The yardsticks used in the very last purchase of an information system may be totally out of date when searching for its replacement. Of equal significance, if the person wielding the yardstick does not know the questions to ask, then the users of the system are deprived of the tools which may significantly improve their personal performance and that of their employing company in the marketplace.
e15199 Background: Inflammation observed in response to some monoclonal antibody drugs and adaptive T-Cell therapies has become a major issue in cancer immunotherapy. Prognostic monitoring of the inflammatory response requires simultaneous measurement of multiple cytokines at widely divergent concentrations. At present, no analytical method, known to us, can provide large dynamic range (> 6 logs), high sensitivity (< 1pg/ml) and high multiplex in a single test. Methods: The NanoMosaic platform is a cytokine quantification technology powered by silicon nanoneedle biosensors that are densely integrated on a plate and manufactured with CMOS-compatible nanofabrication processes. Each nanoneedle is a label-free biosensor, functionalized with capture antibodies. Each analyte specific sensing area consists a total of ~23k nanoneedles divided into a digital region (~20k nanoneedles) and an analog region (~3k nanoneedles), combined to cover the entire range of inflammatory biomarkers from 0.1pg/ml to 1ug/ml. Results: We demonstrated that the digital nanoneedles achieve the single molecule sensitivity. Therefore, at ultra-low concentrations when antigens that are captured by the nanoneedles follow Poisson statistics, the number of antigens can be quantitated by counting the presence or absence of color changes of individual nanoneedles in a binary fashion. As the protein concentrations increase, the binding events increase accordingly and achieve saturation when all nanoneedles capture more than one protein. Above the digital saturation concentration, an adjacent section of analog nanoneedles perform quantitative analysis based on the level of color change, thus providing a wider dynamic range up to 1ug/ml. Each single analyte area, including both digital and analog sensors, is less than 500um. Therefore, high level multiplex can be achieved by duplicating the detection sensor in a microarray format without loss of sensitivity and dynamic range. Conclusions: The CMOS-compatible NanoMosaic technology provides the cost-effectiveness, sensitivity, dynamic range and multiplexing capacity required to fully integrate patient immune response into therapeutic development and decision making.
e15010 Background: Circulating biomarkers have the potential to detect cancer in its earliest stages and monitor patients in remission. The integration of proteogenomics in circulating biomarkers may transform the molecular diagnostics of cancer and accelerate basic and clinical oncology research. Proteomics bridges the gaps of functional information lost due to post-transcriptional and post-translational modifications in a genomic approach. A recent study showed that adding just 8 protein biomarkers to a panel of circulating DNA biomarkers increased the diagnostic accuracy up to 98% sensitivity and 99% specificity. However, the proteogenomic approach normally requires the use of multiple different assay technologies and laboratory workflows, including mass spectrometry. Methods: MosaicNeedles are densely integrated nanoneedle sensors fabricated on a planar substrate that integrates proteogenomic analysis in one platform. 94,000 sensors with more than 2 billion total nanoneedles can be integrated on to a standard SBS plate, which can be configured into 96, 384 or 1536 well format. Each sensor contains an array of nanoneedles, dedicated to detecting one analyte of interest. All nanoneedles comprising the same sensor are functionalized with the same capture probes. The capture probe can be either an antibody for protein detection or an oligonucleotide with a specific target sequence to a DNA fragment, mRNA, or miRNA of interest. Results: At low analyte concentration, the binding of proteins to the nanoneedles follows a Poisson distribution. Therefore, statistically, no more than one molecule is bound per nanoneedle. A further addition of aptamers or antibodies will form a sandwich complex with the target analyte. Since each of the nanoneedles has an intrinsic optical resonance spectrum and will red-shift as the sandwich complex forms on its surface, the number of analytes can be quantitated by simply counting the number of nanoneedles that display a color change. At high analyte concentration, each nanoneedle has more than one analyte, so the number of analytes can be calculated by averaging the spectrum shifts of all nanoneedles. This combined single molecule counting (digital) and spectrum shift (analog) analysis allows the platform to detect both high abundance and low abundance protein analytes in one reaction. A 10,000-plex study can be achieved with a total of 2.5 billion nanoneedles on a 50mm by 50mm consumable. In this consumable, a 2,000-plex proteome and 8,000 cell-free DNA fragments can be detected. Conclusions: A full proteogenomic quantification can be performed on the NanoMosaic platform in one reaction with high sensitivity and large dynamic range. It simplifies the workflow and allows users to integrate proteomic and genomic information to discover new circulating biomarkers.
