Periodic Reporting for period 2 - ANFIBIO (AmplificatioN Free Identification of cancer and viral biomarkers via plasmonic nanoparticles and liquid BIOpsy)
Reporting period: 2022-12-01 to 2024-05-31
The detection of freely circulating disease biomarkers in bodily fluids, also known as liquid biopsy, has taken important strides toward the implementation of truly personalized medicine. However, it still suffers from low sensitivity and high costs, which render its clinical implementation often not practical or affordable. In particular, the identification and quantification of oligonucleotide biomarkers is hampered by the need of technologies that are expensive, require highly trained personnel, and are prone to error. Nonetheless, the recent clinical breakthroughs demonstrating the importance of detecting cancerous or viral biomarker to susceptibility, onset, and aggressiveness of the disease, motivate the need for further research that could render their detection simpler, cheaper, and thus more widely available. Therefore, developing a low-cost, easy to implement, robust and reliable sensing method for the detection of genetic biomarkers of disease would render diagnosis and disease progression monitoring more affordable and thus accessible to all, regardless of their socioeconomic status and geographic location. Similarly, such a method would decrease the testing burden on clinics and hospital, thus allowing to diagnose and follow up on more patients more often and monitor disease spread within the population, which would be particularly important in case of a future pandemic.
Based on these needs and opportunities, ANFIBIO seeks to implement a breakthrough concept of DNA and RNA identification that takes inspiration from sequencing technologies and leverages direct surface enhanced Raman spectroscopy (SERS)-based sensing and machine learning approaches. ANFIBIO will deliver a sensitive, accurate, and low-cost platform for the detection of biomarkers of disease of clinical relevance. Its main concept takes inspiration from the fundamental advantages of both short- and long-read sequencing technologies and aims to overcome their limitations to deliver a clinically relevant diagnostic technology that can be available to all.
Based on the above considerations, the specific and measurable project objectives of ANFIBIO are:
OBJECTIVE 1. To DESIGN and OPTIMIZE bespoke star-shaped gold nanoparticles (i.e. nanostars) to provide SERS signal enhancement and detect key disease biomarkers even at very low concentration.
OBJECTIVE 2. To AMPLIFY the SERS signal of DNA and RNA targets that directly interact with the metallic surface of the nanostars by understanding the key features driving DNA and RNA adsorption and stabilization onto the metallic surface and identifying the key conditions to obtain well-resolved and intense spectra with high reproducibility, so that a machine learning algorithm can be devised to discriminate even seemingly identical spectra. Ideally, spectra obtained from specimens that differ only by one point mutation should be discernible.
OBJECTIVE 3. To DETERMINE the identity and relative position of constituent nucleobases in specific target oligonucleotide biomarkers by employing machine learning algorithms, starting with short reads and then moving on to medium-size reads. Machine learning is new in materials science and chemistry; it has never been employed in SERS as proposed here.
OBJECTIVE 4. To VALIDATE the method and DELIVER a technology for identification and quantification of biomarkers of prostate cancer (PCa) and Influenza A virus (IAV) in bodily fluids of patients.
Based on these needs and opportunities, ANFIBIO seeks to implement a breakthrough concept of DNA and RNA identification that takes inspiration from sequencing technologies and leverages direct surface enhanced Raman spectroscopy (SERS)-based sensing and machine learning approaches. ANFIBIO will deliver a sensitive, accurate, and low-cost platform for the detection of biomarkers of disease of clinical relevance. Its main concept takes inspiration from the fundamental advantages of both short- and long-read sequencing technologies and aims to overcome their limitations to deliver a clinically relevant diagnostic technology that can be available to all.
Based on the above considerations, the specific and measurable project objectives of ANFIBIO are:
OBJECTIVE 1. To DESIGN and OPTIMIZE bespoke star-shaped gold nanoparticles (i.e. nanostars) to provide SERS signal enhancement and detect key disease biomarkers even at very low concentration.
OBJECTIVE 2. To AMPLIFY the SERS signal of DNA and RNA targets that directly interact with the metallic surface of the nanostars by understanding the key features driving DNA and RNA adsorption and stabilization onto the metallic surface and identifying the key conditions to obtain well-resolved and intense spectra with high reproducibility, so that a machine learning algorithm can be devised to discriminate even seemingly identical spectra. Ideally, spectra obtained from specimens that differ only by one point mutation should be discernible.
OBJECTIVE 3. To DETERMINE the identity and relative position of constituent nucleobases in specific target oligonucleotide biomarkers by employing machine learning algorithms, starting with short reads and then moving on to medium-size reads. Machine learning is new in materials science and chemistry; it has never been employed in SERS as proposed here.
OBJECTIVE 4. To VALIDATE the method and DELIVER a technology for identification and quantification of biomarkers of prostate cancer (PCa) and Influenza A virus (IAV) in bodily fluids of patients.
The results obtained so far cover the first half of the project, and in particular Work Package 1 and Work Package 2.
In work package 1 we have studied the parameters that affect the synthesis of star-shaped gold nanoparticles, which are the key materials providing the SERS signal amplification needed to measure the signal of DNA and RNA biomarkers of disease. The principal parameters under study are the optical response of the seeds (which is correlated to their concentration and morphology), the concentration of the surfactant employed to stabilize and shape-direct the nanoparticle morphology, the order of reagents during the growth phase, the age of the seeds, the dependence of the reaction yield to the concentration of the reducing agent, and the humidity of the environment. Understanding how these parameters affect the final outcome is important to make the synthesis reproducible and accurate, so that anyone who is interested in producing these materials can do so. Through our experiments we have determined that temperature and humidity do not directly affect the synthesis but can complicate the synthetic procedures, and are therefore confounding factors in the experiments. We determined that the quality of the water employed to synthesize these materials is key to reproduce the synthesis. We have also observed that the surfactant cannot be measured by volume but rather by weight as it is a very viscous liquid and that wrong determination of its amount can be highly detrimental to the quality of the final product. While we can now consistently reproduce the syntheses carried out in our lab, further systematic analyses are needed to ensure that widespread reproducibility to other labs is ensured.
