Label-free Real-time Monitoring Research Articles

Electrode Droplet Microarray (eDMA): An Impedance Platform for Label-Free Parallel Monitoring of Cellular Drug Response in Nanoliter Droplets.

Label-free real-time monitoring of cellular behavior using impedance spectroscopy is important for drug development and toxicological assessments. Parallelization and miniaturization of such experiments are essential for increasing throughput and enabling experiments with low abundant stem or primary cells. Traditional methods are not miniaturized and require large volumes of reagents and number of cells, limiting their suitability for cost effective high-throughput screening of cells of limited availability. Here, the fabrication, optimization, and application of a bioelectrical signaling monitoring system - electrode droplet microarray (eDMA) are demonstrated. The eDMA platform is based on preparation of a hydrophilic-superhydrophobic patterns covering an array of individually addressable microelectrodes, which confines cells to individual microelectrodes, allowing for parallel, real-time, and label-free detection of cellular responses to drug treatments in nanoliter droplets. The real-time monitoring of cytotoxic effect of an anticancer drug is demonstrated over 48 h with real-time calculation of the half-inhibitory concentration (IC50) values through impedance spectroscopy. This demonstrates eDMA's ability to dynamically assess responses to various drugs in parallel at any given time point, which is crucial for functional personalized oncology. Specifically, the platform can be employed for monitoring anticancer drug toxicity using limited patient samples, where the miniaturization provided by eDMA is essential.

Optical and electronic signal stabilization of plasmonic fiber optic gate electrodes: towards improved real-time dual-mode biosensing

The use of multimodal readout mechanisms next to label-free real-time monitoring of biomolecular interactions can provide valuable insight into surface-based reaction mechanisms. To this end, the combination of an electrolyte-gated field-effect transistor (EG-FET) with a fiber optic-coupled surface plasmon resonance (FO-SPR) probe serving as gate electrode has been investigated to deconvolute surface mass and charge density variations associated to surface reactions. However, applying an electrochemical potential on such gold-coated FO-SPR gate electrodes can induce gradual morphological changes of the thin gold film, leading to an irreversible blue-shift of the SPR wavelength and a substantial signal drift. We show that mild annealing leads to optical and electronic signal stabilization (20-fold lower signal drift than as-sputtered fiber optic gates) and improved overall analytical performance characteristics. The thermal treatment prevents morphological changes of the thin gold-film occurring during operation, hence providing reliable and stable data immediately upon gate voltage application. Thus, the readout output of both transducing principles, the optical FO-SPR and electronic EG-FET, stays constant throughout the whole sensing time-window and the long-term effect of thermal treatment is also improved, providing stable signals even after 1 year of storage. Annealing should therefore be considered a necessary modification for applying fiber optic gate electrodes in real-time multimodal investigations of surface reactions at the solid-liquid interface.

Label-free DNA quantification using isothermal amplification on an exposed core optical fiber microfluidic platform.

Isothermal amplification technology has triggered a surge in research due to its compatibility with small and portable equipment, simplicity, and high efficiency, especially in light of the COVID-19 pandemic where reliable widescale testing is critical to outbreak management. In this paper, a label-free isothermal deoxyribonucleic acid (DNA) amplification method based on refractive index (RI) quantification is proposed and demonstrated for the first time by combining optical fiber sensing, microfluidics, and isothermal amplification. A highly RI-sensitive Mach-Zehnder (MZ) interference is formed by splicing a short length of an exposed-core fiber between two lengths of a single-mode fiber while the microfluidic liquid channel on the exposed side of the ECF is filled with target DNA and the amplification solution. Real-time quantitative measurement of the target DNA is then realized by monitoring the change in RI of the solution during the isothermal DNA amplification process. The experimental results show that the platform successfully realizes real-time label-free monitoring of isothermal amplification of 0.16 aM DNA samples. This method is a breakthrough for applications in the fields of DNA detection and quantification where simple operation, rapid detection, portability, small size, high selectivity, and high sensitivity are required.

