Nucleic Acid Functionalized Gold Nanoparticle Probes

Nucleic Acid Functionalized Gold Nanoparticle Probes

Nucleic acids are one of the primary biomacromolecules within cells, capable of specific binding between nucleotide chains based on the Watson-Crick base-pairing principle, thus transmitting genetic information in organisms. With the development of molecular biology and biochemistry, various artificially synthesized functional nucleic acid molecules, such as deoxyribozymes (DNAzymes), aptamers, and aptazymes, have been obtained through in vitro screening techniques. Unlike endogenous nucleic acids, these functional nucleic acid molecules can interact or bind with specific analytes, leading to conformational changes or catalyzing certain reactions. Additionally, the mature synthesis technology and diverse structures of functional nucleic acids, along with their ease of modification, demonstrate significant potential in biosensing.

Due to the highly challenging process of free nucleic acid molecules entering cells and their susceptibility to enzymatic degradation within cells, they cannot be directly used for in situ detection of intracellular analytes. To address this issue, nucleic acid molecules are combined with nanocarriers to construct composite nanoprobes. Among them, gold nanoparticles have simple synthesis, strong stability, easy modification, and excellent biocompatibility, making them widely used in sensing, diagnostics, therapeutics, and various other fields. Fluorescently labeled nucleic acid functionalized gold nanoparticle probes combine the advantages of multifunctional composite nanomaterials and optical imaging techniques. The synergistic interaction between nucleic acids and gold nanoparticles exhibits many enhanced properties, such as good cell transfection ability, excellent intracellular stability, unique targeting specificity, and remarkable signal amplification capability.

Properties of Gold Nanomaterials

Gold nanomaterials refer to gold particles with at least one-dimensional size smaller than 100 nm, including gold nanoparticles (AuNPs), gold nanorods (AuNRs), gold nanostars (AuNSs), gold nanocages (AuNCs), and various other structures. When exposed to light of specific wavelengths, the surface conduction electrons immediately respond to the electromagnetic field, producing electron cloud oscillations at the same frequency as the incident light, known as localized surface plasmon resonance (LSPR). This process involves scattering and absorption, collectively leading to the attenuation (extinction) of incident light intensity. Additionally, the strong electric field generated on the surface of gold nanostructures significantly enhances or quenches the fluorescence or Raman signals of surface or nearby analytes.

The LSPR properties of gold nanostructures (such as the ratio of scattering to absorption, peak position, etc.) are determined by many parameters, including size, shape, structure, morphology, and the environment surrounding the nanostructure. Generally, when the dielectric constant of the surrounding medium changes, the LSPR peak of gold nanostructures shifts. However, changes in size have minimal effects on the LSPR peak position of gold nanostructures. In biomedicine, there is particular interest in gold nanostructures with LSPR peaks distributed in the near-infrared (NIR) region because water, blood, and soft tissues in the body absorb and scatter less light in this wavelength range, allowing incident light to penetrate easily into soft tissues.

Assembly of Nucleic Acids on Gold Nanoparticles

Currently, there are mainly two methods for attaching nucleic acid chains, such as DNA, to the surface of gold nanoparticles: (1) DNA chains containing thiol or disulfide bonds self-assemble with the gold nanostructure surface through strong Au-S bonds (184 kJ/mol); (2) DNA sequences with multiple repeated A bases at the end serve as anchoring segments and can be strongly adsorbed to the surface of gold nanostructures. Among them, self-assembly based on Au-S bonds is the most commonly used approach. Synthesized gold nanomaterials often carry a large number of protective groups (such as citrate ions), which may cause electrostatic repulsion with the phosphate groups of DNA, preventing dense stacking of DNA chains on the surface of gold nanostructures. To address this issue, the salt aging method is employed, gradually increasing the salt concentration in the reaction system to balance the charge, ensuring that DNA chains can approach the nanostructure surface. Studies have shown that the amount of DNA chains connected to the surface of gold nanomaterials is directly proportional to the final salt concentration of the system until steric hindrance prevents further adsorption. Conversely, dense nucleic acid loading can prevent gold nanomaterials from aggregating at higher salt concentrations, thereby ensuring the stability of the probe solution. Additionally, highly oriented nucleic acid monolayers enable probes to enter cells without the need for transfection reagents, laying the foundation for probe applications in live cells.

