公开(公告)号：US08273578B2

公开(公告)日：2012-09-25

申请号：US13423228

申请日：2012-03-18

申请人： Rastislav Levicky ,  Napoleon Tercero ,  Kang Wong ,  Ping Gong ,  Kenneth Shepard

发明人： Rastislav Levicky ,  Napoleon Tercero ,  Kang Wong ,  Ping Gong ,  Kenneth Shepard

IPC分类号： G01N33/50

CPC分类号： C12Q1/6825 ,  Y10T436/143333 ,  C12Q2525/113

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.

摘要翻译： 使用Morpholinos，一类不带电荷的DNA类似物，用于表面杂交应用。 使用类似于DNA使用的方法制备金支持物上的Morpholino探针的单层，并且用于与靶链有效地和序列特异性地杂交。 用电化学方法分析杂交诱导的界面电荷组织变化，并比较Morpholino和DNA探针单层。 通过与数值泊松 - 玻尔兹曼计算进行比较，可以确定和解释连接表面杂交态与界面电容的分子机制。 取决于由施加电位控制的移动离子的表面种群，对于杂交，阳性以及负电容反应（对比反转）是可能的。 表面电容与目标覆盖（目标/面积）的定量比较显示近似线性关系，并显示pg mm-2范围内的灵敏度（定量限度）。

公开(公告)号：US20100187133A1

公开(公告)日：2010-07-29

申请号：US12362071

申请日：2009-01-29

申请人： Rastislav Levicky ,  Napoleon Tercero ,  Kang Wang ,  Ping Gong ,  Kenneth Shepard

发明人： Rastislav Levicky ,  Napoleon Tercero ,  Kang Wang ,  Ping Gong ,  Kenneth Shepard

IPC分类号： G01N27/26

CPC分类号： C12Q1/6825 ,  Y10T436/143333 ,  C12Q2525/113

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.

摘要翻译： 表面杂交，其中溶液中的核酸分子与固定在表面上的核酸配偶体反应的反应在生命科学研究中广泛应用。 在这些应用中，固定化的配偶体或“探针”通常是单链DNA。 由于DNA是强电荷的，因此需要高盐条件才能使溶液中的分析物核酸（“靶”）与DNA探针结合。 然而，高盐不利于通过表面电场进行无标记监测或控制杂交反应的前景，以及稳定可能干扰探针 - 目标识别的靶物种中的二级结构。 在这项工作中，通过引入Morpholinos（一类不带电荷的DNA类似物）进行表面杂交应用来采取解决这些挑战的最初步骤。 可以用与DNA一起使用的方法类似的方法制备金载体上的Morpholino探针的单层，并且显示与靶链有效地并且序列特异性地杂交。 用电化学方法分析杂交诱导的界面电荷组织变化，并比较Morpholino和DNA探针单层。 通过与数值泊松 - 玻尔兹曼计算进行比较，可以确定和解释连接表面杂交态与界面电容的分子机制。 有趣的是，取决于由施加电位控制的移动离子的表面种群，杂交的阳性和负电容反应（对比反转）是可能的。 表面电容与目标覆盖（目标/面积）的定量比较显示近似线性关系，并显示pg mm-2范围内的灵敏度（定量限度）。

公开(公告)号：US20120211375A1

公开(公告)日：2012-08-23

申请号：US13423228

申请日：2012-03-18

申请人： Rastislav Levicky ,  Napoleon Tercero ,  Kang Wang ,  Ping Gong ,  Kenneth Shepard

发明人： Rastislav Levicky ,  Napoleon Tercero ,  Kang Wang ,  Ping Gong ,  Kenneth Shepard

IPC分类号： G01N33/50 ,  G01N27/26

CPC分类号： C12Q1/6825 ,  Y10T436/143333 ,  C12Q2525/113

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.

摘要翻译： 使用Morpholinos，一类不带电荷的DNA类似物，用于表面杂交应用。 使用类似于DNA使用的方法制备金支持物上的Morpholino探针的单层，并且用于与靶链有效地和序列特异性地杂交。 用电化学方法分析杂交诱导的界面电荷组织变化，并比较Morpholino和DNA探针单层。 通过与数值泊松 - 玻尔兹曼计算进行比较，可以确定和解释连接表面杂交态与界面电容的分子机制。 取决于由施加电位控制的移动离子的表面种群，对于杂交，阳性以及负电容反应（对比反转）是可能的。 表面电容与目标覆盖（目标/面积）的定量比较显示近似线性关系，并显示pg mm-2范围内的灵敏度（定量限度）。