A recent article published in Scientific Reports presents a novel biosensor based on a gold nanoparticle (AuNP) conjugated single-legged DNA walker, designed to detect Klebsiella pneumoniae with high sensitivity and specificity. Using a catalytic hairpin assembly (CHA) mechanism, the biosensor offers a reliable and rapid method for early detection of this pathogen, which is crucial for effective treatment and management of infections.
Study: A gold nanoparticle conjugated single-legged DNA walker driven by catalytic hairpin assembly biosensor to detect a prokaryotic pathogen. Image Credit: Athiyada's/Shutterstock.com
Background
The rise of antibiotic-resistant bacteria poses a significant threat to global public health, highlighting the need for rapid and accurate diagnostic tools. Klebsiella pneumoniae, a common pathogen associated with various infections, is of particular concern due to its increasing antibiotic resistance, making timely detection critical for effective management. Traditional detection methods, such as culture-based techniques, can be slow and may delay treatment.
Advancements in nanotechnology and molecular biology have paved the way for biosensors capable of detecting pathogens swiftly and accurately. AuNPs are especially appealing for biosensing applications due to their unique optical properties, which can be exploited for colorimetric detection. By utilizing the CHA mechanism, which enhances the specificity and sensitivity of nucleic acid detection, this biosensor represents a significant leap forward in diagnosing K. pneumoniae.
The Current Study
In this study, researchers developed a AuNP conjugated DNA walker biosensor using a catalytic hairpin assembly (CHA) mechanism to detect Klebsiella pneumoniae. Specific oligonucleotides were designed to initiate the CHA process, targeting sequences from the K. pneumoniae genome. AuNPs were synthesized and characterized using transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), and dynamic light scattering (DLS) to confirm nanoparticle size and stability.
The conjugation of thiolated oligonucleotides to AuNPs was achieved through salt induction, which improved the stability of the nanoprobe solution. The CHA reaction was carried out at room temperature, where target DNA was mixed with hairpin oligonucleotides. The progress of the reaction was monitored using UV-Vis spectroscopy, specifically measuring absorbance changes at 520 nm, a key indicator of successful hybridization.
The biosensor’s specificity was tested using multiple bacterial strains, while sensitivity was evaluated using serial dilutions of the target DNA. Gel electrophoresis was used to confirm the formation of intermediate secondary structures, validating the effectiveness of the DNA walker system in detecting the pathogen.
Results and Discussion
The study demonstrated that the AuNP-conjugated DNA walker biosensor is highly effective for detecting Klebsiella pneumoniae. Upon the introduction of target DNA, a visible color change occurred, which was quantitatively assessed through UV-Vis spectroscopy. In positive samples, absorbance at 520 nm dropped significantly, from 0.08 to 0.058, while negative samples showed a smaller change, from 0.098 to 0.05. This colorimetric shift confirmed successful hybridization and the formation of the DNA walker structure, validating the sensor's functionality.
TEM imaging provided additional insights, revealing an increase in the distance between AuNPs in positive samples compared to negative controls. This supported the hypothesis that the CHA mechanism effectively facilitated the assembly of the DNA walker, resulting in the spatial rearrangement of nanoparticles. Zeta potential analysis further corroborated these findings, showing a higher charge density around the nanoparticles in the presence of target DNA, which is indicative of successful conjugation and hybridization.
The biosensor displayed excellent sensitivity, capable of detecting DNA concentrations as low as 2.5 nM, making it suitable for early diagnosis in clinical settings. Specificity tests using five different bacterial strains confirmed the biosensor’s accuracy in detecting K. pneumoniae without cross-reactivity, underscoring its potential for practical applications in pathogen detection.
Conclusion
In conclusion, this study successfully developed a gold nanoparticle-based DNA walker biosensor for the rapid and sensitive detection of Klebsiella pneumoniae. Leveraging the catalytic hairpin assembly (CHA) mechanism, the biosensor exhibited exceptional specificity and sensitivity, making it a promising tool for clinical diagnostics. The research highlights the potential of integrating nanotechnology and molecular biology to tackle challenges posed by antibiotic-resistant bacteria.
Future research could focus on optimizing the biosensor for point-of-care applications and expanding its use to detect other pathogens, ultimately contributing to improved public health outcomes. The innovative approach presented in this study represents a significant advancement in biosensing, with the potential to enhance the speed and accuracy of infectious disease diagnosis.
Journal Reference
Shahbazi E., Mollasalehi H. et al. (2024). A gold nanoparticle conjugated single-legged DNA walker driven by catalytic hairpin assembly biosensor to detect a prokaryotic pathogen. Scientific Reports 14, 22980. DOI: 10.1038/s41598-024-74227-5, https://www.nature.com/articles/s41598-024-74227-5