Research has shown that the bases of nucleic acids can adsorb to the surface of gold nanomaterials, with the affinity of different bases ranging from strong to weak in the order of A>C≥G>T. Based on the adsorption of A bases to gold nanoparticles, nucleic acid functionalized gold nanoparticle probes for sensing target DNA sequences are constructed using nucleic acid sequences containing different numbers of A bases at the ends. Furthermore, gold nanoparticle modified with peptides through Au-Se bonds can be used for in situ imaging inside cells. The Au-Se bonds in such probes have stronger thermal stability than Au-S bonds and can resist interference from millimolar levels of glutathione (GSH). This approach is expected to become one of the main assembly methods for nucleic acid functionalized gold nanoparticle probes.

Construction of Nucleic Acid Functionalized Gold Nanoparticle Probes

Nucleic acid functionalized gold nanoparticle probes mainly consist of two parts: the gold nanoparticle carrier and the nucleic acid chains, such as DNA and RNA, surface-modified with them. Typically, these nucleic acid sequences must possess both target recognition and signal transduction functions. When used for fluorescence imaging, the nucleic acid sequences in such probes also need to be labeled with fluorescent groups or quantum dots, etc., as luminescent materials. Changes in the structure of nucleic acids result in variations in the distance between luminescent materials and gold nanomaterials or organic quenching groups, altering the efficiency of resonant energy transfer and thereby exhibiting differences in fluorescent signals.

Classic Nucleic Acid Functionalized Gold Nanoparticle Probes

Fluorescently labeled nucleic acid functionalized gold nanoparticle probes are easy to prepare, respond rapidly, and exhibit good specificity and biocompatibility, making them widely used for detecting and analyzing various substances inside cells, including RNA, telomerase, small molecules, specific ions, etc. The concept of molecular beacons (MBs) was first proposed by Tyagi and Kramer in 1996. Traditional molecular beacons are oligonucleotide chains with a stem-loop structure, each end labeled with a fluorescent group and a quenching group, respectively, possessing unique signal transduction and target recognition capabilities. However, traditional molecular beacons are sensitive to nucleases and background fluorescence cannot be completely quenched, severely limiting their applications in biological systems. Gold nanomaterials, acting as carriers and quenchers, can effectively address these issues. Researchers have modified fluorescently labeled MBs onto AuNP surfaces to construct a type of nanobeacon, used for detecting mRNA inside live cells. When the MB maintains a hairpin conformation, the fluorescently labeled end is close to the AuNP surface, leading to fluorescence quenching. Once the target mRNA hybridizes with the MB, the hairpin opens, and the fluorescent group can move away from the AuNP surface, restoring the fluorescence signal.

Signal Amplification Nucleic Acid Functionalized Gold Nanoparticle Probes

The sensitivity of traditional nucleic acid functionalized nanoparticle probes is limited within a certain range. For some analytes that are crucial in cellular metabolism but have extremely low concentrations, these probes cannot function effectively. Therefore, improved probes based on nucleic acid amplification technology have been developed. These probes can undergo 1:n signal conversion, increasing sensitivity by several orders of magnitude compared to traditional probes. Aptamers and DNAzymes have been hybridized to produce a new class of nucleic acid enzymes, called aptazymes. In these aptazymes, the target recognition event in the aptamer region can be converted into DNAzyme activation, thereby generating detectable optical signals. In 2016, the first AuNP/aptazyme nanocomplex used for sensing ATP inside live cells was reported. The specific binding of aptazymes with ATP promotes the formation of the DNAzyme's active secondary structure. The activated aptazymes catalyze the cleavage of substrate chains in the presence of Mg2+, leading to the release of fluorescently labeled substrate chain fragments from the aptazyme chain and moving away from the AuNP surface. The released active aptazyme chains can further catalyze the cleavage of other substrate chains, achieving signal amplification. Experimental results have shown that aptazyme-functionalized gold nanoparticle probes have higher sensitivity, with detection limits 2-3 orders of magnitude higher than traditional ATP sensors based on aptamers.

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