Highly‐Specific Single‐Stranded Oligonucleotides and Functional Nanoprobes for Clinical Determination of Chlamydia Trachomatis and Neisseria Gonorrhoeae Infections

Introduction: Sexually transmitted infections (STIs) caused by Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are a significant public health concern due to their high prevalence and potential to cause serious health complications. Early and accurate diagnosis is crucial for effective treatment and control of these infections. Traditional diagnostic methods, such as nucleic acid amplification tests (NAATs), are reliable but often require sophisticated laboratory infrastructure, trained personnel, and can be time-consuming. These limitations can delay treatment, contributing to the spread of infections and the development of complications. Rapid point-of-care (POC) diagnostics offer a promising alternative by providing timely and accurate results that can guide immediate clinical decisions. This study introduces a novel diagnostic approach using highly specific single-stranded oligonucleotides (ssDNAs) functionalized with gold nanoparticles (AuNPs). These ssDNAs target conserved genetic regions of CT and NG, ensuring high specificity and sensitivity. The AuNP-ssDNA conjugates undergo aggregation in the presence of target DNA, resulting in a measurable change in absorbance that can be detected using simple spectroscopic techniques. This aggregation-based assay is further adapted to a lateral flow platform, enabling rapid POC testing without the need for DNA extraction and amplification. The simplicity, speed, and accuracy of these assays make them suitable for use in various clinical settings, including emergency departments and primary care clinics, where rapid decision-making is critical. The study highlights the potential of these innovative diagnostic tools to improve STI management and control, reduce inappropriate antibiotic use, and curb the spread of antibiotic-resistant strains of CT and NG.

Materials and

Methods: In this study, we designed a series of new oligonucleotide (ssDNA) probes targeting different genetic segments of CT and NG. Gold nanoparticles (AuNPs) were then synthesized according to previous work. The ssDNA probes were then conjugated on to the surface of gold nanoparticles (AuNPs) and their target binding efficacies were investigated. The ssDNA probes facilitate the aggregation of gold nanoparticles when they are attached to the nanoparticle surface and exposed to the specific target gene sequence. The principle governing the sensing mechanism of the absorbance-based assay lies in the complementary binding between the novel oligonucleotide probes (ssDNA) and their targeted gene sequences unique to CT or NG. The confirmation of the sensing phenomena was achieved by the utilization of various bioanalytical techniques, including UV-Visible spectroscopy, transmission electron microscopy (TEM), Raman spectroscopy, and hyperspectral microscopy.

Results, Conclusions, and Discussions: The study developed highly specific single-stranded oligonucleotides (ssDNAs) to target conserved genetic regions of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). These ssDNAs were functionalized with thiol groups at their 5' and 3' ends and conjugated with gold nanoparticles (AuNPs) to create an innovative absorbance-based assay. This assay facilitated the selective aggregation of AuNPs in the presence of target DNA from CT and NG, resulting in measurable changes in absorbance. The ssDNA-tagged AuNPs demonstrated high sensitivity and specificity, detecting CT and NG with limits of detection (LOD) of 7 and 5 copies/μL, respectively. Validation with 60 anonymized clinical samples achieved a clinical sensitivity of 100% for both pathogens. Additionally, the assay was adapted to a lateral flow platform, enabling rapid point-of-care (POC) detection without the need for DNA extraction. This approach provided significant advantages over conventional diagnostic methods, delivering rapid and accurate results in under 10 minutes, making it suitable for clinical settings. The study concluded that the developed ssDNA-based assays have significant potential for the rapid and accurate diagnosis of CT and NG infections. The assays combine the high accuracy, sensitivity, and specificity of nucleic acid-based tests with the convenience and speed of POC diagnostics. This novel diagnostic tool could enhance infection control measures and appropriate antibiotic use, addressing the challenges of overtreatment, undertreatment, and the emergence of antibiotic-resistant strains. Overall, the study presents a robust framework for developing sensitive and specific diagnostic assays for sexually transmitted infections, emphasizing the importance of targeting conserved genetic regions and utilizing innovative nanotechnology-based approaches.

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