Label-free real-time monitoring of the BCR-triggered activation of primary human B cells modulated by the simultaneous engagement of inhibitory receptors

Today, there is an intense demand for lab-on-a-chip and tissue-on-a-chip applications in basic cell biological research and medical diagnostics. A particular challenge is the implementation of advanced biosensor techniques in point-of-care testing utilizing human primary cells. In this study, a resonant waveguide grating (RWG)-based label-free optical biosensor technique has been applied for real-time monitoring of the integrated responses of primary human tonsillar B cells initiated by B cell receptor (BCR) and modified by FcγRIIb and CR1 engagement. The BCR-triggered biosensor responses of resting and activated B cells were revealed to be specific and dose-dependent, in some cases with strong donor dependency. Targeted inhibition of Syk attenuated the label-free biosensor response upon BCR stimulation. Indifferent protein human serum albumin (HSA) did not interfere with the recorded signal to BCR stimulation. Simultaneous engagement of BCR and FcγRIIb modulated the kinetic signal of the cells. Activated and resting B cells exhibited different response profiles upon simultaneous engagement of BCR and CR1. This advanced approach has the potential to decipher interfering signaling events in human B cells, manage differences between activated and resting B cell states, helping to understand the actual integrated response of these immune cells, and could be useful in the point-of-care diagnostic testing on human primary cells.

An analysis method to detect the presence of DNA using electrochemical impedance spectroscopy (EIS) for real-time PCR

This paper reports a technique for detecting the presence of DNA in real-time PCR using label-free electrochemical impedance spectroscopy. The change in the real and imaginary parts of the impedance with the number of PCR cycles indicates the concentration of amplified DNA. In general, the real part of the impedance tends to increase with the number of PCR cycles over most of the measurement frequency range (100 Hz–1 MHz), whereas the imaginary part of the impedance increases in the negative direction. The optimal frequency for impedance analysis is determined by the performance index (PI), as one of the figures of merit, which quantitatively indicates how monotonously the impedance changes with the PCR cycle number. The detectability of DNA in the sample is scrutinized by analyzing the impedance at the optimal frequency (3.984 kHz and 20.02 kHz for the real and imaginary parts of the impedance, respectively) obtained from the performance index. The proposed technique can be used to quantitatively analyze PCR by applying simple electrochemical impedance spectroscopy (EIS) technology so that a label-free real-time monitoring system has high potential for a portable PCR system.

Detection of Cancer Exosomes By a Localized Surface Plasmon Resonance Method Using Gold Nano-Islands

IntroductionNoble metals have a most important property, the Localized Surface Plasmon Resonance (LSPR) that can be tuned by changing the size and shape of the nanoparticles. This property is useful to perform any real-time label-free monitoring of biomolecular interactions, by measuring the shift of the Plasmon band towards longer wavelengths due to the change in the local refractive index upon molecule binding [1, 2]. Exosomes are nano-sized particles (30-100 nm) released to the extracellular space by all cells in large numbers, prospective biomarkers for early-stage cancer diagnosis [3-5]. The aim of the present study is the detection and capture of exosomes, by using (1) a synthetic peptide and (2) antibodies specific to the surface proteins of exosomes. In both approaches, the biomolecules are immobilized on gold (Au) nano-islands. The first approach makes use of a synthetic polypeptide Vn96 (Venceremin) that has a high affinity towards the heat shock proteins present on the surface of exosomes. The second approach is based on the antibodies (CD63, CD81 and CD9) that are specific to the proteins present on the surface of exosomes.MethodThe gold nano-islands are fabricated by the thermal convection of ex-situ synthesized gold nanoparticles, followed by the heat treatment of substrates at 560oC to tune the size and shape of the particles. Au nano-islands are initially functionalized by 11-mercaptoundecanoic acid (Nanothink) to form a self-assembled monolayer. This layer is activated, using a cross-linker, which is a mixture of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) in the ratio of 1:1. The activated layer is coated with streptavidin to bind, biotinylated Vn96 peptide to capture exosomes whereas, in the antibody-based approach, streptavidin-biotin step is replaced with the antibodies. To the immobilized molecules of polypeptide/antibody (anti-CD63/81), MCF7 (breast cancer cell line) cell culture conditioned media, containing EVs/exosomes is added. At each successive functionalization and immobilization step, the position of the Au plasmon band in the UV-Visible spectrum is measured and the corresponding shift is calculated.ResultsThe preliminary results obtained by using the two protocols indicate that both approaches have the capability to detect and capture the exosomes for cancer diagnosis. The wavelength shift observed in the antibody-based approach, is relatively higher, compared to the one observed when Vn96 was used to capture the exosomes. By using specifically, the anti-CD63 antibody, there is at least an appreciable increase in the wavelength shift, compared to Vn96 polypeptide. In addition, the sensing protocol can be simplified and the detection can be carried out in a shorter time.ConclusionsThe quantification of exosomes has been performed by using two different LSPR methods. One approach uses the Vn96 peptide for the capture of exosomes, while the other, makes use of the antibodies corresponding to surface proteins. Both methods provided reliable results. However, the method using antibodies results in a larger shift of the Plasmon band showing a stronger interaction and the method can be carried out in a shorter time.References Nath and A. Chilkoti, “Label-free biosensing by surface plasmon resonance of nanoparticles on glass: optimization of nanoparticle size," Anal. Chem., vol. 76, pp. 5370-5378, 2004Nath and A. Chilkoti, “Label free colorimetric biosensing using nanoparticles,” J. Fluoresc., vol. 14, pp. 377-389, 2004.María Yáñez-Mó, Pia R.-M. Siljander, Zoraida Andreu, Apolonija Bedina Zavec, Francesc E. Borràs, et al., “Biological properties of extracellular vesicles and their physiological functions,” Journal of Extracellular Vesicles, vol.4: 27066, 2015.Gregor Fuhrmann, Inge K. Herrmann, and Molly M. Stevensa, “Cell-derived vesicles for drug therapy and diagnostics: Opportunities and challenges,” Nano Today, vol.10, pp.397—409, 2015.Bathini, D. Raju, S. Badilescu, et al., “Nano–Bio Interactions of Extracellular Vesicles with Gold Nanoislands for Early Cancer Diagnosis,” Research, vol. 2018, Article ID 3917986, 10 pages, 2018.

