Oligonucleotide-Conjugated Gold Nanoparticles for Application on Lateral Flow Biosensors: Evaluation and Optimization of Low pH and Salt-Aging Conjugation Methods

Paper-based lateral flow biosensors (LFBs) have been shown to be optimal for rapid analysis, since they exhibit user-friendly format, low analysis times, and no need for specialized instrumentation or highly trained personnel. Low detection limits are desirable for a variety of diagnostic applications, indicating the need for optimal detection labels primarily based on gold nanoparticles. Oligonucleotide-functionalized gold nanoparticles have attained great interest as LFB detection components and study of efficient conjugation methods is ongoing. However, a comparative investigation of oligonucleotide–gold nanoparticle conjugates for application as LFBs has not been performed. Therefore, the goal of the present study was the evaluation of two oligonucleotide–gold nanoparticles conjugation methods, and optimization of the most promising reaction. An LFB for nucleic acids detection was utilized for the evaluation study. Two conjugation protocols were tested: the pH 3 citrate method and the salt-aging method. The prepared conjugates were evaluated in terms of particle color and signal intensity, with a reference oligonucleotide that was used as a model system. Subsequently, the salt-aging protocol, which resulted in better oligonucleotide–gold nanoparticle conjugates, was optimized regarding several reaction parameters. Finally, the conjugate detection sensitivity and reproducibility were assessed.