Label-free Detection Research Articles

Microcavity Array-Based Digital SERS Chip for Rapid and Accurate Label-free Quantitative Detection of Live Bacteria.

In this study, we developed a novel digital surface-enhanced Raman spectroscopy (SERS) chip that integrates an inverted pyramid microcavity array, a microchannel cover plate, and a multilayer gold nanoparticle (AuNP) SERS substrate. This innovative design exploits the synergistic effects of the microcavity array and the microchannel to enable rapid and large-scale digital discretization of bacterial suspensions. The concentration effect of the picoliter cavities, combined with the superior Raman enhancement effect of the multilayer AuNP SERS substrate, allows for the precise identification of live bacteria within the microcavities through in situ and label-free SERS testing after a short incubation period. By counting the resulting positive or negative signals, the concentration of the target analyte can be directly determined via Poisson statistics. Experimental results demonstrate that this method enables the accurate quantification of Escherichia coli (E. coli) BL21 within a 4-h incubation period. Compared with traditional analog SERS detection methods, our proposed digital SERS detection strategy reduces the impact of signal intensity fluctuations, thereby significantly improving detection efficiency and accuracy. We believe that this digital SERS chip has great application prospects in the fields of bacterial detection, antibiotic resistance analysis, and cellular dynamics monitoring.

Developments in Localized Surface Plasmon Resonance

AbstractLocalized surface plasmon resonance (LSPR) is a nanoscale phenomenon associated with noble metal nanostructures that has long been studied and has gained considerable interest in recent years. These resonances produce sharp spectral absorption and scattering peaks, along with strong electromagnetic near-field enhancements. Over the past decade, advancements in the fabrication of noble metal nanostructures have propelled significant developments in various scientific and technological aspects of LSPR. One notable application is the detection of molecular interactions near the nanoparticle surface, observable through shifts in the LSPR spectral peak. This document provides an overview of this sensing strategy. Given the broad and expanding scope of this topic, it is impossible to cover every aspect comprehensively in this review. However, we aim to outline major research efforts within the field and review a diverse array of relevant literature. We will provide a detailed summary of the physical principles underlying LSPR sensing and address some existing inconsistencies in the nomenclature used. Our discussion will primarily focus on LSPR sensors that employ metal nanoparticles, rather than on those utilizing extended, fabricated structures. We will concentrate on sensors where LSPR acts as the primary mode of signal transduction, excluding hybrid strategies like those combining LSPR with fluorescence. Additionally, our examination of biological LSPR sensors will largely pertain to label-free detection methods, rather than those that use metal nanoparticles as labels or as means to enhance the efficacy of a label. In the subsequent section of this review, we delve into the analytical theory underpinning LSPR, exploring its physical origins and its dependency on the material properties of noble metals and the surrounding refractive index. We will discuss the behavior of both spherical and spheroidal particles and elaborate on how the LSPR response varies with particle aspect ratio. Further, we detail the fundamentals of nanoparticle-based LSPR sensing. This includes an exploration of single-particle and ensemble measurements and a comparative analysis of scattering, absorption, and extinction phenomena. The discussion will extend to how these principles are applied in practical sensing scenarios, highlighting the key experimental approaches and measurement techniques.

Terahertz cancer cell sensor based on plasmonic toroidal metasurface

This paper investigates a plasmonic toroidal metasurface cancer cell sensor capable of highly sensitive, cost-effective, and label-free detection. Utilizing titanium and gold split ring resonators, the metasurface unit are formed. Numerical findings indicate a significant resonance frequency shift of the metasurface upon changes in the measured substance, enabling accurate detection of various cancer cell types, such as blood cancer cell and cervical cancer cell in the 2.5 THz-4.5 THz band, with sensitivity reaching 1.075 THz/RIU. Moreover, the metasurface sensor demonstrates superior angular stability and high sensitivity in detecting cyanide and heavy metal-contaminated water. These research outcomes lay a theoretical foundation for the development and application of highly sensitive cancer cell sensors.

Construction of G-quadruplex/thioflavin T fluorescent probe for label-free detection of malachite green

Construction of G-quadruplex/thioflavin T fluorescent probe for label-free detection of malachite green

Label-free detection and simultaneous viability determination of CTCs by lens-free imaging cytometry.

