Periodic Reporting for period 1 - MI-SCAN (A rapid non-invasive on-chip miRNA-based Sputum Assay for early stage Lung Cancer Screening)
Periodo di rendicontazione: 2021-01-01 al 2022-12-31
The all-embracing aim of MI-SCAN is to get up and go beyond current abilities to advance a novel disruptive technology to drive lung cancer screening toward personalized treatment, with possible applications to fast-track the discovery of therapeutic targets and biomarkers for lung cancer through the study of practicable miRNA and Surface enhancer Raman Scattering (SERS) technology application.
The objective of MI-SCAN is to develop a portable point-of-care (PoC) device with the capacity to detect multiple miRNA biomarkers present in the Sputum sample of lung cancer patients. The developed methodology will open the way to develop a clinically validated approach for early-stage diagnosis of lung cancer.
• What is the problem/issue being addressed?
Lung cancer is one of the leading causes of cancer mortality in the world accounting serious number deaths, every year. It is well known that genetic damages could result in lung tumorigenesis. Despite years of research, the survival rate of the patients has not been markedly improved. Due to lack of high sensitivity and specificity in early-stage diagnostic tests, just about 15–20% of lung cancer cases are discovered prior to progression of the disease. The proposed research addressed the issue by application of Micro-RNA (miRNA) bioassay on a microfluidic chip combined with Surface Enhanced Raman Scattering (SERS) spectroscopy. The goal of the project is achieved by developing the methodology for screening of miRNA biomarker on-chip and screening out the responsible target biomarker for lung cancer. The developed approach has the potential to address the issue of early diagnosis and With proper validation, it can be applied to the public system for annual health checks.
• Why is it important for society?
Presently in our hospitals gold standard diagnostic methods for lung cancer are used such as X-ray, low-dose CT scan, and sputum cytology, which are not very efficient in diagnosing lung cancer at early stages. Other techniques like, fluorescence bronchoscopy improves the detection of lung tumor, but it needs expert doctors and is an invasive method that cannot be applied for regular checkups. The developed approach uses a non-invasive sample that can easily obtained from patients or from health personnel. It does not require any very technical and expert person to operate and need no hospital facility or admission. This will help society to be implied for regular health checks and easy access to high-end early-stage cancer detection.
• What are the overall objectives?
The objectives of MI-SCAN were to develop a novel technology to perform early-stage diagnostics towards personalized diagnostics, with possible applications to fast-track cancer screening and treatment.
The major goals are addressed by individual objectives in consecutive steps:
• Selection of best panel miRNA biomarkers for early-stage lung cancer identification.
• Development of a microfluidic chip for the detection of target miRNA biomarkers in sputum samples using SERS detection,
• Optimize miRNA detection using SERS.
• Validation of developed microfluidic chip for early-stage detection and patient stratification for lung cancer.
The objective of MI-SCAN is to develop a portable point-of-care (PoC) device with the capacity to detect multiple miRNA biomarkers present in the Sputum sample of lung cancer patients. The developed methodology will open the way to develop a clinically validated approach for early-stage diagnosis of lung cancer.
• What is the problem/issue being addressed?
Lung cancer is one of the leading causes of cancer mortality in the world accounting serious number deaths, every year. It is well known that genetic damages could result in lung tumorigenesis. Despite years of research, the survival rate of the patients has not been markedly improved. Due to lack of high sensitivity and specificity in early-stage diagnostic tests, just about 15–20% of lung cancer cases are discovered prior to progression of the disease. The proposed research addressed the issue by application of Micro-RNA (miRNA) bioassay on a microfluidic chip combined with Surface Enhanced Raman Scattering (SERS) spectroscopy. The goal of the project is achieved by developing the methodology for screening of miRNA biomarker on-chip and screening out the responsible target biomarker for lung cancer. The developed approach has the potential to address the issue of early diagnosis and With proper validation, it can be applied to the public system for annual health checks.
• Why is it important for society?
Presently in our hospitals gold standard diagnostic methods for lung cancer are used such as X-ray, low-dose CT scan, and sputum cytology, which are not very efficient in diagnosing lung cancer at early stages. Other techniques like, fluorescence bronchoscopy improves the detection of lung tumor, but it needs expert doctors and is an invasive method that cannot be applied for regular checkups. The developed approach uses a non-invasive sample that can easily obtained from patients or from health personnel. It does not require any very technical and expert person to operate and need no hospital facility or admission. This will help society to be implied for regular health checks and easy access to high-end early-stage cancer detection.
• What are the overall objectives?
The objectives of MI-SCAN were to develop a novel technology to perform early-stage diagnostics towards personalized diagnostics, with possible applications to fast-track cancer screening and treatment.
The major goals are addressed by individual objectives in consecutive steps:
• Selection of best panel miRNA biomarkers for early-stage lung cancer identification.
• Development of a microfluidic chip for the detection of target miRNA biomarkers in sputum samples using SERS detection,
• Optimize miRNA detection using SERS.
• Validation of developed microfluidic chip for early-stage detection and patient stratification for lung cancer.
• Step-1: The project started with the finding of the suitable miRNA biomarker panel as available in the literature. With the selection of the miRNA biomarkers panel (miRNA-21, 31, 200b, and 210).
• Step 2: The initial mechanism and miRNA capturing probe immobilization on the surface (direct UV assisted immobilization) and optimization were carried out. Followed by the capturing of target probes and recognition probes hybridization was also optimized and characterized under fluorescence microscopy.
• Step-3: After optimization of all the steps with validation and developed protocol this step was repeated on the microfluidic chip surface for characterization under SERS.