Facing the Challenges of Pharmaceutical Research Using Whispering Gallery Modes-Based Biosensors

During the last decade, different Whispering Gallery Modes-based biosensors (WGMS) have been proposed as bioanalytical platforms. These systems allow for a label-free real-time monitoring of a wide range of molecules, including DNA, proteins and polymers. With the advancement of new technologies and the increasing complexity of novel biotherapeutics, the development of versatile tools is of great relevance to meet the needs of the pharmaceutical research. Throughout this review, the role of WGMS in the drug development process and their ability to face the current challenges and limitations in this field will be discussed.

Fast, sensitive and selective determination of herbicide glyphosate in water samples with a White Light Reflectance Spectroscopy immunosensor

An optical immunosensor based on White Light Reflectance Spectroscopy is described for the determination of the herbicide glyphosate in drinking water samples. The biosensor allows for the label-free real-time monitoring of biomolecular interactions taking place onto a SiO2/Si chip by transforming the shift in the reflected interference spectrum caused by the immunoreaction to effective biomolecular adlayer thickness. Glyphosate determination is accomplished by functionalizing the chip with a protein conjugate of the herbicide followed by a competitive immunoassay format. Prior to the assay, glyphosate derivatization in the calibrators and/or the samples was performed through reaction with succinic anhydride. Under the optimized assay protocol, a detection limit of 10 pg mL−1 was achieved. Recovery values ranging from 90.0 to 110% were determined in spiked bottled and tap water samples, demonstrating the accuracy of the method. In addition, the sensor could be regenerated and re-used for at least 14 times without statistically significant effect on the assay sensitivity and accuracy. The excellent analytical performance and short analysis time (approx. 25 min), combined with the small sensor size, should be helpful for the fast on-site determination of glyphosate in drinking water samples.

Unveil early-stage nanocytotoxicity by a label-free single cell pH nanoprobe.

Single-cell analysis is an emerging research area that aims to reveal delicate cellular status and underlying mechanisms by conquering the intercellular heterogeneity. Current single-cell research methods, however, are highly dependent on cell-destructive protocols and cannot sequentially display the progress of cellular events. A recently developed pH nanoprobe in our lab conceptually showed its ability to detect intracellular pH (pHi) without cell labeling or disruption. In the present study, we took the cytotoxicity of nanoparticles (NPs) as a typical example of cell heterogeneity, to testify the practicality of the pH nanoprobe in interpreting cell status. Three types of NPs (CeO2, TiO2, and SiO2) were employed to generate varied toxic effects. Results showed that the traditional assays - including cell viability, intracellular ROS generation, and mitochondrial inner membrane depolarization - not only failed to report the nanotoxicity accurately and timely, but also drew confusing or misleading conclusions. The pH nanoprobe revealed explicit pHi changes induced by the NPs, which corresponded well with the cell damages found by the transmission electron microscopic (TEM) imaging. Besides, our results unveiled an unexpectedly devastating effect of SiO2 NPs on cells during the early stage NP-cell interaction. The developed novel pH nanoprobe demonstrated a rapid sensing capability at single-cell resolution with minimum invasiveness. Therefore, it may become a promising alternative for a wide range of applications in areas such as single-cell research and precision medicine.