The detection of extremely rare circulating tumor cells (CTCs) in peripheral blood and simultaneously identifying their viabilities are significant for cancer diagnosis and prognosis as well as monitoring the efficacy of personalized treatment. A lens-free imaging system features high-resolution images taken over a large field of view (FOV), which has great potential for CTC detection and viability determination. But current still lens-free systems restrict the application for CTC detection in real samples due to the inherent limitations of lens-free technology: (1) the location of cells in the FOV will affect the imaging; (2) the extremely rare CTCs probably did not exist in one observation. In this paper, we realized the detection of CTCs in whole blood and the simultaneous determination of their viabilities by lens-free imaging cytometry. Our in-flow system plus a large FOV range of lens-free imaging highly increased the detection rate of rare CTCs with a high throughput of 150,000 cells per minute and improved the recognition efficiency for blood cells, living/dead CTCs by using a cell tracing-assisted deep learning algorithm. With this method, the average precision of blood cells, living/dead lung cancer cells A549, and living/dead colon cancer cells SW620 reached 98.80%, 97.88%, 97.93%, 97.72%, and 98.60%, respectively. Our system got a highly consistent result with the manual counting method using fluorescent staining (Pearson's r 99.93% for SW620) and can easily detect as few as 10 dead or living CTCs from 100,000 white blood cells (WBCs). Finally, real clinical samples were detected in our system. Both dead and living CTCs were found in all six advanced-stage cancer patients, and the number of living CTCs per million WBCs ranged from 13 to 39, more than that of the dead CTCs (5 to 25), while none of the CTCs were detected in six healthy control subjects. Moreover, we also found that CTCs died very quickly after leaving the human body, indicating that CTCs should be studied as soon as possible after sampling. Although this method is implemented for CTCs, it can also be used for the detection of other rare cells.

Dielectric Surface-Based Biosensors for Enhanced Detection of Biomolecular Interactions: Advances and Applications

Surface plasmon resonance (SPR) biosensors are extensively utilized for analyzing molecular interactions due to their high sensitivity and label-free detection capabilities. Recent innovations in surface-sensitive biosensors with dielectric surfaces address the inherent limitations associated with traditional gold surfaces, such as thermal effects and biocompatibility issues, which can impede broader applications. This review examines state-of-the-art biosensor configurations, including total internal reflection, optical waveguide, photonic crystal resonators, Bloch surface wave biosensors, and surface electrochemical biosensors, which can enhance analyte signals and augment the molecular detection efficiency at the sensor interface. These technological advancements not only improve the resolution of binding kinetics analysis and single-molecule detection but also extend the analytical capabilities of these systems. Additionally, this review explores prospective advancements in augmenting field enhancement and incorporating multimodal sensing functionalities, emphasizing the significant potential of these sophisticated biosensing technologies to profoundly enhance our understanding of molecular interactions.

Hydrogel interfaced glass nanopore for high-resolution sizing of short DNA fragments

Solid-state nanopores, known for their label-free detection and operational simplicity, face challenges in accurately sizing the short nucleic acids due to fast translocation and a lack of enzyme-based control mechanisms as compared to their biological counterparts with sizing resolutions still limited to ≥100 bp. Here, we present a facile polyethylene glycol-dimethacrylate (PEG-DMA) hydrogel interfaced glass nanopore (HIGN) system by inserting glass nanopore into the hydrogel to achieve sub-100 base pair (bp) resolution in short DNA sizing analysis. We systematically investigated the effects of hydrogel mesh size, spatial configurations of glass nanopores about the hydrogel, applied bias voltage, and analyte concentration on the transport dynamics of 200 bp double-stranded DNA (dsDNA). A 7.5 w/v% PEG-DMA hydrogel induced ∼11x increase in the mean dwell times compared with bare solution nanopore system. The insertion locations and depths of the glass nanopore into the hydrogel resulted in 7.16% and 5.28% coefficients of variation (CV) for mean normalized event frequencies. This enhancement of dwell times and invariability in translocation characteristics enables precise sizing of dsDNA fragments under 400 bp using HIGN, with an achieved size resolution of 50 bp with observable mean normalized peak amplitude (ΔI/Io) of ∼0.005. Furthermore, we have demonstrated the capability of HIGN to perform multiplex detection of influenza A virus (IAV) and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) through reverse transcriptase-polymerase chain reaction (RT-PCR). These results demonstrated the potential of HIGN as a versatile tool in nucleic acid analysis and multiplexed label-free molecular diagnostics.