In work package 2 we have studied how DNA and RNA strands bind to the gold nanoparticles and how the resulting SERS spectra are affected by the conformation of these molecules onto the metallic surface. We have explored some of the factors that have a direct effect on the binding of oligonucleotides to gold nanoparticles such as pH, temperature and salt concentration. The protonation or deprotonation of individual bases at various pH governs the overall interaction of the oligonucleotide chain to the nanoparticle. We also observed the differences in behaviour on lowering the incubation temperature to 4 °C and freezing (-20 °C). We believe that the binding at -20 °C is mostly caused by the phase transition from liquid to solid and the restricted space of interaction of oligonucleotides with the nanoparticles. We have also determined that, given the same nucleobases, the behavior of the oligonucleotides, as they interact with the nanoparticles, changes with the position of the bases in the strand.
In work package 1 we have studied the parameters that affect the synthesis of star-shaped gold nanoparticles, which are the key materials providing the SERS signal amplification needed to measure the signal of DNA and RNA biomarkers of disease. The principal parameters under study are the optical response of the seeds (which is correlated to their concentration and morphology), the concentration of the surfactant employed to stabilize and shape-direct the nanoparticle morphology, the order of reagents during the growth phase, the age of the seeds, the dependence of the reaction yield to the concentration of the reducing agent, and the humidity of the environment. Understanding how these parameters affect the final outcome is important to make the synthesis reproducible and accurate, so that anyone who is interested in producing these materials can do so. Through our experiments we have determined that temperature and humidity do not directly affect the synthesis but can complicate the synthetic procedures, and are therefore confounding factors in the experiments. We determined that the quality of the water employed to synthesize these materials is key to reproduce the synthesis. We have also observed that the surfactant cannot be measured by volume but rather by weight as it is a very viscous liquid and that wrong determination of its amount can be highly detrimental to the quality of the final product. While we can now consistently reproduce the syntheses carried out in our lab, further systematic analyses are needed to ensure that widespread reproducibility to other labs is ensured.
In work package 2 we have studied how DNA and RNA strands bind to the gold nanoparticles and how the resulting SERS spectra are affected by the conformation of these molecules onto the metallic surface. We have explored some of the factors that have a direct effect on the binding of oligonucleotides to gold nanoparticles such as pH, temperature and salt concentration. The protonation or deprotonation of individual bases at various pH governs the overall interaction of the oligonucleotide chain to the nanoparticle. We also observed the differences in behaviour on lowering the incubation temperature to 4 °C and freezing (-20 °C). We believe that the binding at -20 °C is mostly caused by the phase transition from liquid to solid and the restricted space of interaction of oligonucleotides with the nanoparticles. We have also determined that, given the same nucleobases, the behavior of the oligonucleotides, as they interact with the nanoparticles, changes with the position of the bases in the strand.
Based on the current results, ANFIBIO will deliver a new diagnostic concept for genetic biomarkers of disease, in which costly and lengthy protocols will not be needed while, at the same time, the ability to detect target mutations will be maintained. This method will be extremely important in the diagnosis and management of cancer and on the monitoring of viral diseases. The results expected until the end of the project will be:
1) The optimization of the synthesis of gold nanostars so that each of them can produce the same SERS amplification regardless of the target interacting with it. This achievement is extremely important to ensure accuracy and reproducibility among different diagnostic labs using the technology;
2) The identification of the optimal conditions of analyte (i.e. DNA and RNA) extraction from bodily fluids, incubation with nanoparticles, and SERS experiments, which will become part of a standard research protocol to employ by all labs around the world that will be implementing our method;
3) The development of a machine learning algorithm that can analyze long DNA and RNA strands up to hundreds of nucleotides and determine whether they belong to the same sequence or not. This aspect will be important especially because in many cases clinicians want to know whether a genetic biomarker obtained from a patient has the sequence it should have, or not.
4) The launch of a prospective study with prostate cancer patients and a comparison with the data already available from retrospective studies and biobanks, to assess whether our method can be validated in the clinic.
Overall, it is our expectation that the results obtained with ANFIBIO will bring important changes in how diagnostics is done, both in the fields of oncology and virology.
1) The optimization of the synthesis of gold nanostars so that each of them can produce the same SERS amplification regardless of the target interacting with it. This achievement is extremely important to ensure accuracy and reproducibility among different diagnostic labs using the technology;
2) The identification of the optimal conditions of analyte (i.e. DNA and RNA) extraction from bodily fluids, incubation with nanoparticles, and SERS experiments, which will become part of a standard research protocol to employ by all labs around the world that will be implementing our method;
3) The development of a machine learning algorithm that can analyze long DNA and RNA strands up to hundreds of nucleotides and determine whether they belong to the same sequence or not. This aspect will be important especially because in many cases clinicians want to know whether a genetic biomarker obtained from a patient has the sequence it should have, or not.
4) The launch of a prospective study with prostate cancer patients and a comparison with the data already available from retrospective studies and biobanks, to assess whether our method can be validated in the clinic.
Overall, it is our expectation that the results obtained with ANFIBIO will bring important changes in how diagnostics is done, both in the fields of oncology and virology.