Leptospirosis is a bacterial disease that infects both humans and animals. This zoonotic disease is more common in tropical countries. In a recent ACS Applied Nano Materials study, researchers demonstrated the fabrication of an immunosensor that can rapidly and accurately detect leptospira in clinical samples.
Study: Au Single-use electrochemical sensor based on nanoparticles for the detection of leptospirosis in clinical samples. Image credit: Jarun Ontakrai/Shutterstock.com
This disposable immunosensor has been developed using paper electrodes (PPE) modified with gold nanoparticles (AuNp) together with carbon nanotubes (CNTs) labeled with monoclonal anti-LipL32 (e-LipL32).
What do we know about leptospirosis?
Among zoonotic diseases, leptospirosis is a leading cause of morbidity and mortality worldwide, with approximately 1.03 million cases each year. This disease is common in populations with limited resources, specifically in low- and middle-income countries. The global impact of leptospirosis is not well understood because it is one of those diseases that has been neglected globally.
Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. It is extremely difficult to differentiate between pathogenic and non-pathogenic spirochete species based on morphological and biochemical characteristics.
Some common factors leading to the worldwide spread of leptospirosis are climate change, globalization, and a wide range of mammalian hosts. Leptospirosis is often misdiagnosed due to various non-specific symptoms. Therefore, a more accurate diagnostic method is needed to aid in early diagnosis, so that appropriate treatment can begin immediately.
Available diagnostic methods for leptospirosis lack accuracy, sensitivity, portability, and repeatability. Mainly for the clinical diagnosis of the disease, macroscopic agglutination test (MAT), conventional polymerase chain reaction (PCR), culture test, enzyme-linked immunosorbent assay (ELISA) IgM, fusion analysis of ‘high resolution (HRM) and real analyses. real time PCR (RT-PCR) are used.
Among these methods, MAT is considered the gold standard for the diagnosis of leptospirosis. However, this method is time-consuming, requires rare instruments, maintenance of several live cultures, skilled personnel and is limited to specific laboratories. For the early detection and management of leptospirosis, it is essential to develop a new sensitive, cost-effective, rapid and easy-to-use detection system.
Nano-based electrochemical immunosensor for the diagnosis of leptospirosis
Recently, a highly sensitive, selective, disposable electrochemical sensor based on gold nanoparticles has been developed to detect leptospirosis in serum samples. For the fabrication of e-LipL32 in AuNp-modified disposable PPE, a sandwich-type electrochemical immunoassay combining LipL32, anti-LipL32, and carbon nanotube-tagged anti-LipL32 (CNT-Ab) was applied.
AuNp significantly increased the electron transfer rate and also aided the immobilization of specific molecules for the fabricated immunosensor. Glutathione (GSH) was used to promote the formation of an elaborately ordered self-assembled monolayer (SAM) on AuNp, via S-Au covalent bonding. GSH SAMs allowed the addition of an appropriate amount of carboxylic acid (functional group) as well as monoclonal antiLipL32 immobilization. The biological activity of the immobilized proteins was preserved by the SAMs.
Better immunorecognition was facilitated by the presence of the CNT tag, which acted as a signal enhancer by increasing the rate of electron transfer. Therefore, the CNT tag improved the selectivity and sensitivity of the eLipL32 immunosensor.
Analytical techniques, such as X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) analysis, were used to validate the successful fabrication of the e-LipL32 sensor. Surface morphological studies confirmed the immobilization of GSH and mAb on PPE-Au.
The concentration of GSH and the time required to form GSH SAMs on the surface of the AuNp-based electrode were optimized. Ten mM GSH was selected as the optimal concentration because it exhibited a maximum redox potential due to the electrostatic interaction of the carboxyl group of GSH with the cationic probe. Since the best oxidation current response was obtained at 10-6 g/mL antibody, this was selected to be the optimal antibody concentration to fabricate the new e-LipL32 sensor.
Importantly, the single-use e-LipL32 sensor showed higher sensitivity to LipL32, with the upper limit of detection estimated at 1 μg/mL and the lower limit of detection found to be 348 fg/ ml. Furthermore, this electrochemical immunosensor showed excellent selectivity, reproducibility, and acceptable long-term stability.
Clinical validation of the immunosensor
For the clinical validation of the e-LipL32 sensor, 20 serum samples from individuals suspected of leptospirosis and other febrile diseases were used. Clinical samples from patients with febrile illness showed a small increase in current, while leptospirosis samples showed a significant intensification of the peak current, nearly threefold. This finding indicated the high specificity of the e-LipL32 immunosensor towards LipL32 protein present in clinical samples. The new AuNp-based immunosensor was able to detect LipL32 in clinical samples within 60 minutes.
To further confirm the detection efficacy of the e-LipL32 sensor, the results of the tested clinical samples were compared with gold standard MAT, IgM spot test and IgM ELISA. In addition, to differentiate the positive diagnosis of leptospirosis from other diseases, a box plot was calculated based on the mean peak currents of all clinical samples. A current greater than 9.8 μA was determined to be the threshold for a positive diagnosis of leptospirosis, which was confirmed by the MAT test.
In conclusion, the e-Lip32 sensor can be used to accurately detect leptospirosis, in its early phase of infection, under both resource-rich and resource-limited conditions.
reference
Kannan, S. et al. (2022) Au Nanoparticle-Based Disposable Electrochemical Sensor for Detection of Leptospirosis in Clinical Samples. Nanomaterials applied ACS. https://doi.org/10.1021/acsanm.2c0197
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