Fast and label-free detection of procalcitonin in human serum for sepsis using a WTex-based extended-gate field-effect transistor biosensor

In this article, we present the first instance of depositing WTex-sensitive films with varying thicknesses (3, 4, and 5 nm) onto flexible polyimide substrates using radio-frequency sputtering. These films were used to create an extended-gate field-effect transistor (EGFET) for pH sensing and detecting procalcitonin (PCT) in the sera of patients with sepsis or bacterial infections. Among the films, the 4 nm WTex film exhibited high sensitivity (59.57 mV/pH), minimal hysteresis (∼0.8 mV), and a low drift rate (0.14 mV/h). Additionally, this WTex-based EGFET sensor retained a pH sensitivity of 59.2 mV/pH even after 180 days of operation and exhibited excellent mechanical flexibility, enduring 500 bending cycles without degradation. Moreover, PCT antibodies, activated using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide, were immobilized on the WTex film functionalized with 3-aminopropyl triethoxysilane. This effective immobilization enabled the specific binding of PCT antigens. The WTex-based EGFET biosensor demonstrated high sensitivity (18.12 mV/pCPCT) across a wide dynamic range (1 fg/mL to 1 μg/mL). Furthermore, the PCT concentrations in patient sera, whether from individuals with or without sepsis or bacterial infections, measured by our biosensor were comparable to results obtained using clinical enzyme-linked immunosorbent assay kits.

Electrokinetic Preconcentration and Label-Free Electrical Detection of SARS-CoV-2 RNA at a Packed Bed of Bioconjugated Microspheres

Electrokinetic Preconcentration and Label-Free Electrical Detection of SARS-CoV-2 RNA at a Packed Bed of Bioconjugated Microspheres

A Novel PCR-Free Ultrasensitive GQD-Based Label-Free Electrochemical DNA Sensor for Sensitive and Rapid Detection of Francisella tularensis

Biological warfare agents are infectious microorganisms or toxins capable of harming or killing humans. Francisella tularensis is a potential bioterrorism agent that is highly infectious, even at very low doses. Biosensors for biological warfare agents are simple yet reliable point-of-care analytical tools. Developing highly sensitive, reliable, and cost-effective label-free DNA biosensors poses significant challenges, particularly when utilizing traditional techniques such as fluorescence, electrochemical methods, and others. These challenges arise primarily due to the need for labeling, enzymes, or complex modifications, which can complicate the design and implementation of biosensors. In this study, we fabricated Graphene Quantum dot (GQD)-functionalized biosensors for highly sensitive label-free DNA detection. GQDs were immobilized on the surface of screen-printed gold electrodes via mercaptoacetic acid with a thiol group. The single-stranded DNA (ssDNA) probe was also immobilized on GQDs through strong π−π interactions. The ssDNA probe can hybridize with the ssDNA target and form double-stranded DNA, leading to a decrease in the effect of GQD but a positive shift associated with the increase in DNA concentration. The specificity of the developed system was observed with different microorganism target DNAs and up to three-base mismatches in the target DNA, effectively distinguishing the target DNA. The response time for the target DNA molecule is approximately 1010 s (17 min). Experimental steps were monitored using UV/Vis spectroscopy, Atomic Force Microscopy (AFM), and electrochemical techniques to confirm the successful fabrication of the biosensor. The detection limit can reach 0.1 nM, which is two–five orders of magnitude lower than previously reported methods. The biosensor also exhibits a good linear range from 105 to 0.01 nM and has good specificity. The biosensor’s detection limit (LOD) was evaluated as 0.1 nM from the standard calibration curve, with a correlation coefficient of R2 = 0.9712, showing a good linear range and specificity. Here, we demonstrate a cost-effective, GQD-based SPGE/F. tularensis DNA test suitable for portable electrochemical devices. This application provides good perspectives for point-of-care portable electrochemical devices that integrate sample processing and detection into a single cartridge without requiring a PCR before detection. Based on these results, it can be concluded that this is the first enzyme-free electrochemical DNA biosensor developed for the rapid and sensitive detection of F. tularensis, leveraging the nanoenzyme and catalytic properties of GQDs.

Bifunctional MoO3-x/CuS Heterojunction Nanozyme-Driven "Turn-On" SERS Signal for the Sensitive Detection of Cerebral Infarction Biomarker S100B.

