摘要:
The present invention is directed to methods for immobilizing molecules on siliceous and metallic surfaces. Molecules are immobilized on siliceous or metallic surfaces by stable covalent linkages that are capable of withstanding prolonged use and elevated temperatures. Further, the methods of the present invention describe less-complicated chemistries for the immobilization of molecules that will benefit the reproducibility, efficiency and effectiveness of applications in sensing, chromatography, medical diagnostics and related areas where specific recognition between immobilized and free molecules provides diagnostic information or serves as part of a purification or separations process.
摘要:
The sequence determination, detection, and quantification of nucleic acid molecules through sequence-specific binding (hybridization) on a solid support, specifically when Morpholinos are used as the surface-immobilized probe species in surface-based nucleic acid assays, and the assays as disclosed herein.
摘要:
Use of Morpholinos, a class of uncharged DNA analogues, for surface-hybridization applications. Monolayers of Morpholino probes on gold supports are fabricated with methods similar to those employed with DNA, and are used to hybridize efficiently and sequence-specifically with target strands. Hybridization-induced changes in the interfacial charge organization are analyzed with electrochemical methods and compared for Morpholino and DNA probe monolayers. Molecular mechanisms connecting surface hybridization state to the interfacial capacitance are identified and interpreted through comparison to numerical Poisson-Boltzmann calculations. Positive as well as negative capacitive responses (contrast inversion) to hybridization are possible, depending on surface populations of mobile ions as controlled by the applied potential. Quantitative comparison of surface capacitance with target coverage (targets/area) reveals a near-linear relationship, and demonstrates sensitivities (limits of quantification) in the pg mm−2 range.
摘要:
Surface hybridization, a reaction in which nucleic acid molecules in solution react with nucleic acid partners immobilized on a surface, is widely practiced in life science research. In these applications the immobilized partner, or “probe”, is typically single-stranded DNA. Because DNA is strongly charged, high salt conditions are required to enable binding between analyte nucleic acids (“targets”) in solution and the DNA probes. High salt, however, compromises prospects for label-free monitoring or control of the hybridization reaction through surface electric fields, as well as stabilizes secondary structure in target species that can interfere with probe-target recognition. In this work, initial steps toward addressing these challenges are taken by introducing Morpholinos, a class of uncharged DNA analogues, for surface-hybridization applications. Monolayers of Morpholino probes on gold supports can be fabricated with methods similar to those employed with DNA, and are shown to hybridize efficiently and sequence-specifically with target strands. Hybridization-induced changes in the interfacial charge organization are analyzed with electrochemical methods and compared for Morpholino and DNA probe monolayers. Molecular mechanisms connecting surface hybridization state to the interfacial capacitance are identified and interpreted through comparison to numerical Poisson-Boltzmann calculations. Interestingly, positive as well as negative capacitive responses (contrast inversion) to hybridization are possible, depending on surface populations of mobile ions as controlled by the applied potential. Quantitative comparison of surface capacitance with target coverage (targets/area) reveals a near-linear relationship, and demonstrates sensitivities (limits of quantification) in the pg mm−2 range.
摘要:
Use of Morpholinos, a class of uncharged DNA analogues, for surface-hybridization applications. Monolayers of Morpholino probes on gold supports are fabricated with methods similar to those employed with DNA, and are used to hybridize efficiently and sequence-specifically with target strands. Hybridization-induced changes in the interfacial charge organization are analyzed with electrochemical methods and compared for Morpholino and DNA probe monolayers. Molecular mechanisms connecting surface hybridization state to the interfacial capacitance are identified and interpreted through comparison to numerical Poisson-Boltzmann calculations. Positive as well as negative capacitive responses (contrast inversion) to hybridization are possible, depending on surface populations of mobile ions as controlled by the applied potential. Quantitative comparison of surface capacitance with target coverage (targets/area) reveals a near-linear relationship, and demonstrates sensitivities (limits of quantification) in the pg mm−2 range.
摘要:
An active CMOS biosensor chip for fluorescent-based detection is provided that enables time-gated, time-resolved fluorescence spectroscopy. In one embodiment, analytes are loaded with fluorophores that are bound to probe molecules immobilized on the surface of the chip. Photodiodes and other circuitry in the chip are used to measure the fluorescent intensity of the fluorophore at different times. These measurements are then averaged to generate a representation of the transient fluorescent decay response unique to the fluorophores. In addition to its low-cost, compact form, the biosensor chip provides capabilities beyond those of macroscopic instrumentation by enabling time-gated operation for background rejection, easing requirements on optical filters, and by characterizing fluorescence lifetime, allowing for a more detailed characterization of fluorophore labels and their environment. The biosensor chip can be used for a variety of applications including biological, medical, in-the-field applications, and fluorescent lifetime imaging applications.
摘要:
An active CMOS biosensor chip for fluorescent-based detection is provided that enables time-gated, time-resolved fluorescence spectroscopy. Analytes are loaded with fluorophores that are bound to probe molecules immobilized on the surface of the chip. Photodiodes and other circuitry in the chip are used to measure the fluorescent intensity of the fluorophore at different times. These measurements are then averaged to generate a representation of the transient fluorescent decay response unique to the fluorophores. In addition to its low-cost, compact form, the biosensor chip provides capabilities beyond those of macroscopic instrumentation by enabling time-gated operation for background rejection, easing requirements on optical filters, and by characterizing fluorescence lifetime, allowing for a more detailed characterization of fluorophore labels and their environment. The biosensor chip can be used for a variety of applications including biological, medical, in-the-field applications, and fluorescent lifetime imaging applications.
摘要:
Methods for analysis of interactions between nucleic acid-binding agents (BAs) and nucleic acids (NAs) by performance of nucleic acid denaturation assays on solid supports. Typically, BA is a small molecule less than 1000 g/gmol in molecular weight. The methods provide quantitative thermodynamic and kinetic analysis of BA-NA interaction; for example, in the form of free energies, enthalpies, and entropies of BA-NA binding in case of thermodynamic analysis, or in the form of rate constants and activation energies of BA-NA binding in the case of kinetic analysis. Examples of BAs of interest include transcription regulators and other NA-recognition molecules such as dyes and drug potentiators, DNA-targeted therapeutic agents including anticancer, antibiotic, antiviral, and antitrypanosomal compounds, carcinogens, and any other molecules whose interaction with DNA may, or is suspected to, lead to a biologically-relevant consequence. BA may bind to NA either through physical interactions or through formation of covalent adducts.
摘要:
A method to monitor the progress of hybridization between nucleic acid strands in solution and Morpholino strands immobilized on a solid support such as a working electrode in-situ, in real-time, and using label-free electrochemical measurements sensitive to hybridization-induced changes in the near-surface dielectric constant and charge organization.
摘要:
An active CMOS biosensor chip for fluorescent-based detection is provided that enables time-gated, time-resolved fluorescence spectroscopy. In one embodiment, analytes are loaded with fluorophores that are bound to probe molecules immobilized on the surface of the chip. Photodiodes and other circuitry in the chip are used to measure the fluorescent intensity of the fluorophore at different times. These measurements are then averaged to generate a representation of the transient fluorescent decay response unique to the fluorophores. In addition to its low-cost, compact form, the biosensor chip provides capabilities beyond those of macroscopic instrumentation by enabling time-gated operation for background rejection, easing requirements on optical filters, and by characterizing fluorescence lifetime, allowing for a more detailed characterization of fluorophore labels and their environment. The biosensor chip can be used for a variety of applications including biological, medical, in-the-field applications, and fluorescent lifetime imaging applications.