(Nanowerk Spotlight) The detection of genetic mutations in circulating tumor DNA (ctDNA) has long been a critical challenge in cancer diagnostics and personalized medicine. Traditional methods like polymerase chain reaction (PCR) and next-generation sequencing have limitations in sensitivity and accuracy, especially when dealing with minute quantities of genetic material or distinguishing single-base mutations. These constraints have hampered early cancer detection and the development of targeted therapies. The COVID-19 pandemic further complicated matters by disrupting routine cancer screenings, leading to delayed diagnoses and potentially worse outcomes for patients.
In recent years, researchers have explored various technologies to enhance genetic detection capabilities. Surface plasmon resonance (SPR) biosensors emerged as a promising approach due to their label-free, real-time, and high-throughput characteristics. However, SPR biosensors faced challenges in discriminating between similar nucleic acid sequences and detecting low-abundance targets in complex biological samples. Concurrently, advancements in DNA nanotechnology, particularly DNA origami, opened new possibilities for precise molecular engineering at the nanoscale. The CRISPR gene-editing system also demonstrated remarkable specificity in recognizing DNA sequences.
Against this backdrop, a team led by Professor Zhang Han and Associate Professor Chen Zhi from Shenzhen University in China , has demonstrated an innovative approach that combines DNA origami, CRISPR technology, and surface plasmon resonance sensing to achieve unprecedented levels of sensitivity and specificity in genetic mutation detection. This novel method addresses longstanding challenges in the field and offers potential breakthroughs in early cancer diagnosis and personalized treatment strategies.
The research, detailed in a recent paper in Laser & Photonics Reviews (“Ultrasensitive DNA Origami Plasmon Sensor for Accurate Detection in Circulating Tumor DNAs”), introduces a sophisticated biosensing platform that leverages the structural precision of DNA origami, the single-base discrimination capability of CRISPR-Cas12a, and the high sensitivity of SPR detection. The system is designed to detect specific mutations in the EGFR and KRAS genes, which are crucial biomarkers for non-small cell lung cancer (NSCLC).
Schematic of the design of surface plasmon resonance (SPR) biosensing incorporating DNA origami and DNA scissors. (Reprinted with permission by Wiley-VCH Verlag)
At the heart of the technology is a carefully engineered DNA origami structure that serves as a probe. This three-dimensional DNA assembly is designed to overcome limitations of conventional single-stranded DNA probes, such as entanglement and surface-laying anomalies. The DNA origami provides a stable scaffold for attaching gold nanoparticles (AuNPs) at precise locations, creating a uniform distribution across the sensor surface.
The researchers first validated 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. The integration of an additional DNA strand with a poly(A) tail allowed for the attachment of AuNPs, completing the probe design.
The CRISPR-Cas12a system plays a crucial role in the detection mechanism. The researchers demonstrated its ability to discriminate 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 cleavage event is then detected by the SPR sensor as a change in the local refractive index.
The integration of these technologies resulted in a detection system with remarkable sensitivity. The researchers reported a limit of detection in the zeptomolar range (10-21 moles per liter) for both EGFR and KRAS gene mutations. This level of sensitivity surpasses that of conventional PCR-based methods by several orders of magnitude.
To validate the clinical utility of their approach, the team tested the system using samples from NSCLC patients. The results closely aligned with those obtained from quantitative PCR (qPCR) analysis. Importantly, the new method successfully detected mutations in samples that had been falsely identified as negative by PCR. This demonstrates the potential of the technology to reduce false-negative results in clinical settings, potentially leading to earlier and more accurate cancer diagnoses.
On-device measurement with the DNA scissors and origami SPR. a) The change in wavelength (δW) for chips immobilized with either ssDNA or DNA origami probes, alongside the calculated relative wavelength change (Rw) values following trans-cleavage. b) Surface plasmon resonance (SPR) signals obtained using EGFR and KRAS gene templates. The ability for single-base discrimination was validated by CRISPR reactions, establishing a threshold for positive signal identification. c and d) Correlation between concentration levels of the EGFR gene ranging from 10 zm to 100 pm and their respective Rw signals, demonstrating a direct relationship between concentration and signal response. The limit of detection was calculated for each gene sequence. (Reprinted with permission by Wiley-VCH Verlag)
The success of this approach lies in the synergistic combination of its component technologies. The DNA origami provides a stable and precisely engineered platform for probe immobilization, overcoming issues of probe entanglement and irregular distribution that plague conventional SPR biosensors. The CRISPR-Cas12a system offers unparalleled specificity in recognizing target sequences, enabling single-base mutation discrimination. Finally, the SPR sensing platform provides a sensitive and label-free detection method that can transduce molecular binding events into measurable signals.
This integrated approach addresses several key challenges in genetic mutation detection. The zeptomolar-level sensitivity allows for the detection of extremely low concentrations of ctDNA, which is crucial for early-stage cancer diagnosis. The single-base resolution enables accurate identification of specific mutations, which is essential for guiding targeted therapies in personalized medicine approaches. Moreover, the label-free nature of SPR sensing eliminates the need for complex sample preparation or amplification steps, simplifying the detection process and potentially reducing turnaround times in clinical settings.
The implications of this technology extend beyond lung cancer diagnostics. The platform’s high sensitivity and specificity make it potentially applicable to a wide range of genetic disorders and infectious diseases where the detection of low-abundance nucleic acids or single-nucleotide polymorphisms is critical. It could find applications in prenatal genetic testing, monitoring of minimal residual disease in cancer patients, and rapid detection of emerging pathogen variants.
However, as with any new technology, there are likely challenges to be addressed before widespread clinical adoption. These may include issues of scalability, cost-effectiveness, and integration into existing diagnostic workflows. Further validation studies with larger patient cohorts will be necessary to establish the robustness and reliability of the method across diverse clinical scenarios.
The development of this ultrasensitive DNA detection platform represents a significant advancement in the field of molecular diagnostics. By harnessing the strengths of DNA nanotechnology, gene-editing tools, and advanced biosensing techniques, the team has created a system that pushes the boundaries of what is possible in genetic mutation detection. As this technology continues to be refined and validated, it has the potential to transform early cancer diagnosis, enable more precise treatment selection, and ultimately improve patient outcomes in the era of personalized medicine.
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