The use of nanozymes has become a promising auxiliary approach to "turn on" surface-enhanced Raman scattering (SERS) signals for the label-free detection of disease markers. Nevertheless, there are still major challenges to develop bifunctional nanomaterials with both excellent enzyme-like activity and high SERS performance. To this end, a novel Z-scheme MoO3-x/CuS heterojunction was first constructed as a powerful "two-in-one" substrate, which can not only catalyze leucomalachite green (LMG) to SERS-active malachite green (MG) but also serve as an efficient substrate to amplify the SERS signal of catalysate. Due to the strong interfacial coupling effect between the MoO3-x and CuS nanomaterial, which promoted the separation and transport of carriers in the heterojunction, the MoO3-x/CuS heterojunction showed higher peroxidase-like activity compared to individual components and the previously reported heterojunction nanozymes. Inspired by these results, a sandwich-type SERS immunoassay for the detection of the cerebral infarction biomarker S100 calcium-binding protein (S100B) was proposed based on the output signal of MG at 1620 cm-1. Furthermore, introducing the antifouling material chitosan on the surface of the MoO3-x/CuS heterojunction can effectively resist nonspecific protein adsorption and significantly improve the detection accuracy of the immunoassay. Therefore, the SERS immunoassay based on the MoO3-x/CuS heterojunction realized highly sensitive and selective detection of S100B in the concentration range of 0.001 to 100 ng/mL, with a low limit of detection of 0.47 pg/mL. The developed method has been successfully used for the accurate detection of S100B in clinical serum. The results showed that the level of S100B in the serum of cerebral infarction patients can be distinguished from those of healthy individuals and intracranial tumor patient controls. In addition, the acquired values of S100B in the serum of cerebral infarction patients based this strategy were well consistent with the results of electrochemiluminescence (ECL) detection with a relative error of less than ±7.3. It is expected that this work may open up a paradigm for improving detection sensitivity and accuracy for the early diagnosis and treatment monitoring of cerebral infarction in the clinic.

Split G-Quadruplex Programmed Recyclable AIE-Biosensor for Label-Free Detection of miRNA in Acute Kidney Injury.

We herein rationally designed a target-recyclable AIE-biosensor based on a split G-quadruplex for label-free detection of miRNA in acute kidney injury. Initially, the PG was in an "OFF" state, and the two split segments (G4-a and G4-b) of G4 were tethered at the two terminals of P1 and far away from each other due to the rigid duplex structure formed by the partially complementary intermediate sequences of P2 and P1, bringing MG with quenched fluorescence. In the presence of target, the 5'-PO4 P2 was displaced from PG probe and competitively hybridized with target, which led to G4-a and G4-b tending to form an intact intermolecular G-quadruplex, providing sites for MG intercalation, thus generating an activated "ON" fluorescence signal due to the restriction of intermolecular motion. Successively, relying on the λ-exonuclease (λ-Exo) cleavage reaction-assisted target recycling, more amounts of targets will be liberated, accompanied by forming more G-quadruplex and binding more MG, resulting in a strong fluorescence signal, further realizing the sensitive detection of the targets. As a proof of concept, miRNA-21 was chosen as the model target. Endowing with the precise target recognition and efficient cleavage activity of λ-Exo, the AIE-biosensor exhibited excellent detection sensitivity and specificity, which could quantitatively detect miRNA-21 down to 10.36 fM with a single mismatch specificity. The results revealed that the distinctive attributes of noninvasive, simple, and efficient in this G-quadruplex-based AIE-biosensor offered promising prospects for extensive applications in AKI screening and early clinical diagnosis.

Development of graphene oxide-based biosensing platforms for label-free bioelectronic detection of pathogenic microorganisms

Development of graphene oxide-based biosensing platforms for label-free bioelectronic detection of pathogenic microorganisms

An Ultrahigh Affinity DNA Aptamer for Quinine and Its Intrinsic Fluorescence Based Label-free Detection.

