Periodic Reporting for period 1 - BIoRead (Reading DNA in real time for medical applications)
Reporting period: 2022-10-01 to 2024-03-31
The proposed methodology, “Laser-Assisted DNA Optical Mapping (LADOM)”, allows retrieving the barcode of single molecules of DNA in real time, as they flow through a nanochannel in a fluidic device. It combines a cheap device fabrication, flexible DNA labelling (customizable for different applications), microscope- and camera-free set up, a read-out sensitive to single molecules, very high throughput (hundreds of molecules per minute), ability to detect very small fragments, and no limitation for the maximum molecule’s length, what is especially interesting for applications dealing with ultra-long intact, genomic DNA. The simple volumen used is just a few uL (typically 3 uL, could be even less), and, since the analysis is performed at the single molecule level, the amount of material needed to obtain preliminary trends is also very low.
One of the potential application fields of this technology is liquid biopsy. Liquid biopsy is a powerful, emerging method, which will be the future of cancer monitoring. It holds the promise to replace conventional biopsy, where a needle is used to extract tissue from the affected organ. Instead, in liquid biopsy, the material used for the screening is obtained from body fluids –mainly blood- where it is possible to find circulating tumor material (vesicles, cells or DNA and RNA). But this liquid biopsy still has (technical) challenges to overcome before it can be routinarily used in the clinics. One of the main technical challenges is the very low amount of material available for the analysis, which comes surrounded by plenty “noise DNA” from healthy cells. And the DNA analysis still depends on expensive methods, not easily accessible.
In this project, our goal is to show the proof of concept for using our single molecule analysis method to analyze the DNA of samples with the potential of being used for cancer monitoring.
One of the potential application fields of this technology is liquid biopsy. Liquid biopsy is a powerful, emerging method, which will be the future of cancer monitoring. It holds the promise to replace conventional biopsy, where a needle is used to extract tissue from the affected organ. Instead, in liquid biopsy, the material used for the screening is obtained from body fluids –mainly blood- where it is possible to find circulating tumor material (vesicles, cells or DNA and RNA). But this liquid biopsy still has (technical) challenges to overcome before it can be routinarily used in the clinics. One of the main technical challenges is the very low amount of material available for the analysis, which comes surrounded by plenty “noise DNA” from healthy cells. And the DNA analysis still depends on expensive methods, not easily accessible.
In this project, our goal is to show the proof of concept for using our single molecule analysis method to analyze the DNA of samples with the potential of being used for cancer monitoring.
Methylation profiling of DNA can be an indicator of the pathogenety of tumor cells. One of our main activities was on the development of a low-cost, simple labelling method, which allows us to obtain the methylation profiling of the single molecules of DNA as they flow through the nanochannels. This method is based on a protein, expressed and synthesized in-house, which simplifies the overall methodology.
Our method allows to retrieve the signal of thousands of individual molecules per test. To improve the throughput, we have developed a software which allows for the automated and fast analysis of the signals. Several iterations have been developed, and the use of machine learning has also been explored.
One of the advantages of our method is that the DNA flows spontaneously along the nanochannels, meaning there is no need for the use of external driving forces, like pumps or electrodes for electrophoresis. This is very conventient to achieve our goal of making a portable technology, but we observed flow drifts along time. We developed a read out method, based on two laser spots, that we have used to understand the flow behavior of the molecules along the time needed for the tests (which can be from a few minutes and up to one hour). With this, we have improved the reproducibility and worked towards standardization the methodology, by doing systematic flow tests and by measuring the changes in the flow patterns for different conditions, like nanochannel cross section and buffer concentration.
We have also developed a lab-on-a-chip which can be used to extract DNA on-chip from biological fluids. This lab-on-a-chip has been integrated with the nanochannels, allowing us to detect the signal of individual molecules of DNA extracted directly from saliva or plasma.
Our method allows to retrieve the signal of thousands of individual molecules per test. To improve the throughput, we have developed a software which allows for the automated and fast analysis of the signals. Several iterations have been developed, and the use of machine learning has also been explored.
One of the advantages of our method is that the DNA flows spontaneously along the nanochannels, meaning there is no need for the use of external driving forces, like pumps or electrodes for electrophoresis. This is very conventient to achieve our goal of making a portable technology, but we observed flow drifts along time. We developed a read out method, based on two laser spots, that we have used to understand the flow behavior of the molecules along the time needed for the tests (which can be from a few minutes and up to one hour). With this, we have improved the reproducibility and worked towards standardization the methodology, by doing systematic flow tests and by measuring the changes in the flow patterns for different conditions, like nanochannel cross section and buffer concentration.
We have also developed a lab-on-a-chip which can be used to extract DNA on-chip from biological fluids. This lab-on-a-chip has been integrated with the nanochannels, allowing us to detect the signal of individual molecules of DNA extracted directly from saliva or plasma.
Preliminary tests of using our methodology for the analysis of tumor DNA are very promising. The variety of the new, open applications highlights the versatility of the technology.
Further research is needed to show the proof of concept with enough statistics for each one of the explored applications, including the methylation profiling, or the analysis of (very small amounts of) DNA from biological fluids.
Further research is needed to show the proof of concept with enough statistics for each one of the explored applications, including the methylation profiling, or the analysis of (very small amounts of) DNA from biological fluids.