Periodic Reporting for period 1 - NoOne (A binary sensor with single-molecule digit to discriminate biofluids enclosing zero or at least one biomarker)
Reporting period: 2022-04-01 to 2024-09-30
Biomarkers are measurable indicators of a particular disease state of an organism. There has been an increasing demand for diagnostic markers, enabling reliable and non-invasive screening of peripheral biofluids. The NoOne project aims at conceiving, engineering, fabricating and validating a ground-breaking platform based on a single-molecule binary bioelectronic sensor, capable to reliably discriminate biofluid samples enclosing zero biomarkers from those containing just one. The technology can be used for ultimate binary sensing of both proteins/peptides and genomic markers to enable the reliable screening of diseases such as cancer as well as viral and bacterial infections. The NoOne binary platform is designed to be portable, cost-effective, easy to operate, and with a time-to-results within one hour; hence it is the ideal candidate for point-of-care applications. The prototype will enable to identify the set of samples that are totally free from a protein, peptide or genomic marker as well as from a pathogen (virus or bacteria), from those enclosing at least one with a confidence level of 99%. This makes the NoOne platform the best-performing ever in enabling a fast, highly reliable, cost-effective identification of the subset of biological samples belonging to the potentially diseased part of a population. This is of paramount importance for the predictive screening of humans, plants, or animals. NoOne will demonstrate its effectiveness in key relevant applications such as the binary detection of pancreatic cancer biomarkers, SARS-CoV-2 virus, the Xylella-Fastidiosa bacterium, and the assay of post-translational peptides evidencing the phosphorylated forms regulating crosstalk with oncogenic signaling pathways.
The NoOne assay is not conceived to be quantitative but to discriminate a sample containing not even one biomarker from those with at least one. Reliability is guaranteed by operating at the limit-of-identification (LOI = n + 6·sigma) that assures a level of confidence larger than 99 % with false positives lower than 1% and false negatives lower than 1%. The NoOne concept, that will be pursued through the following main objectives.
O1: Fabrication of stable and cost-effective biofunctionalized FETs and amplifying circuits integrated into 3D structures
The label-free single-molecule bioelectronic sensing devices will be based on an Electrolyte-Gated Field-Effect Transistor (EGFET) or on a CMOS compatible silicon-based electrolyte extended gate FET (EEGFET) both endowed with a biofunctionalized millimeter-wide (0.5 cmˆ2) gate. They will be both integrated into ad hoc designed circuits and totally innovative potentiometric 3D architectures with no need of a reference electrode as electrochemical reactions are avoided.
O2:Reliable and fast binary sensing of zero or one marker -
The second main objective concerns conceptualizing, operating and modelling a binary sensor capable to assess the total absence - zero - or the presence of just a single-molecule - one - of a marker of a progressive diseases or of a pathogen in sampled biofluids. This extremely challenging task is accomplished by discriminating the two-fold response of the NoOne bioelectronic sensor at the limit-of-identification, LOI = n + 6·sigma, hence in a very reliable fashion as the level of confidence is higher than 99 % while false negative and the false positive errors will be less than 1%.
O3:Multiscale amplification effects
Assessing the amplified mechanisms that enable to reliably measure a single-molecule binary response to one single analyte on a millimeter-wide NoOne transistor gate, is the highly challenging third objective. This is accomplished assaying a biological fluid, a complex matrix full of much more abundant interferents. A reliable sensing signal will be measured thanks to the design of a bioelectronic system endowed with three signal-enhancing steps at a multiscale level, namely: (i) the amplification occurring in the biolayer upon selective binding of the marker (bio-material amplification); (ii) the optimized capacity coupling between the biolayer and the FET channel (FET-amplification); (iii) the designed amplifier circuit type (amplification circuit).
O4: Engineering applications for early diagnosis
Engineering a widely-applicable binary single-molecule technology platform comprising four different sensing devices developed as lab-prototypes. The applications will provide compelling evidences of the strategic relevance of a binary single-molecule sensor for screening tests supporting an ultimately early diagnosis. Two of the applications will involve the binary detection of the SARS-CoV-2 virus (with Prof. Maria Chironna – University of Bari) and the Xylella Fastidiosa bacterium (Dr. Pasquale Saldarelli, National Research Council - Bari).
Other applications will involve the detection of a protein or genomic marker for the early detection of cancer. The NoOne sensor will provide a novel and complementary approach by assaying only the absence/presence at the single-molecule level of markers such as KRAS (genomic) and MUC1 (protein) that can support clinicians in the early diagnosis of pancreatic cancer (with Prof. Irene Esposito of Dusseldorf University).
