Single-molecule sensitivity can be nowadays achieved when trying to characterize minute amounts of macromolecules on an environmental or diagnostic specimen in a research laboratory setting. The sensitivity is significantly worse when working in the field and trying to assess the presence of a pollutant or a pathogen as quickly as possible. Point-of-care testing is the realm of biosensors and the trend of research is towards simplicity of use, low cost, reliability and, last but not least, sensitivity. In the detection of nucleic acids, the hybridization of an oligonucleotide probe with its poly-nucleotidic target is monitored by changes in physico-chemical properties of an interface, commonly induced by the use of a specific label. The probe, often bound to an oligonucleotide itself, can be a fluorescent dye, an enzyme, an electroactive moiety or other functional element. Such probes impart specificity and sensitivity to the assay, but make it drift far from simplicity of use (and low cost). Recently, label-free techniques are developing in which the presence of the analyte macromolecule itself can induce detectable physico-chemical changes. The struggle is to make such techniques sufficiently sensitive and specific. In our work, we are trying to implement DNA-based surface-bound amplification strategies that can serve to amplify the read-out signal of DNA hybridization in a label-free biosensor, such as those measuring the electrochemical properties of an electrode interface. The ‘hybridization chain reaction’ is an isothermal enzyme-free strategy to trigger polymerization of oligonucleotides into a long double-stranded DNA. We have demonstrated that the ‘hybridization chain reaction’ can be also implemented in a surface-bound configuration, leading to the self-assembly of many copies of oligonucleotides on a target DNA bound on an oligonucleotide self-assembled monolayer. In another attempt, we have shown that terminal transferase (a template-free DNA polymerase) can be used to build a long polynucleotide out of the target DNA that is bound to its immobilized oligonucleotide probe. Both such strategies will lead to the accumulation of nucleic acids at the solid-liquid interface when triggered by the probe-target recognition. This produces an amplification of the signal of a label-free biosensor. Even though the amplification factors of these implementations are still as low as 10, their further development is still possible, with the hope of avoiding or reducing the need of polymerase chain reaction in the detection of low concentrations of nucleic acids.

DNA self-assembly or enzymatic reactions toward the amplification of biosensor signals

ONOFRI, MANUELE;VINELLI, ALESSANDRA;ZUCCHERI, GIAMPAOLO;SAMORI', BRUNO
2010

Abstract

Single-molecule sensitivity can be nowadays achieved when trying to characterize minute amounts of macromolecules on an environmental or diagnostic specimen in a research laboratory setting. The sensitivity is significantly worse when working in the field and trying to assess the presence of a pollutant or a pathogen as quickly as possible. Point-of-care testing is the realm of biosensors and the trend of research is towards simplicity of use, low cost, reliability and, last but not least, sensitivity. In the detection of nucleic acids, the hybridization of an oligonucleotide probe with its poly-nucleotidic target is monitored by changes in physico-chemical properties of an interface, commonly induced by the use of a specific label. The probe, often bound to an oligonucleotide itself, can be a fluorescent dye, an enzyme, an electroactive moiety or other functional element. Such probes impart specificity and sensitivity to the assay, but make it drift far from simplicity of use (and low cost). Recently, label-free techniques are developing in which the presence of the analyte macromolecule itself can induce detectable physico-chemical changes. The struggle is to make such techniques sufficiently sensitive and specific. In our work, we are trying to implement DNA-based surface-bound amplification strategies that can serve to amplify the read-out signal of DNA hybridization in a label-free biosensor, such as those measuring the electrochemical properties of an electrode interface. The ‘hybridization chain reaction’ is an isothermal enzyme-free strategy to trigger polymerization of oligonucleotides into a long double-stranded DNA. We have demonstrated that the ‘hybridization chain reaction’ can be also implemented in a surface-bound configuration, leading to the self-assembly of many copies of oligonucleotides on a target DNA bound on an oligonucleotide self-assembled monolayer. In another attempt, we have shown that terminal transferase (a template-free DNA polymerase) can be used to build a long polynucleotide out of the target DNA that is bound to its immobilized oligonucleotide probe. Both such strategies will lead to the accumulation of nucleic acids at the solid-liquid interface when triggered by the probe-target recognition. This produces an amplification of the signal of a label-free biosensor. Even though the amplification factors of these implementations are still as low as 10, their further development is still possible, with the hope of avoiding or reducing the need of polymerase chain reaction in the detection of low concentrations of nucleic acids.
proceedings of FUN-M-ARCH FUNctional Multiscale ARCHitectures (European Science Foundation)
M. Onofri; A. Vinelli; G. Zuccheri; B. Samorì
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/96891
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