Description:Description:
Field of the Invention:
This invention relates to the field of biomedical diagnostics, particularly to the development of DNA-based electrochemical biosensors. Specifically, it pertains to a label-free DNA origami biosensor for the rapid and sensitive detection of lung cancer-specific microRNAs in serum samples.

Background:
Cancer is one of the leading causes of mortality worldwide, with lung cancer being the most common cause of cancer-related deaths. Early detection of lung cancer remains challenging due to the absence of symptoms at initial stages and the limitations of conventional diagnostic methods, such as sputum cytology, bronchoscopy, and imaging techniques. In recent years, microRNAs (miRNAs) have emerged as potential biomarkers for early cancer detection due to their regulatory roles in oncogenesis. However, there is an unmet need for a highly sensitive, label-free, and rapid biosensing method to detect lung cancer-specific miRNAs. DNA origami nanostructures provide an innovative platform for constructing biosensors with enhanced sensitivity, stability, and user-defined functionality, offering a promising solution to address the existing diagnostic challenges.

Object of the Invention:
The primary objective of this invention is to develop a label-free DNA origami biosensor capable of high-throughput detection of lung cancer-specific microRNAs in serum samples with enhanced sensitivity, specificity, and ease of operation.

Summary of the Invention:
This invention involves a DNA origami-based biosensor designed for the rapid and label-free detection of lung cancer-specific miRNAs. The biosensor employs DNA origami nanostructures as a platform for ssDNA probe immobilization, leveraging the advantages of DNA origami, including high surface area, stability, and nanometer-scale customization. The biosensor detects target miRNAs through electrochemical signals, enabling precise and sensitive quantification of lung cancer biomarkers.

Detailed Description:

Synthesis and Characterization of ssDNA Probe-Functionalized DNA Origami:
The DNA origami nanostructures are synthesized using the Rothemund protocol. In this approach, a long circular DNA scaffold strand (M13mp18) is combined with a set of short, thiol-functionalized ssDNA staple strands. The synthesis is carried out in a TAE buffer containing magnesium ions, which facilitate the folding process through a thermal annealing cycle. The resulting nanostructures are purified using centrifugal filters to remove excess staples and buffer components. To ensure the structural integrity and successful folding of the DNA origami, transmission electron microscopy (TEM) and atomic force microscopy (AFM) are employed for characterization.

Fabrication on Gold Electrode:
The purified DNA origami nanostructures are immobilized on the surface of a polished gold electrode using thiol-gold chemistry. The gold electrode is pre-cleaned and polished to ensure proper surface adhesion. Thiol groups on the DNA origami form strong bonds with the gold electrode, ensuring stable immobilization. After the immobilization, the electrode is treated with hexanethiol, which passivates the unreacted areas of the gold surface and provides a uniform coating. This process minimizes non-specific interactions and enhances the specificity of the biosensor. The modified electrodes are characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) to confirm the successful attachment of DNA origami structures and evaluate their surface properties.

miRNA Detection:
The DNA origami-functionalized gold electrode is utilized for the detection of lung cancer-specific microRNAs (miRNAs). Serum samples containing the target miRNAs are incubated with the biosensor to allow hybridization between the miRNAs and the complementary ssDNA probes immobilized on the DNA origami. Non-hybridized miRNAs are removed through a systematic washing process. Control samples are subjected to the same procedure to validate the specificity and reliability of the detection system.

Electrochemical Analysis:
After hybridization, electrochemical measurements are conducted using CV and EIS techniques to assess the interaction between the miRNAs and the DNA origami-based biosensor. The hybridization of target miRNAs results in measurable changes in the electrochemical properties of the biosensor. CV provides information about the redox behavior of the electrode, while EIS measures the impedance changes at the electrode-solution interface. These changes are used to confirm the presence and determine the concentration of lung cancer-specific miRNAs in the serum samples. This label-free detection approach ensures high sensitivity, reproducibility, and cost-effectiveness for miRNA analysis.

Brief Description of the Drawing:
1. DNA Origami Synthesis: Thiol-functionalized single-stranded DNA (ssDNA) probes are combined with a scaffold strand (M13mp18) to form DNA origami nanostructures.
2. Gold Electrode Preparation: The gold electrode surface is cleaned and polished for immobilization.
3. Immobilization of DNA Origami: Thiol-gold chemistry is used to attach the DNA origami nanostructures to the gold electrode.
4. Surface Modification: Hexanethiol is applied to passivate the surface, minimizing non-specific binding and ensuring uniformity.
5. Electrochemical Characterization: The modified gold electrode is tested using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) to confirm the successful attachment of the DNA origami structures.
6. Incubation with Samples: Serum samples containing target miRNAs (test) or control samples are incubated with the DNA origami-modified electrode.
7. Electrochemical Measurements: CV and EIS techniques are used to detect hybridization events, indicating the presence and concentration of lung cancer-specific miRNAs in the test samples.
, Claims:1. A label-free biosensor for detecting lung cancer-specific microRNAs, comprising:
- DNA origami nanostructures functionalized with immobilized single-stranded DNA (ssDNA) probes, designed to specifically hybridize with target microRNAs.
- A gold electrode as the substrate for biosensor assembly.
- Thiol-gold chemistry for the immobilization of DNA origami nanostructures on the electrode surface.
- Hexanethiol for passivating the non-reactive areas of the gold electrode surface to minimize non-specific binding.
- Electrochemical techniques, including cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), for the detection and quantification of lung cancer-specific microRNAs.
2. The biosensor as claimed in Claim 1, wherein the DNA origami nanostructures are assembled using an M13mp18 scaffold and functionalized staples with thiol and ssDNA probes.
3. The biosensor as claimed in Claim 1, wherein the gold electrode surface is chemically modified to enhance the immobilization of DNA origami and the sensitivity of the biosensor.
4. A method for fabricating the biosensor as claimed in Claim 1, comprising the steps of:
- Preparing DNA origami nanostructures functionalized with ssDNA probes and thiol modifications.
- Immobilizing the DNA origami on a gold electrode surface via thiol-gold bonding.
- Passivating the gold electrode surface using hexanethiol.
- Conducting electrochemical characterization of the biosensor to confirm its readiness for detection.
5. A method for detecting lung cancer-specific microRNAs in serum samples using the biosensor as claimed in Claim 1, comprising the steps of:
- Incubating the serum sample with the fabricated biosensor under optimal conditions for target microRNA hybridization.
- Performing electrochemical measurements using CV and EIS to detect and quantify the hybridization of microRNAs on the biosensor surface.
- Comparing the obtained electrochemical signals against control samples to ensure specificity and accuracy.
6. The method as claimed in Claim 5, wherein the biosensor is reusable after proper dehybridization and cleaning procedures.