In a recent article published in the journal Scientific Reports, researchers presented a novel biosensor based on a gold nanoparticle (AuNP) conjugated single-legged DNA walker, designed to detect K. pneumoniae with high sensitivity and specificity. By employing a catalytic hairpin assembly (CHA) mechanism, the biosensor aims to provide a reliable method for the 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 emergence of antibiotic-resistant bacteria poses a significant threat to public health, necessitating the development of rapid and accurate diagnostic tools. Klebsiella pneumoniae, a common pathogen associated with various infections, has garnered attention due to its increasing resistance to antibiotics. K. pneumoniae can cause severe infections, particularly in immunocompromised individuals. Traditional methods for detecting K. pneumoniae often involve culture-based techniques, which can be time-consuming and may not provide timely results.
Recent advancements in nanotechnology and molecular biology have paved the way for the development of biosensors that can detect pathogens rapidly and accurately. Gold nanoparticles are particularly attractive for biosensing applications due to their unique optical properties, which can be exploited for colorimetric detection. The CHA mechanism enhances the specificity and sensitivity of nucleic acid detection by facilitating the hybridization of DNA strands, making it an ideal approach for developing a biosensor targeting K. pneumoniae.
The Current Study
The study developed a gold nanoparticle (AuNP) conjugated DNA walker biosensor utilizing a catalytic hairpin assembly (CHA) mechanism for the detection of Klebsiella pneumoniae. Specific oligonucleotides were designed to facilitate the CHA process, with the target sequence selected 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 their size and stability.
The conjugation of thiolated oligonucleotides to AuNPs was achieved through salt induction, enhancing the stability of the nanoprobe solution. The CHA reaction was conducted at room temperature, where the target DNA was mixed with the hairpin oligonucleotides in a controlled environment. The reaction's progress was monitored through UV-Vis spectroscopy, focusing on absorbance changes at 520 nm, indicative of successful hybridization.
The specificity of the biosensor was assessed using various bacterial strains, while sensitivity was determined through serial dilutions of the target DNA. The formation of intermediate secondary structures was confirmed via gel electrophoresis, validating the effectiveness of the CHA-DNA walker system in detecting the pathogen.
Results and Discussion
The results of this study indicate that the AuNP conjugated DNA walker biosensor is a highly effective tool for the detection of Klebsiella pneumoniae. Upon introducing the target DNA, a distinct color change was observed, which was quantitatively assessed through UV-Vis spectroscopy. The absorbance at 520 nm decreased significantly, shifting from 0.08 to 0.058 for positive samples, while negative samples showed a lesser change from 0.098 to 0.05. This colorimetric shift is indicative of successful hybridization and the formation of the DNA walker structure, confirming the operational efficacy of the biosensor.
Transmission electron microscopy (TEM) imaging provided further insights, revealing that the distance between AuNPs increased in positive samples compared to negative controls. This observation supports the hypothesis that the CHA mechanism effectively facilitated the assembly of the DNA walker, leading to a spatial rearrangement of the nanoparticles. Zeta potential analysis corroborated these findings, demonstrating a higher charge density around the nanoparticles in the presence of the target DNA, which is indicative of successful conjugation and hybridization.
The biosensor exhibited remarkable sensitivity, capable of detecting target DNA concentrations as low as 2.5 nM, which is crucial for early diagnosis in clinical settings. Specificity tests against five different bacterial strains confirmed that the biosensor accurately identified 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. By leveraging the CHA mechanism, the biosensor demonstrated remarkable specificity and sensitivity, making it a promising tool for clinical diagnostics. The findings underscore the importance of integrating nanotechnology and molecular biology to address the 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 the field of 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