(Nanowork Spotlight) Detection of genetic mutations in circulating tumor DNA (ctDNA) has long been an important challenge in cancer diagnosis and personalized medicine. Traditional methods such as polymerase chain reaction (PCR) and next-generation sequencing have limitations in sensitivity and accuracy, especially when dealing with trace amounts of genetic material or distinguishing single base mutations. These limitations have hindered early cancer detection and the development of targeted treatments. The COVID-19 pandemic further complicates the problem by disrupting routine cancer screening, which could delay diagnosis and lead to worse patient outcomes.
In recent years, researchers have explored a variety of techniques to improve gene detection capabilities. Surface plasmon resonance (SPR) biosensors have emerged as a promising approach due to their label-free, real-time, and high-throughput properties. However, SPR biosensors have struggled to distinguish between similar nucleic acid sequences and detect low abundance targets in complex biological samples. At the same time, advances in DNA nanotechnology, especially DNA origami, have opened up new possibilities for precise molecular engineering at the nanoscale. The CRISPR gene editing system has also shown remarkable specificity in recognizing DNA sequences.
Against this backdrop, a team led by Professor Zhang Han and Associate Professor Chen Zhi from Shenzhen University, China, demonstrated an innovative approach that combines DNA origami, CRISPR technology and surface plasmon resonance sensing to achieve unprecedented levels of sensitivity and specificity in genetics. I did. Mutation detection. This new method addresses long-standing challenges in the field and offers a potential breakthrough in early cancer diagnosis and personalized treatment strategies.
The study, detailed in a recent paper, Lasers and Photonics Reviews (“Ultrasensitive DNA origami plasmonic sensors for accurate detection in circulating tumor DNA”) introduces a sophisticated biosensing platform that leverages the structural precision of DNA origami, the single base identification capability of CRISPR-Cas12a, and the high sensitivity of SPR detection. do. This system is designed to detect specific mutations in the EGFR and KRAS genes, which are important biomarkers for non-small cell lung cancer (NSCLC).
![Surface plasmon resonance (SPR) biosensing design schematic integrating DNA origami and DNA scissors.](https://www.nanowerk.com/spotlight/id65422_1.jpg)
The core of the technology is a carefully designed DNA origami structure that acts as a probe. This three-dimensional DNA assembly is designed to overcome the limitations of conventional single-stranded DNA probes, such as entanglement and surface placement abnormalities. DNA origami provides a stable support for attaching gold nanoparticles (AuNPs) to precise locations, creating a uniform distribution across the sensor surface.
The researchers first verified the successful assembly of the DNA origami probes using polyacrylamide gel electrophoresis and transmission electron microscopy. They observed distinct bands corresponding to different stages of the assembly process and confirmed the triangular shape of the final structure. Incorporation of additional DNA strands and poly(A) tails allowed attachment of AuNPs, completing the probe design.
The CRISPR-Cas12a system plays an important role in the sensing mechanism. The researchers demonstrated the ability to distinguish between wild-type and mutant gene sequences with single-base precision. When the target DNA sequence is present, the activated Cas12a enzyme cleaves the DNA origami probe, releasing the attached AuNPs. This splitting event is detected by the SPR sensor as a change in local refractive index.
The integration of these technologies results in a detection system with remarkable sensitivity. The researchers found that the zeptomol range (10-21 moles per liter for both EGFR and KRAS gene mutations). This level of sensitivity exceeds that of conventional PCR-based methods by several orders of magnitude.
To validate the clinical utility of the approach, the team tested the system using samples from NSCLC patients. The results were in close agreement with those obtained from quantitative PCR (qPCR) analysis. Importantly, the new method successfully detected mutations in samples incorrectly identified as negative by PCR. This demonstrates the potential of the technology to reduce false-negative results in clinical settings, potentially enabling faster and more accurate cancer diagnosis.
![In-device measurements using DNA scissors and origami SPR](https://www.nanowerk.com/spotlight/id65422_2.jpg)
The success of this approach lies in the synergistic combination of component technologies. DNA origami provides a stable and precisely designed platform for probe immobilization, overcoming the problems of probe entanglement and irregular distribution that plague conventional SPR biosensors. The CRISPR-Cas12a system provides unparalleled specificity in recognizing target sequences, enabling the identification of single base mutations. Finally, the SPR sensing platform provides a sensitive, label-free detection method that can convert molecular binding events into measurable signals.
This integrated approach addresses several key challenges in genetic mutation detection. Sensitivity at zeptomole levels allows detection of very low concentrations of ctDNA, which is important for early cancer diagnosis. Single-base resolution allows accurate identification of specific mutations, which is essential for guiding targeted therapy in personalized medicine approaches. Additionally, the label-free nature of SPR detection eliminates the need for complex sample preparation or amplification steps, simplifying the detection process and potentially reducing turnaround time in clinical settings.
The implications of this technology extend beyond lung cancer diagnosis. The high sensitivity and specificity of the platform makes it potentially applicable to a wide range of genetic diseases and infectious diseases where detection of small amounts of nucleic acids or single nucleotide polymorphisms is important. This could have applications in prenatal genetic testing, minimal residual disease monitoring in cancer patients, and rapid detection of new pathogen strains.
However, as with any new technology, there may be challenges that need to be addressed before widespread clinical adoption. These may include issues of scalability, cost-effectiveness, and integration with existing diagnostic workflows. Additional validation studies with larger patient populations will be needed to establish the robustness and reliability of the method in different clinical scenarios.
The development of this ultrasensitive DNA detection platform represents a significant advance in the field of molecular diagnostics. Leveraging the strengths of DNA nanotechnology, gene editing tools, and advanced biosensing technologies, the team created a system that pushes the boundaries of genetic mutation detection. As this technology continues to be improved and validated, it has the potential to transform early cancer diagnosis, enable more accurate treatment selection, and ultimately improve patient outcomes in the era of personalized medicine.
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– Michael is the author of three books published by the Royal Society of Chemistry. Nano-Society: Pushing the Boundaries of Technology, Nanotechnology: The Future is Tiny, Nanoengineering: The Skills and Tools Making Technology Invisible Copyright ©
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