Monitoring kinetics reveals critical parameters of IgA-dependent granulocyte-mediated anti-tumor cell cytotoxicity

Human IgA antibodies effectively engage myeloid cells for the FcαRI-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells. Established methods to investigate ADCC are the 51chromium and Calcein release assays. Their critical limitations are the end-point measurement, the unspecific release of the probes, the requirement of target cells in suspension and thus do not reflect physiologic conditions of adherently growing cells. Here we report the label-free real-time monitoring of granulocyte-mediated ADCC using an impedance-based method. We investigated the efficacy of an engineered epidermal growth factor receptor (EGFR)-directed IgA2 antibody to engage neutrophils for ADCC against a panel of adherently growing EGFR-expressing cancer cell lines majorly head and neck squamous cell carcinoma (HNSCC). The impedance assay allowed the documentation of the IgA-neutrophil-and FcαRI-signaling dependent ADCC of adherently growing target cells. While at a short-term it provided comparable results to release assays, in the long run real time monitoring also revealed cell-line specific kinetics and long-term efficacy. Although short-term results may depend on EGFR expression, long-term efficacy did not correlate with the surface level of EGFR nor of the myeloid checkpoint CD47 pointing to additional critical parameters to predict the treatment efficacy. Real-time monitoring of neutrophil-mediated ADCC allowed documenting effector cell activity and exhaustion. Along with excess expression of Mac-1 ligands, which may explain the target cell resistance, this eventually leads to tumor cell outgrowth at later time points. In conclusion, the impedance assay provides valuable information on the kinetics, effector cell performance, efficacy and critical parameters of IgA-dependent granulocyte-mediated cytotoxicity and is expected to become an important tool in its evaluation.

Simultaneous determination of paraquat and atrazine in water samples with a white light reflectance spectroscopy biosensor

An optical immunosensor based on White Light Reflectance Spectroscopy for the simultaneous determination of the herbicides atrazine and paraquat in drinking water samples is demonstrated. The biosensor allows for the label-free real-time monitoring of biomolecular interactions taking place onto a SiO2/Si chip by transforming the shift in the reflected interference spectrum due to reaction to effective biomolecular layer thickness. Dual-analyte determination is accomplished by functionalizing spatially distinct areas of the chip with protein conjugates of the two herbicides and scanning the surface with an optical reflection probe. A competitive immunoassay format was adopted, followed by reaction with secondary antibodies for signal enhancement. The sensor was highly sensitive with detection limits of 40 and 50 pg/mL for paraquat and atrazine, respectively, and the assay duration was 12 min. Recovery values ranging from 90.0 to 110% were determined for the two pesticides in spiked bottled and tap water samples, demonstrating the sensor accuracy. In addition, the sensor could be regenerated and re-used at least 20 times without significant effect on the assay characteristics. Its excellent analytical performance and short analysis time combined with the small sensor size should be helpful for fast on-site determinations of these analytes.

Real-time monitoring of amyloid fibrillation by electrical impedance spectroscopy

Electrical impedance spectroscopy (EIS) appears a promising label-free methodology for the investigation of processes related to the aggregation of macromolecules in solution. Here, we explore the EIS technique as a convenient tool for studying the irreversible aggregation of human insulin and describing its corresponding fibrillation kinetics. The in situ measurement of the electrical response of pure insulin solutions at 60°C allows for the real-time monitoring of the protein fibrillation as a function of the incubation time. The fitting of the EIS data through an equivalent circuit based on a constant phase element provides a simple set of electric parameters whose abrupt changes can be associated to transitions occurring in the organization of the macromolecules. For establishing the reliability of the method proposed, we have compared the protein aggregation profile collected from the EIS data to that obtained from a conventional fluorescence methodology where Thioflavin T (ThT) is used as a dye probe. The description of the fibrillation process is quite similar in both cases, since characteristic times of the same order were found for the consecutive processes associated to the initial lag phase of insulin fibrillation, to the rapid growth of amyloidal aggregates and to the final saturation step. Our results suggest that in situ EIS can be considered as a promising approach for the real-time label-free monitoring of protein fibril formation.