O1: Fabrication of stable and cost-effective biofunctionalized FETs and amplifying circuits integrated into 3D structures
The label-free single-molecule bioelectronic sensing devices will be based on an Electrolyte-Gated Field-Effect Transistor (EGFET) or on a CMOS compatible silicon-based electrolyte extended gate FET (EEGFET) both endowed with a biofunctionalized millimeter-wide (0.5 cmˆ2) gate. They will be both integrated into ad hoc designed circuits and totally innovative potentiometric 3D architectures with no need of a reference electrode as electrochemical reactions are avoided.
O2:Reliable and fast binary sensing of zero or one marker -
The second main objective concerns conceptualizing, operating and modelling a binary sensor capable to assess the total absence - zero - or the presence of just a single-molecule - one - of a marker of a progressive diseases or of a pathogen in sampled biofluids. This extremely challenging task is accomplished by discriminating the two-fold response of the NoOne bioelectronic sensor at the limit-of-identification, LOI = n + 6·sigma, hence in a very reliable fashion as the level of confidence is higher than 99 % while false negative and the false positive errors will be less than 1%.
O3:Multiscale amplification effects
Assessing the amplified mechanisms that enable to reliably measure a single-molecule binary response to one single analyte on a millimeter-wide NoOne transistor gate, is the highly challenging third objective. This is accomplished assaying a biological fluid, a complex matrix full of much more abundant interferents. A reliable sensing signal will be measured thanks to the design of a bioelectronic system endowed with three signal-enhancing steps at a multiscale level, namely: (i) the amplification occurring in the biolayer upon selective binding of the marker (bio-material amplification); (ii) the optimized capacity coupling between the biolayer and the FET channel (FET-amplification); (iii) the designed amplifier circuit type (amplification circuit).
O4: Engineering applications for early diagnosis
Engineering a widely-applicable binary single-molecule technology platform comprising four different sensing devices developed as lab-prototypes. The applications will provide compelling evidences of the strategic relevance of a binary single-molecule sensor for screening tests supporting an ultimately early diagnosis. Two of the applications will involve the binary detection of the SARS-CoV-2 virus (with Prof. Maria Chironna – University of Bari) and the Xylella Fastidiosa bacterium (Dr. Pasquale Saldarelli, National Research Council - Bari).
Other applications will involve the detection of a protein or genomic marker for the early detection of cancer. The NoOne sensor will provide a novel and complementary approach by assaying only the absence/presence at the single-molecule level of markers such as KRAS (genomic) and MUC1 (protein) that can support clinicians in the early diagnosis of pancreatic cancer (with Prof. Irene Esposito of Dusseldorf University).
A plethora of different molecular biomarkers’ screening tests are available for handling early-stage disease in an asymptomatic population. A basic classification divides them into immunoassays, targeting proteins (antigens, antibodies) and genomic assays of nucleic acids. To the best of our knowledge, a commercial platform that can handle, simultaneously, the ultra-sensitive assay of both protein and genomic markers, is not yet available. Screening tests are further classified into qualitative and quantitative tests. A qualitative test assays the smallest amount of a targeted marker that can be distinguished from the level of the noise (random error) at an acceptable level of confidence. This is addressed as limit-of-detection, LOD = noise-average-level (n) + 3 times the noise-standard-deviation (3·sigma), and results in false positives lower than 1% but false negatives up to 50%. Point-of-care, single use, lateral-flow, rapid screening tests generally work at the LOD. These are largely immuno-tests, but genomic markers can be targeted too, with time-to-results of tens of minutes. Drawbacks include large errors and LODs in the nM range, often too high to effectively serve in early diagnosis. Quantitative tests work beyond the limit-of-quantification (LOQ = n + 9·sigma) to quantify an analyte with an acceptable precision. i.e. both false positive and false negative lower than 1%, and gamma-error (overlap of gaussian error distributions of the LOQ and the LOD – type II error) lower than 1%. Yet, drawbacks are that the LODs do not always reach the single-molecule-level. Also, the need for a labelling step that contributes to the very long time-to results (several hours). Moreover, none can simultaneously handle assays for both protein and genomic markers. This is limiting the development of innovative and more efficient diagnostic protocols. This makes them inherently unsuited for detection at concentrations lower than pM. The LOD is the cut-off concentration below which the screening test becomes blind returning a negative (zero) response while yet, up to 100 million (ELISA) or thousands (Simoa) protein markers can be present in a 100 ul sample.