Measuring quinine is critical for the detection of its overdose, understanding its pharmacological and toxicological effects, and monitoring its pollution. While a previously reported aptamer named MN4 can bind quinine, it was not selected for it, leading to compromised binding affinity and specificity. In this work, a new quinine aptamer was isolated using the library immobilization capture-SELEX technique. The Q1 aptamer has a Kd value of 10 nM determined by an isothermal titration calorimetry experiment and 45 nM in a fluorescence binding assay. A 3.5 nM quinine limit of detection was obtained based on the aptamer binding-induced quenching of the intrinsic fluorescence of quinine. A large blue shift in fluorescence was observed for quinine upon binding to Q1, whereas binding to MN4 led to a very small red shift, indicating different ways of quinine binding by these two aptamers. Q1 did not bind cocaine based on NMR spectroscopy and fluorescence assays also indicated excellent selectivity against other tested molecules. This work has supplied a high affinity aptamer for quinine that can be useful for its detection and fundamental aptamer binding studies. It also highlights the advantages of using capture-SELEX to isolate aptamers for small molecules.

Surface Blocking-Based Potentiometric Biosensor for Detection of E. coli ATCC 15597 Using Phage MS2 as a Receptor.

Nowadays, using a potentiometric ion sensor to achieve detection of biological analytes is still a big challenge, since these analytes usually do not yield a measurable ion signal. To address this challenge, a simple and robust potentiometric sensing protocol based on a delayed Nernstian response is proposed for the label-free detection of biological analytes. The proposed sensor platform is composed of two layers: the surface recognition layer and the indicator-ion-selective electrode (ISE) membrane layer. It is based on a surface blocking mechanism in which the surface recognition interactions between the surface recognition element and the target can impede the diffusion of the indicator ion from the aqueous phase to the sensing membrane phase to reach the final Nernstian-response equilibrium, thus resulting in a delayed potential change. Such a potential change could be used to measure the concentration of a biological target in the sample. Thus, a sensing system was designed by using phage MS2, its host bacterium Escherichia coli ATCC 15597(abbreviated as E. coli H), and a solid-contact butyrylcholine ISE as a surface recognition element, a target, and an indicator electrode, respectively. This new concept offers a simple, sensitive, and extremely selective potentiometric method for detection of E. coli H with a detection limit of 100 CFU mL-1. It can be expected that the present approach may pave the way to using ISEs to detect various important nonionic biological targets in clinical and environmental applications.

A novel approach ofdifferentiation of adenoma and carcinoma in lung cancer based on biogenic in situ synthesis of gold nanostructures on various oligonucleotide motifs.

Aunique approach is introducedfor constructing gold nanocrystals (AuNCs) with RNA motif-directed morphologies in a sequence-independent manner and itsapplications in theclinical areaare described. By using this method, a label-free LSPR-based detection method for the SOX2OT transcript, long non-coding RNAs (lncRNAs), which is a prognostic indicator of poor survival in lung cancer patientsis presented. For the first time, we examined how the structural changes of RNA after the heteroduplex formation with a specific DNA probe can change the morphology and LSPR band of AuNCs. Using this method, is was possible to differentiate lung squamous cell carcinoma from adenocarcinoma samples without a need for a prior amplification of the target lncRNA. The approach of using specific DNA probe enables the in situ synthesis of nanocrystals in a different way and expands this method for future translational medicine, particularly detection of specific RNA.

Dielectric Modulated Nanotube Tunnel Field-Effect Transistor with Core-Shell Cavity as a Label-Free Biosensor: Proposal and Analysis.

Dielectric Modulated Field-Effect Transistors (DMFETs) have emerged as promising candidates for label-free bioanalyte detection. However, the inherent short-channel effects in conventional DMFETs increase their static power dissipation significantly and limit their scalability and sensitivity. Therefore, FETs based on alternate conduction mechanism such as tunneling (TFETs), which are immune to the short-channel effects, appear to be a lucrative alternative to the MOSFETs for biosensing application. In this work, we propose a novel Dual Cavity Dielectric Modulated Nanotube Tunnel FET (DCDM NTTFET)-based label-free biosensor consisting of a Ge source and nanocavities within the core as well as a shell gate stack, which not only outperforms the conventional MOSFET and advanced nanowire (NW) TFET-based biosensors in terms of energy-efficiency and scalability but also exhibits a significantly high drain current sensitivity (SION = 2.9 × 108) and a threshold voltage sensitivity (SVth = 0.85), and a considerably high selectivity of more than 6 orders of magnitude. We also perform a comprehensive design space exploration for the proposed DCDM NTTFET and provide necessary design guidelines to further improve its performance considering the practical artifacts such as steric hindrance.

Blood Biomarker Detection Using Integrated Microfluidics with Optical Label-Free Biosensor.