Label-free visualization and quantification of single cell signaling activity using metal-clad waveguide (MCWG)-based microscopy

Label-free biosensing methods are very effective for studying cell signaling cascade activation induced by external stimuli. Assays generally involve a large number of cells and rely on the underlying assumption that cell response is homogeneous within a cell population. However, there is an increasing body of evidence showing that cell behavior may vary significantly even among genetically identical cells. In this paper, we demonstrate the use of metal-clad waveguide (MCWG)-based microscopy for label-free real-time monitoring of signaling activity and morphology changes in a small population of cells, with the ability to resolve individual cells. We demonstrate the potential of this approach by quantifying apoptosis-induced intracellular activity in individual cells following exposure to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and by visualizing and quantifying extracellular changes in endothelial cell layer integrity following the activation of the proteinase-activated receptor 1 (PAR1) by thrombin. Results show that averaged signals obtained from a cell population may incorrectly reflect the actual distribution of morphology and kinetics parameters across a cell population by a significant margin.

ICELLigence real-time cell analysis system for examining the cytotoxicity of drugs to cancer cell lines.

The recently developed iCELLigence™ real-time cell analyzer (RTCA) can be used for the label-free real-time monitoring of cancer cell proliferation, viability, invasion and cytotoxicity. The RTCA system uses 16-well microtiter plates with a gold microelectrode biosensor array that measures impedance when cells adhere to the microelectrodes causing an alternating current. By measuring the electric field generated in this process, the RTCA system can be used for the analysis of cell proliferation, viability, morphology and migration. The present review aimed to summarize the working method of the RTCA system, in addition to discussing the research performed using the system for various applications, including cancer drug discovery via measuring cytotoxicity.

Real-Time Label-Free Monitoring of Nanoparticle Cell Uptake.

The surface plasmon resonance technique in combination with whole cell sensing is used for the first time for real-time label-free monitoring of nanoparticle cell uptake. The uptake kinetics of several types of nanoparticles relevant to drug delivery applications into HeLa cells is determined. The cell uptake of the nanoparticles is confirmed by confocal microscopy. The cell uptake of silica nanoparticles and polyethylenimine-plasmid DNA polyplexes is studied as a function of temperature, and the uptake energies are determined by Arrhenius plots. The phase transition temperature of the HeLa cell membrane is detected when monitoring cell uptake of silica nanoparticles at different temperatures. The HeLa cell uptake of the mesoporous silica nanoparticles is energy-independent at temperatures slightly higher than the phase transition temperature of the HeLa cell membrane, while the uptake of polyethylenimine-DNA polyplexes is energy-dependent and linear as a function of temperature with an activation energy of Ea = 62 ± 7 kJ mol-1 = 15 ± 2 kcal mol-1 . The HeLa cell uptake of red blood cell derived extracellular vesicles is also studied as a function of the extracellular vesicle concentration. The results show a concentration dependent behavior reaching a saturation level of the extracellular vesicle uptake by HeLa cells.

A novel label-free cell-based assay technology using biolayer interferometry

Biolayer interferometry (BLI) is a well-established optical label-free technique to study biomolecular interactions. Here we describe for the first time a cell-based BLI (cBLI) application that allows label-free real-time monitoring of signal transduction in living cells. Human A431 epidermoid carcinoma cells were captured onto collagen-coated biosensors and serum-starved, followed by exposure to agonistic compounds targeting various receptors, while recording the cBLI signal. Stimulation of the epidermal growth factor receptor (EGFR) with EGF, the β2-adrenoceptor with dopamine, or the hepatocyte growth factor receptor (HGFR/c-MET) with an agonistic antibody resulted in distinct cBLI signal patterns. We show that the mechanism underlying the observed changes in cBLI signal is mediated by rearrangement of the actin cytoskeleton, a process referred to as dynamic mass redistribution (DMR). A panel of ligand-binding blocking and non-blocking anti-EGFR antibodies was used to demonstrate that this novel BLI application can be efficiently used as a label-free cellular assay for compound screening and characterization.

