DNA/RNA-conjugated nanozymes represent a cutting-edge convergence of nanotechnology, enzymology, and molecular biology, offering powerful tools for biosensing applications. Among these, peroxidase-mimic cerium oxide (CeO2) nanoparticles stand out due to their robust catalytic activity, stability, and biocompatibility. When functionalized with DNA or RNA probes, these nanozymes enable highly sensitive and sequence-specific detection of nucleic acid targets, making them ideal for point-of-care diagnostics, including HIV viral load testing. The integration of catalytic signal amplification with target recognition allows for visual readouts, simplifying diagnostics in resource-limited settings.

Peroxidase-mimic CeO2 nanoparticles catalyze the oxidation of chromogenic substrates, such as 3,3',5,5'-tetramethylbenzidine (TMB), in the presence of hydrogen peroxide (H2O2), producing a color change detectable by the naked eye. This catalytic activity mimics natural peroxidases but with greater stability under harsh conditions. By conjugating these nanoparticles with single-stranded DNA or RNA probes, the system gains the ability to recognize specific nucleic acid sequences. Hybridization between the probe and target DNA/RNA triggers changes in the nanozyme's activity or accessibility to substrates, modulating the colorimetric signal. This dual functionality—target recognition and signal amplification—forms the basis of highly sensitive biosensors.

The mechanism of signal amplification in DNA/RNA-conjugated CeO2 nanozymes hinges on the catalytic turnover of substrates. Each nanoparticle can catalyze multiple reactions, generating an amplified signal even at low target concentrations. For example, in HIV viral load testing, a DNA probe complementary to a conserved region of the HIV genome is attached to the CeO2 surface. In the presence of viral RNA, hybridization occurs, altering the nanozyme's catalytic efficiency. The degree of signal change correlates with the target concentration, enabling quantitative assessment. The visual readout, often a blue-to-yellow transition with TMB, allows for immediate interpretation without sophisticated instrumentation.

One critical challenge in colorimetric biosensing is interference from endogenous peroxidases present in biological samples, such as blood or saliva. These enzymes can catalyze the same reactions as nanozymes, leading to false-positive signals. To mitigate this, selective inhibitors like sodium azide or heat treatment can be employed to deactivate natural peroxidases without affecting CeO2 nanozymes. Additionally, careful optimization of pH and temperature conditions can enhance the selectivity of nanozyme activity. For instance, CeO2 nanoparticles exhibit optimal peroxidase-like activity at acidic pH, whereas many natural peroxidases function at neutral pH, allowing for selective signal generation under controlled conditions.

Point-of-care HIV viral load testing benefits significantly from this technology. Traditional methods, such as reverse transcription-polymerase chain reaction (RT-PCR), require expensive equipment and skilled personnel, limiting accessibility in low-resource regions. In contrast, DNA-conjugated CeO2 nanozyme biosensors offer a low-cost, rapid, and equipment-free alternative. A typical assay involves mixing the sample with probe-functionalized nanoparticles, adding H2O2 and TMB, and observing the color change. The intensity of the color correlates with viral RNA concentration, providing semiquantitative results within minutes. This simplicity is particularly advantageous in decentralized healthcare settings.

The specificity of DNA/RNA-conjugated nanozymes is another key advantage. By designing probes targeting unique HIV sequences, cross-reactivity with other pathogens or human RNA is minimized. Mismatched sequences do not hybridize effectively, leaving the nanozyme's catalytic activity unaltered. This high specificity ensures accurate diagnosis, even in complex biological matrices. Furthermore, the modular design allows for adaptation to other targets by simply changing the probe sequence, demonstrating versatility beyond HIV testing.

Despite these advantages, several factors influence the performance of nanozyme-based biosensors. Nanoparticle size, surface charge, and probe density affect hybridization efficiency and catalytic activity. Smaller CeO2 nanoparticles generally exhibit higher surface-to-volume ratios, enhancing probe loading and signal generation. However, excessive probe density can sterically hinder catalysis, necessitating optimization. Similarly, the length and secondary structure of DNA/RNA probes impact binding kinetics. Shorter probes may offer faster hybridization but reduced specificity, while longer probes enhance specificity at the cost of slower kinetics.

Stability is another critical consideration. CeO2 nanoparticles are inherently stable under a wide range of temperatures and pH levels, making them suitable for storage and transport in challenging environments. However, the conjugated DNA/RNA probes may degrade over time, especially in humid or nuclease-rich conditions. Encapsulation in protective matrices or chemical modification of nucleic acids can enhance probe stability, ensuring long-term functionality of the biosensor.

Future developments in DNA/RNA-conjugated nanozymes may focus on multiplexing capabilities, enabling simultaneous detection of multiple HIV strains or co-infections. By functionalizing different nanoparticles with distinct probes and using chromogenic substrates with unique color outputs, a single assay could provide comprehensive diagnostic information. Additionally, integrating smartphone-based colorimetric analysis could enhance quantitative accuracy while retaining point-of-care simplicity.

In summary, DNA/RNA-conjugated CeO2 nanozymes represent a transformative approach to colorimetric biosensing, combining catalytic signal amplification with sequence-specific recognition. Their application in HIV viral load testing demonstrates the potential to democratize diagnostics, offering rapid, visual, and equipment-free solutions. Addressing challenges like endogenous peroxidase interference through selective inhibition ensures reliable performance in real-world settings. As research advances, these systems may expand to broader applications, further bridging the gap between laboratory-based diagnostics and global healthcare needs.