Point of care nucleic acid test using Single Stranded DNAs paper microfluidics for differential detection of Mpox Virus Clades under low resource setting : Development of a ssDNAs-based molecular assay for the simultaneous detection and differentiation of Mpox virus Clade I and II Satheesh Natarajan, Ketan Dighe, David Skrodzki, Dipanjan Pan 2025 IEEE Biosensors Conference Biosensors 2025 Conferece Proceedings, 2025 Since 2022, the Mpox outbreak has resulted in multiple fatalities and has spread to 107 countries across all six World Health Organization (WHO) regions. In response, the WHO declared it a Public Health Emergency of International Concern, emphasizing the urgent need for rapid and precise detection of different Mpox virus (Mpox) clades. Early diagnosis, particularly through point-of-care molecular testing (POCT), is crucial for controlling its transmission. Here, we present the development of a colorimetric lateral flow assay detectable by the naked eye, utilizing gold nanoparticles (AuNPs) capped with single-stranded DNAs (ssDNAs) to specifically identify and differentiate Mpox Clades I and II. The ssDNAs, functionalized with thiol groups and conjugated to AuNPs, selectively agglomerate in the presence of target Mpox DNA, inducing a shift in surface plasmon resonance. This optimized assay achieves a detection limit of 100 copies/μl across all clades and exhibits high specificity for Mpox, with no cross-reactivity to other pathogens. These assays hold great potential for clinical applications, enabling rapid and reliable viral detection.
Fabrication of Immunobiosensors by 2D Borophene-Induced Covalent and Oriented Immobilization of Antibody for Ultrasensitive Antigen Detection : 2D Borophene to achieve oriented immobilization of an antibody for better antigen-binding in Menstrual effluent Satheesh Natarajan, Ketan Dighe, Teresa Aditya, David Skrodzki, Purva Gupta, Pranay Saha, Nivetha Gunaseelan, Dipanjan Pan 2025 IEEE Biosensors Conference Biosensors 2025 Conferece Proceedings, 2025 Lateral flow immunoassays (LFIAs) are essential tools for the swift detection of clinically significant biomarkers, playing a vital role in point-of-care diagnostics. However, their effectiveness is often compromised due to the improper orientation of immobilized antibodies on nitrocellulose membranes, leading to diminished colorimetric signals. Enhancing the performance of LFIAs hinges on achieving precise antibody orientation, as correctly aligned antibodies exhibit superior antigen-binding capabilities compared to their randomly oriented counterparts. To address this issue, we employed borophene nanosheets—a two-dimensional nanomaterial—and functionalized them with antibodies using a photoinduced immobilization technique (PIT) to ensure optimal orientation. This method involves utilizing UV light to selectively break antibody disulfide bonds, revealing thiol groups that form stable covalent boron-sulfur bonds with the borophene surface. The resultant densely packed antibody layer maintains antigen-binding activity and enhances target interaction. We tested the effectiveness of this approach by applying antibody-coated borophene nanosheets onto a nitrocellulose membrane, creating a portable lateral flow assay aimed at detecting HMGB-1—a potential biomarker for endometriosis—in menstrual effluent. The assay delivered results in under 10 minutes and achieved a detection threshold of 40 pg/mL, markedly improving upon the sensitivity of traditional LFIAs. Specificity tests confirmed no cross-reactivity with common blood proteins such as albumin, fibrinogen, and gamma globulin, underscoring the assay's reliability and clinical relevance. This testing platform can also be adapted to identify other analytes, indicating its potential applicability across various women's health-related conditions that currently lack adequate diagnostic tools.
Ultrasensitive aptasensors for the evaluation of food safety utilising the lateral flow assay Satheesh Natarajan, Maria C. DeRosa Aptasensors for Food Safety Fundamentals and Applications, 2024 Antibodies are commonly employed in lateral flow assays, yet they come with various drawbacks such as production challenges and inconsistent batch quality. To overcome these limitations, researchers turn to small single-stranded nucleic acids known as DNA or RNA aptamers. These aptamers are an appealing alternative due to their simplicity, cost-effective large-scale synthesis, robustness at high temperatures, and strong binding affinity and specificity for diverse analytes. Unlike antibodies, aptamers also avoid triggering immune responses. The process for discovering these aptamers is called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). It is an iterative combinatorial screening method conducted in vitro, leading to the identification of nucleic acid sequences that can selectively and tightly bind to specific molecular targets within a given matrix. Since their inception in the early 1990s, aptamers have played a crucial role in detecting analytes relevant to health and ensuring food safety. A particular focus of this chapter is aptamer-based biosensors, which offer an effective means of assessing food safety. These biosensors operate on lateral flow assay principles and have shown promise in detecting microbial proteins and small molecules in food samples. Despite their potential, there are challenges that must be addressed to transition these innovative technologies from the research laboratory to the commercial market. These hurdles are discussed within the chapter, shedding light on the journey from development to practical application.
Enhancing the Sensitivity of Lateral Flow Assay with Europium Nanoparticles for Accurate Human IgG Quantification Satheesh Natarajan, Aashish Priye Micromachines, 2023 Accurate quantification of immunoglobulin G (IgG) levels is vital for understanding immune status and diagnosing various medical conditions. Lateral flow assays (LFAs) offer rapid and convenient diagnostic tools, but their sensitivity has been a limitation. Our research introduces a refined method incorporating europium nanoparticles, enhancing both sensitivity and accuracy of LFAs in human IgG measurement. Utilizing a unique sandwich format, carboxylate-modified polystyrene Eu (III) chelate microparticles (CM-EUs) acted as the primary reporters. The concentrations of both detection and capture antibodies on the strip were optimized to bolster the LFA’s quantitative performance. The subsequent calibration curve between the IgG concentration and the measured intensity ratio (VR) established the linearity and analytical sensitivity of our method with a high correlation coefficient (r = 0.99) and an impressively low limit of detection (LoD = 0.04 ng/mL). Our precision assessment, segmented into intra-assay and inter-assay evaluations, showcases the method’s consistency and reproducibility. The LFA assay’s stability was established by demonstrating its resistance to degradation and affirming its potential for extended storage without a dip in performance. The study’s findings underscore the potential of this method to contribute to diagnostic medicine and improve patient care.
Graphene oxide coatings enhance fluorescence signals in a lateral flow immunoassay for the detection of UCH-L1, a marker for trauma brain injury Satheesh Natarajan, Jayaraj Joseph, Duarte Miguel França Prazeres Sensors and Actuators B Chemical, 2023 We present a lateral flow immunoassay (LFA) for the quantitative, fluorescence-based detection of the trauma brain injury (TBI) biomarker ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1) that features a layer of graphene oxide (GO) particles over the test zone of nitrocellulose (NC) strips. The introduction of 80 ng of GO at test lines increased fluorescence signals 2–3-fold. This was attributed to an increase in the immobilization of capture antibodies in the top layer of NC, which increased the density of recognition complexes at the surface and hence the signals reaching the reader used. A linear concentration–response relationship was observed in the 0–200 pg/mL UCH-L1 range with an inter assay CoV of 2.65 %. The LOD and LOQ values of 11.1 pg/mL and 33.5 pg/mL were compatible with threshold levels of UCH-L1. Finally, mock plasma samples with UCH-L1 levels characteristic of TBI patients with negative (60 pg/mL) and positive (130 pg/mL) CT scans were successfully analyzed. Storage stability testing further showed that GO did not affect the biological activity of the capture antibody. In summary, we demonstrate that the use of GO particles in the top layers of the test zone of NC strips improves signal intensity, which could potentially enhance the accuracy and efficiency of LFA-based diagnosis.