A novel 384-multiwell microelectrode array for the impedimetric monitoring of Tau protein induced neurodegenerative processes

Over the last decades, countless bioelectronic monitoring systems were developed for the analysis of cells as well as complex tissues. Most studies addressed the sensitivity and specificity of the bioelectronic detection method in comparison to classical molecular biological assays. In contrast, the up scaling as a prerequisite for the practical application of these novel bioelectronic monitoring systems is mostly only discussed theoretically.In this context, we developed a novel 384-multiwell microelectrode array (MMEA) based measurement system for the sensitive label-free real-time monitoring of neurodegenerative processes by impedance spectroscopy. With respect to the needs of productive screening systems for robust and reproducible measurements on high numbers of plates, we focused on reducing the critical contacting of more than 400 electrodes for a 384-MMEA. Therefore, we introduced an on top array of immersive counter electrodes that are individually addressed by a multiplexer and connected all measurement electrodes on the 384-MMEA to a single contact point. More strikingly, our novel approach provided a comparable signal stability and sensitivity similar to an array with integrated counter electrodes. Next, we optimized a SH-SY5Y cell based tauopathy model by introducing a novel 5-fold Tau mutation eliminating the need of artificial tauopathy induction. In combination with our novel 384-MMEA based measurement system, the concentration and time dependent neuroregenerative effect of the kinase inhibitor SRN-003-556 could be quantitatively monitored. Thus, our novel screening system could be a useful tool to identify and develop potential novel therapeutics in the field of Tau-related neurodegenerative diseases.

A biolayer interferometry-based assay for rapid and highly sensitive detection of biowarfare agents

Biolayer interferometry (BLI) is an optical technique that uses fiber-optic biosensors for label-free real-time monitoring of protein–protein interactions. In this study, we coupled the advantages of the Octet Red BLI system (automation, fluidics-free, and on-line monitoring) with a signal enhancement step and developed a rapid and sensitive immunological-based method for detection of biowarfare agents. As a proof of concept, we chose to demonstrate the efficacy of this novel assay for the detection of agents representing two classes of biothreats, proteinaceous toxins, and bacterial pathogens: ricin, a lethal plant toxin, and the gram-negative bacterium Francisella tularensis, the causative agent of tularemia. The assay setup consisted of biotinylated antibodies immobilized to the biosensor coupled with alkaline phosphatase-labeled antibodies as the detection moiety to create nonsoluble substrate crystals that precipitate on the sensor surface, thereby inducing a significant wavelength interference. It was found that this BLI-based assay enables sensitive detection of these pathogens (detection limits of 10 pg/ml and 1 × 104 pfu/ml ricin and F. tularensis, respectively) within a very short time frame (17 min). Owing to its simplicity, this assay can be easily adapted to detect other analytes in general, and biowarfare agents in particular, in a rapid and sensitive manner.

Label-Free Real-Time Monitoring of Reactions Between Internalin A and Its Antibody by an Oblique-Incidence Reflectivity-Difference Method

Surface protein internalin (InlA) is a major virulence factor of the food-borne pathogen L. monocytogenes. It plays an important role in bacteria crossing the host's barrier by specific interaction with the cell adhesion molecule E-cadherin. Study of this protein will help to find better ways to prevent listeriosis. In this study, a monoclonal antibody against InlA was used to detect InlA. The reaction was label-free and monitored in real time with an oblique-incidence reflectivity-difference (OI-RD) technique. The kinetic constants k on and k off and the equilibrium dissociation constant K d for this reaction were also obtained. These parameters indicate that the antibody is capable of detecting InlA. Additionally, the results also demonstrate the feasibility of using OI-RD for proteomics research and bacteria detection.

Surface Plasmon Resonance Based Label-Free Detection of Salmonella using DNA Self Assembly

Typhoid is re-emerging as a biggest health threat to third world countries. One of the major challenge is false negative diagnosis using existing immunodiagnostic methods due to overlapping symptoms of other infections (like brucellosis, malaria, hepatitis) that mimic this enteric fever (typhoid). Surface plasmon resonance (SPR) based DNA hybridisation biosensor has been fabricated by generating self-assembled monolayer of 5'-thiolated single-stranded DNA (ssDNA) probe onto gold surface. Highly specific DNA probe has been selected from conserved Vi capsular antigen gene of Salmonella enterica serovars typhi. This DNA biosensor has been investigated for label-free real-time monitoring of Salmonella. Interestingly, 0.019 ng mL(-1) (2 fM) is the lowest detected concentration in PBS with association (k a) and dissociation (k d) rate constants to be k a (M(-1) s(-1)) = 6.68 × 10(4) ± 2.3 and k d (s(-1)) = 5.6 × 10(-3) ± 0.01, respectively. This biosensor was successfully demonstrated to distinguish complementary, non-complementary and one-base mismatch sequences and reusability up to 40 hybridisation cycles at room temperature. Successful results were obtained for hybridisation studies of genomic DNA isolated from spiked urine sample. Performance characteristics of this biosensing device suggested further scope to fine-tune such DNA-based assays to be implied for clinical, food and environmental applications.