e15205 Background: Existing drug development programs are represented by only a few hundred protein targets. A large subset of the ~20,000 proteins encoded by the human genome remain undiscovered. Proteome-wide “druggability” screening may lead to new targets for therapeutics. Methods: The NanoMosaic platform is a digital immunoassay technology that achieves sub-pg/ml level sensitivity, whole-proteome level multiplexing capability, and 7 logs of dynamic range. The platform overcomes the sensitivity and dynamic range limitations of traditional protein arrays and mass spectrometry. Results: The NanoMosaic technology is powered by silicon nanoneedle biosensors that are densely integrated on a plate and manufactured with CMOS-compatible nanofabrication processes. Each nanoneedle is a label-free biosensor, functionalized with capture antibodies. Its scattering spectrum changes when an antigen binds to its surface. Each analyte specific sensing area consists a total of ~23k nanoneedles divided into a digital region (~20k nanoneedles) and an analog region (~3k nanoneedles). The digital nanoneedles provide the single molecule sensitivity. Therefore, at ultra-low concentration when antigens that are captured by the nanoneedles follow Poisson statistics, the number of antigens can be quantitated by counting the presence or absence of color changes of individual nanoneedles in a binary fashion. As the protein concentrations increase, the binding event counts increase accordingly and achieve saturation when all nanoneedles capture more than one protein. Above the digital saturation concentration, an adjacent section of analog nanoneedles perform quantitative analysis based on the level of color change, thus providing a wider dynamic range up to 1ug/ml. Ultrahigh level multiplex can be achieved by parallelizing the detection in a microarray format without loss of the sensitivity and dynamic range. A 20,000-plex proteome-wide study can be achieved with a total of 5 billion nanoneedles on a ~70mm by 70mm chip. Conclusions: In conclusion, proteome-wide biomarker quantification and target discovery can be performed on the NanoMosaic platform at higher sensitivity, wider dynamic range, lower cost and higher throughput than is currently possible by mass spectrometry or traditional immunoassays.
e15019 Background: Liquid biopsy has evolved to be an important method complementary to tissue biopsy. It is not only non-invasive, but also has the potential to detect cancer in its earliest stages and monitor patients in remission. The integration of proteomics into liquid biopsy may transform the molecular diagnostics of cancer and accelerate basic and clinical oncology research. A recent study showed that adding just 8 protein biomarkers to a panel of circulating DNA biomarkers increased the diagnostic accuracy up to 98% sensitivity and 99% specificity. Proteomics also bridges the gaps of functional information lost due to post-transcriptional and post-translational modifications in the genomic approach. However, the proteogenomic approach normally requires the use of multiple different assay technologies and laboratory workflows, including mass spectrometry. Methods: NanoMosaic’s Tessie platform employs a densely integrated nanoneedle sensor array (thus named MosaicNeedles) which can be used to detect both nucleic acids and proteins in a single assay process with reduced workflow complexity, without the need for mass spectrometry. Results: The NanoMosaic platform is a label-free, digital, single molecule counting technology using nanoneedles. It achieves sub-pg/ml (̃fM) level sensitivity with 7 logs of dynamic range. An array of nanoneedles is densely integrated and manufactured with CMOS-compatible nanofabrication processes. Each nanoneedle is a single molecule biosensor that is functionalized with capture probes. The capture probe can be either an antibody for protein detection or an oligonucleotide with a specific target sequence to a DNA fragment, mRNA, or miRNA of interest. The scattering spectrum of each nanoneedle changes when an analyte binds to its surface. At low abundance, analytes that are captured can be quantitated by counting the presence or absence of a color change on each individual nanoneedle in a binary fashion. As an analyte concentration increases the binding events increase accordingly and achieve saturation. In this range, an analog analysis on the spectrum shift will be performed, thus providing a wider dynamic range, up to 7 logs. Ultrahigh level multiplex can be achieved by parallelizing each analyte specific sensing area without loss of sensitivity or dynamic range. A 10,000-plex study can be achieved with a total of 2.5 billion nanoneedles on a 50mm by 50mm consumable. In this consumable, a 2,000-plex proteome and 8,000 cell-free DNA fragments can be detected. Conclusions: In conclusion, a full proteogenomic quantification can be performed on the NanoMosaic platform in one reaction, with higher sensitivity, lower cost and higher throughput than is currently possible by traditional methods. In addition, the high-plexibility of the NanoMosaic platform allows the discovery of new biomarkers across the whole proteome without the need for mass spectrometry.
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