(352b) Facile and Quantitative Detection of Nucleic Acid Markers Via Conformational Change of Gold Nanoparticle Assemblies

Nucleic acid is a promising biomarker for clinical diagnostics. In recent years, the relationship between various types of nucleic acids, such as messenger RNA (mRNA), micro RNA (miRNA), cell free DNA (cfDNA), and diseases has been revealed. In addition, since nucleic acids with short length, such as miRNAs, are contained in various body fluid including saliva and blood, they can be easily collected from the body. These characteristics makes nucleic acids as attractive target for diagnostics.

Polymerase Chain Reaction (PCR) is one of the most widely used nucleic acid detection methods. However, although PCR can quantify target nucleic acids with high sensitivity, it requires complicated temperature operations. As a more facile method, use of surface plasmon coupling of gold nanoparticles (Au NPs) has attracted attentions. Previous studies reported that by modifying AuNP surface with single stranded DNA that is complementary to target nucleic acid markers, the existence of target markers triggers their aggregation by inter-particle crosslinking via double helix formation, resulting in the color change from red to purple by plasmon coupling [1]. This strategy enables simple and rapid detection of nucleic acid markers by just mixing the sample. However, since large scale aggregation of AuNPs induces their sedimentation, absolute quantification of target concentration via optical absorption is difficult. Therefore, the development of a rapid, facile, and quantitative nucleic acid detection method is expected.

In this study, to achieve plasmon coupling between AuNPs without causing sedimentation, we focused on DNA-mediated assembly of gold nanoparticles into superstructures. We previously reported that by using complementary DNA as a linker, single stranded DNA-modified AuNPs can be assembled into core-satellite type superstructure. The assembled superstructure can then be transformed into different conformation via reconstruction of DNA double helix with another specific DNA, called as toehold-mediated strand displacement [2]. By using this assembly technology, in this study, we aimed to design AuNP assemblies so that they show purple color via close inter particle distance without causing sedimentation at the initial state, whereas they dissociate into individual AuNPs in response to specific nucleic acid target via toe hold displacement, resulting in the color shift into red. It is expected that by changing the strategy from ‘dispersion to large aggregation’ to ‘controlled aggregation to dispersion’, facile and quantitative nucleic acid detection can be achieved.

Experimental

AuNPs with sizes of 8, 15, 40, and 90 nm were synthesized by the reduction of tetrachloro gold(III) acid with citric acid, tannic acid and hydroquinone. The surface of the obtained AuNPs were modified with thiol-terminated single stranded DNA. DNA-modified AuNPs were further assembled into core-satellite superstructure by mixing with complementary DNA linkers, which was designed to dissociate in response to target nucleic acid sequence via toehold-mediated strand displacement. Structure and optical property of AuNP assembly before and after the reaction with target nucleic acids were evaluated by TEM and UV-vis spectroscopy, respectively.

Results and Discussion

Core-satellite superstructure was assembled using AuNPs with different size combinations: 15nm core-8nm satellite, 40nm core-15nm satellite, and 90nm core-40nm satellite. Successful assembly was confirmed by TEM observation. Red shift of the absorption wavelength induced by the superstructure formation was observed for all size combination of building block AuNPs. Comparing between different sizes, use of larger AuNPs resulted in longer shift in absorption wavelength, which was consistent with the theory of surface plasmon coupling. Since 90nm core-40nm satellite assemblies possibly cause gravitational sedimentation during the following assay due to their large size, 40nm core-15nm satellite assemblies were used as a representative formulation hereafter.

To test the dissociation of AuNP assembly in response to specific nucleic acid target, we first prepared the model target DNA with different length and complementary position. By the addition of the model target DNA, AuNP assembly was dissociated into individual building block particles, regardless of the types of model target DNA. Furthermore, this conformational change induced distinct blue shift of the absorption wavelength up to 11 nm, suggesting the possibility for colorimetric target nucleic acid quantification. Whereas final structure and consequent absorption wavelength of AuNP assembly was same between model target DNA with different length and complementary position, this difference significantly altered the dissociation kinetics. In the case of short target DNA (36 base), absorbance change at 525 nm, which corresponds to the degree of AuNP dissociation, rapidly occurred within 40 min. The time course also followed the second order reaction kinetics, estimated by the theoretical model for toehold-mediated strand displacement of DNA. On the other hand, if the model DNA became longer or the complementary position in the model DNA became far from the terminal end, the dissociation reaction kinetics became slower and apart from the theoretical line. This delay would be attributed to the steric hinderance of model DNA when approaching to the linker DNA strand surrounded by many AuNP satellites. From these results, we selected miRNA, which has relatively short length (ca. 22 base) compared with other nucleic acid marker, as a representative marker for the following quantification assay.

To investigate if the constructed AuNP assembly can be used for the quantification of actual nucleic acid biomarker for diagnostics, cancer marker miR-21 was selected as a target. miR-21-responsive AuNP assemblies were designed similarly with the above experiments, and then exposed to miR-21 with various concentrations. As a result, dissociation of AuNPs occurred in dose-dependent manner, faster with increasing miR-21 concentration. Reaction rate at the initial 10 min showed linear relationship with miR-21 concentration, demonstrating that our AuNP assembly system can achieve colorimetric, absolute quantification of cancer miRNA marker by just mixing without causing aggregation-induced sedimentation.

Conclusions

We developed AuNP assembly-based assay system for facile and quantitative detection of nucleic acid biomarkers. AuNP assembly dissociated into individual particles in response to target nucleic acids, leading to the shit in absorption wavelength via change in surface plasmon coupling. The kinetics of AuNP dissociation was dependent on the length and complementary position of target nucleic acid, due to steric hinderance. The absolute quantification of cancer biomarker using the developed assay was demonstrated using miR-21 as a target.

Acknowledgement

This work was supported by JST PRESTO.

References

1. A. Reynolds, C. A. Mirkin, and R. L. Letsinger: J. Am. Chem. Soc., 2000, 122, 3795.
2. S. Ohta, D. Glancy, and W. C. W. Chan: Science, 2016, 351, 841.