In this study, we developed an optofluidic chip consisting of a guided-mode resonance (GMR) sensor incorporated into a microfluidic chip to achieve simultaneous blood plasma separation and label-free albumin detection. A sedimentation chamber is integrated into the microfluidic chip to achieve plasma separation through differences in density. After a blood sample is loaded into the optofluidic chip in two stages with controlled flow rates, the blood cells are kept in the sedimentation chamber, enabling only the plasma to reach the GMR sensor for albumin detection. This GMR sensor, fabricated using plastic replica molding, achieved a bulk sensitivity of 175.66 nm/RIU. With surface-bound antibodies, the GMR sensor exhibited a limit of detection of 0.16 μg/mL for recombinant albumin in buffer solution. Overall, our findings demonstrate the potential of our integrated chip for use in clinical samples for biomarker detection in point-of-care applications.

Hierarchical Au@Pt nanoparticle/amino benzoic acid polymer-based hybrid material for labeled and label-free detection of interleukin-6: a comparative assessment.

Interleukin-6 (IL6) is a cytokine mainly involved in inflammatory processes associated with various diseases, from rheumatoid arthritis and pathogen-caused infections to cancer, where malignant cells exhibit high proliferation and overexpression of cytokines, including IL6. Furthermore, IL6 plays a fundamental role in detecting and differentiating tumor cells, including colorectal cancer (CRC) cells. Therefore, given its range of biological activities and pathological role, IL6 determination has been claimed for the diagnosis/prognosis of immune-mediated diseases. Herein, a comparative study is presentedof labeled and label-free electrochemical immunosensors involving a hierarchical Au@Pt nanoparticle/polymer hybrid material for detecting IL6. The electrochemical immunosensors were independently coupled to the surface of screen-printed carbon electrodes (SPCEs) previously modified with polymeric layers. While in the label-free immunosensor, an anti-IL6 antibody (IL6-Ab) was covalently bound to the modified SPCE surface, in the sandwich-like amperometric immunosensor, an anti-biotinylated-IL6 antibody (B-IL6-Ab) was attached to the electrode through biotin-avidin affinity interactions. The label-free format employed a straightforward detection of IL6 by differential pulse voltammetry (DPV). The resulting electrochemical immunosensors exhibited a linear dynamic range from 50 to 750pg/mL IL6, with detection limits (LOD) of 14.4 and 6.0pg/mL for label-free and sandwich-like immunosensors, respectively. This outstanding performance makes them versatile platforms for clinical analysis of a panel of biomarkers for early diagnosis/prognosis of inflammatory processes associated with oncological diseases, among other pathologies.

A bioinspired porous and electroactive reduced graphene oxide hydrogel based biosensing platform for efficient detection of tumor necrosis factor-α.

Oral cancer is one of the leading cancer types, which is frequently diagnosed at an advanced stage, giving patients a poor prognosis and fewer therapeutic choices. To address this gap, exploiting biosensors utilizing anti-biofouling hydrogels for early-stage oral cancer detection in non-invasive body fluids is gaining utter importance. Herein, we have demonstrated the fabrication of an innovative electrochemical immunosensor for the rapid, label-free, non-invasive, and affordable detection of tumor necrosis factor-α (TNF-α), a biomarker associated with oral cancer progression in artificial saliva samples. The gold screen-printed electrodes (gSPEs) are modified with a green synthesized porous and electroactive reduced graphene oxide (rGO) hydrogel utilizing L-cystine (L-cys) as both in situ reducing and surface functionalization agent, followed by covalent immobilization of anti-TNF-α and blocking of residual sites with bovine serum albumin (BSA) to fabricate the BSA/anti-TNF-α/L-cys_rGO hydrogel/gSPE immunosensing platform. The fabricated platform demonstrates excellent performance, with a low limit of detection of 1.20 pg mL-1, a broad linear range from 1 to 200 pg mL-1, and a high sensitivity of 2.10 μA pg-1 mL cm-2 carried out with differential pulse voltammetry (DPV) technique. Moreover, it exhibits specificity towards TNF-α, even in the presence of potential interferents and other cancer biomarkers. Besides, the biosensor showed good reproducibility and repeatability with a relative standard deviation (%RSD) of 5.11% and 1.85%, respectively. Thus, integrating the L-cys_rGO hydrogel in the immunosensor design offers enhanced performance, paving the way for its application in early-stage oral cancer diagnosis.