of the sensor, induce the HP1-HP2 double chain structure, make the surface of the sensor negatively charged, and adsorb a large number of Ag ions. After chemical reduction with hydroquinone, the Ag NPs formed provide signal tracers for electrochemical dissolution analysis of the target. The Ag NPs formed by chemical reduction of hydroquinone can provide signal traces for electrochemical stripping analysis of target thrombin. The linear range of this method is from 10 pg mL−1 to 1000 ng mL−1, and the detection limit is 5.1 pg mL−1. This enzyme-free and labeling detection method provides a new strategy for rapid clinical detection of Apo-A4 and accurate identification of depression.

Microprobes for Label-Free Detection of Short Tandem Repeats: An Insight into Alleviating Secondary Structure Effects.

Overgrowth of short tandem repeat sequences in our genes can cause various neurodegenerative disorders. Such repeat sequences are ideal targets for the label-free electrochemical detection of such potential expansions. However, their length- and sequence-dependent secondary structures may interfere with the interfacial charge transfer of a detection platform, making them complex targets. In addition, the gene contains sporadic repeats that may result in false-positive signals. Therefore, it is necessary to design a platform capable of mitigating these effects and ultimately enhancing the specificity of tandem repeats. Here, we analyzed three different backbones of nucleic acid microprobes [DNA, peptide nucleic acid, and lock-nucleic acid (LNA)] to detect in vitro transcribed RNA carrying CAG repeats, which are associated with Huntington's disease, based on the charge-transfer resistance of the interface. We found that the LNA microprobe can distinguish lengths down to the attomolar concentration level and alleviate the effect of secondary structures and sporadic repeats in the sequence, thus distinguishing the "tandem repeats" specifically. Additionally, the control experiments conducted with and without Mg2+ demonstrated the LNA microprobe to perform better in the presence of the divalent cation. The results suggest that the LNA-based platform may eventually lead to the development of a reliable and straightforward biosensor for genetic neurodegenerative disorders.

Tungsten disulfide nanotubes enhanced nanocomposite paper-based aptasensor for label-free electrochemical detection of interferon-gamma

Interferon-Gamma (IFN-ɣ), a measurable biomarker is known to be related to Tuberculosis which is one of the major sanitation issues in the world. It is also a crucial indicator of the immune response to many infections and diseases. Measuring the concentration of IFN-γ can assist in disease diagnosis. Therefore, we have developed a cost-efficient, disposable, and highly sensitive paper-based electrochemical biosensor with probing aptamer immobilization that can detect IFN-ɣ with high sensitivity and selectivity. WS2 nanotubes (NTs) were modified on the nanocomposite conductive paper electrode to provide a large surface area, facilitating the efficient recognition of aptamer with IFN-ɣ, and yielded a high resistance to adsorption of non-targeted molecules. The step-wise modification of the sensing surface was characterized and then the electrode was examined by using differential pulse voltammetry (DPV) in different concentrations of IFN-ɣ samples. The peak current generated by the proposed sensor demonstrated a linear range from 3.125 to 100 pg mL−1, and the detection limit for IFN-ɣ was determined to be 1.13 pg mL−1. Moreover, this novel WS2 NTs enhanced nanocomposite conductive paper-based electrochemical biosensor was qualified to successfully measure IFN-ɣ in human serum samples, demonstrating its potential application as an efficient tool for Tuberculosis diagnosis.