• Step 4: A laser-assisted approach was applied to fabricate a microfluidic chip in a clean room condition (Certified ISO-7 clean room for bio application). The fabricated chip does need not any further treatment for the application of biomolecules (miRNA). The overall process is very fast (15 min per chip) and can be further optimized for industrial scaleup (1 min per chip).
• Step-5: The fabricated chip was further applied to perform the detection of miRNA biomarkers (known samples) inflow conditions and all steps on the chip for validation of prototype working. SERS detection was performed for 2 different miRNA biomarkers (miRNA-U6 control, and miRNA-21 cancer biomarker).
• Step-6: The miRNA biomarkers are detected first for SERS peaks and then a calibration curve is drawn for various concentrations of the target miRNA. The limit of detection of the approach was found around 10 pico-Molar.
• Step-7: Ethical approval and collaboration with the Hospital HM La Esperanza (Santiago de Compostela, Spain) was obtained for the use of real samples from the lung cancer patients.
• Step 2: The initial mechanism and miRNA capturing probe immobilization on the surface (direct UV assisted immobilization) and optimization were carried out. Followed by the capturing of target probes and recognition probes hybridization was also optimized and characterized under fluorescence microscopy.
• Step-3: After optimization of all the steps with validation and developed protocol this step was repeated on the microfluidic chip surface for characterization under SERS.
• Step 4: A laser-assisted approach was applied to fabricate a microfluidic chip in a clean room condition (Certified ISO-7 clean room for bio application). The fabricated chip does need not any further treatment for the application of biomolecules (miRNA). The overall process is very fast (15 min per chip) and can be further optimized for industrial scaleup (1 min per chip).
• Step-5: The fabricated chip was further applied to perform the detection of miRNA biomarkers (known samples) inflow conditions and all steps on the chip for validation of prototype working. SERS detection was performed for 2 different miRNA biomarkers (miRNA-U6 control, and miRNA-21 cancer biomarker).
• Step-6: The miRNA biomarkers are detected first for SERS peaks and then a calibration curve is drawn for various concentrations of the target miRNA. The limit of detection of the approach was found around 10 pico-Molar.
• Step-7: Ethical approval and collaboration with the Hospital HM La Esperanza (Santiago de Compostela, Spain) was obtained for the use of real samples from the lung cancer patients.
MiRNA based screening for lung cancer can provide valuable insight into disease screening and can potentially transform personalized treatment. Conventional methods for diagnostic of lung cancer biomarkers still face important limitations of multiplexing capabilities, low cost, portability, and non-invasive diagnostic. MI-SCAN presents an innovative approach and capabilities in cost cost-effective way. Hence, upon successful implementation, MI-SCAN will result in a robust device, suitable for low-cost implementation, with ultrasensitive capabilities, at early-stage detection beyond what is available, and being considered the gold standard.
MI-SCAN initiated a radically new approach to unsolved diagnostic and healthcare challenges. Non-invasive lung tumor diagnostics is an unmet challenge. To date, all techniques for the analysis based on miRNA identification require a previous amplification step. So far, early detection of lung cancer using miRNA from sputum samples by PCR is limited by its selectivity and sensitivity (85-90%) below the clinical requirement (above 95%).
Beyond the state-of-art, the project applies a narrow SERS signal with a single molecular sensitivity approach with an expectation to achieve high-quality multiplex detection of a panel of miRNA biomarkers to fulfill the clinical requirement for PoC system. The interdisciplinary methodology applies the chemical synthesis of applied gold nanoparticles and the application of high-end laser physics to fabricate ready-to-use biosafe chip. The MI-SCAN stands out to explore connections between disciplines to develop a potential clinical diagnostic tool that can become the core for new companies, industries, and radically new ways of tackling human health challenges.
MI-SCAN delivers a solution for early-stage cancer diagnosis at the molecular level, flagging the technique for effective economical diagnosis. Furthermore, MI-SCAN idea is not only a possibility to improve the lives of people but also to lower inclusive healthcare costs. Moreover, MI-SCAN impact will not be restricted to cancer, but this technology could further be developed for monitoring various other cancer types and diseases as well as recognition of infectious pathogens (viruses, bacteria, etc.), and translated to other areas such as environmental, food, defense, and security.
MI-SCAN initiated a radically new approach to unsolved diagnostic and healthcare challenges. Non-invasive lung tumor diagnostics is an unmet challenge. To date, all techniques for the analysis based on miRNA identification require a previous amplification step. So far, early detection of lung cancer using miRNA from sputum samples by PCR is limited by its selectivity and sensitivity (85-90%) below the clinical requirement (above 95%).
Beyond the state-of-art, the project applies a narrow SERS signal with a single molecular sensitivity approach with an expectation to achieve high-quality multiplex detection of a panel of miRNA biomarkers to fulfill the clinical requirement for PoC system. The interdisciplinary methodology applies the chemical synthesis of applied gold nanoparticles and the application of high-end laser physics to fabricate ready-to-use biosafe chip. The MI-SCAN stands out to explore connections between disciplines to develop a potential clinical diagnostic tool that can become the core for new companies, industries, and radically new ways of tackling human health challenges.
MI-SCAN delivers a solution for early-stage cancer diagnosis at the molecular level, flagging the technique for effective economical diagnosis. Furthermore, MI-SCAN idea is not only a possibility to improve the lives of people but also to lower inclusive healthcare costs. Moreover, MI-SCAN impact will not be restricted to cancer, but this technology could further be developed for monitoring various other cancer types and diseases as well as recognition of infectious pathogens (viruses, bacteria, etc.), and translated to other areas such as environmental, food, defense, and security.