NanoBioSensor Initiative

DNA微阵列是在转录水平上高通量监测基因表达、确定全基因组DNA拷贝数变化、识别转录因子的靶标、测序以及最近用于分析癌症中微小RNA(MiRNA)水平的强大工具。第一个报道的DNA微阵列是用cDNA克隆在尼龙膜上制作的，并使用放射性标记的靶标进行检测。从那时起，许多大规模的DNA微阵列平台相继问世，其中包括双链cDNA、单链短25聚体(Affymetrix)、中型30聚体(CombiMatrix)或长50-70聚体(NimbleGen或Agilent)寡核苷酸。所有这些方法都依赖于各种组合的核酸酶扩增和目标的荧光标记、杂交和信号放大，然后由光学扫描仪进行检测。虽然基于荧光的DNA微阵列技术已经取得了重大进展，但这些方法往往很耗时，而且依赖于荧光强度的测定，因此灵敏度受到检测少量光子的能力的限制。为了克服这些障碍，我们提出了一种基于晶体管的高灵敏度系统，用于核酸(NA)杂交的电子检测。这项技术类似于微阵列的概念，即每个晶体管都与一个不同的寡核苷酸相关联，预计它将在灵敏度、易用性和小型化能力方面远远超过现有技术。另一个显著的优点是，所提出的方法不需要对被检测的核酸进行化学或酶处理。在场效应晶体管中，源极和漏极之间的电流-电压特性或跨导强烈地依赖于在基极(B)端子上累积的总电荷。这种效应是可以很好地理解的，人们可以通过测量跨导曲线来准确地估计基极的电荷量。在基极没有束缚电荷的情况下，如第一块图所示，在任何电压下都不会有电流流动，因此跨导曲线会画出一条水平线。在中间的图中，我们假设单链DNA寡聚体固定在碱基上。这将导致来自磷酸盐骨架的净负电荷在碱基上积累。由此产生的跨导曲线将表现出轻微但可测量的偏离水平线性。最后，如果单链DNA与其互补序列杂交，碱基上的电荷将净加倍。这将驱动晶体管进入正向偏置的全工作状态，并将产生大幅度和高度非线性的跨导曲线。将开发的主要方法是利用碳纳米管(CNT)的晶体管。同时，将与美国宇航局和天主教大学的斯蒂芬妮·盖蒂博士和冈特·克莱茨卡博士合作进行一项初步研究，以开发硅纳米线(SiNW)晶体管。两者的工作原理都非常类似于硅基金属氧化物场效应晶体管(MOSFET)。值得注意的是，研究表明，碳纳米管具有比硅更高的本征迁移率，这导致了作为晶体管使用时的电荷灵敏度要高得多。与传统MOSFET中使用的平面(平板)几何结构相比，这些纳米材料(圆柱体)的几何形状可以更好地聚焦电场，从而增加了纳米器件的灵敏度。然而，碳纳米管系统带来了挑战，如电子性质的纳米管之间的差异和高温加工，这些可以通过使用SiNW来缓解。

DNA microarrays are powerful tools for high throughput monitoring of gene expression at the transcription level, determining genome wide DNA copy number changes, identifying targets of transcription factors, sequencing and more recently for profiling the micro RNA (miRNA) levels in cancer. The first reported DNA microarray was fabricated on nylon membranes using cDNA clones and used radioactively labeled targets for detection. Since then, many large-scale DNA microarray platforms have been developed, which includes, double-stranded cDNA, single stranded short 25mers (Affymetrix), mid-sized 30mer (Combimatrix) or long 50-70mers (Nimblegen or Agilent) oligonucleotides. All these methods rely upon various combinations of enzymatic amplification of the nucleic acid and fluorescently labeling of targets, hybridization, and amplification of signal followed by detection by optical scanners. While significant strides have been made in fluorescent-based DNA microarray technology, the methodologies are often time-consuming and in addition rely on the determination of fluorescence intensity and the sensitivity is thus limited by the ability to detect small numbers of photons. To overcome these barriers, we propose a highly sensitive system, based on transistors for the electronic detection of nucleic acid (NA) hybridization. The technique is analogous to the microarray concept, in which each transistor is associated with a distinct oligonucleotide, and is projected to far surpass existing technology in sensitivity, ease of use and in the capability toward miniaturization. Another significant advantage is that the method proposed does not require chemical or enzymatic manipulation of the nucleic acid being detected. In field effect transistors, the current versus voltage characteristics or transconductance between the source and drain electrodes are strongly dependent upon the total charge accumulated at the base (B) terminal. This effect is well understood and one can accurately estimate the amount of charge at the base from measurement of the transconductance curve. In the absence of bound charges at the base, as shown in the first panel, no current will flow at any voltage so that the transconductance curve traces a horizontal line. In the middle panel, we assume that single stranded DNA oligomers are immobilized at the base. This will cause a net negative charge from the phosphate backbone to accumulate at the base. The resulting transconductance curve will exhibit slight but measurable departure from horizontal linearity. Finally, if the single stranded DNA were hybridized with its complementary sequence, a net doubling of charge will occur at the base. This will drive the transistor into forward biased full operation and will produce large amplitude and highly nonlinear transconductance curves. The primary method that will be developed are transistors that utilize carbon nanotubes (CNTs). In parallel, a pilot study will be performed in collaboration with Dr. Stephanie Getty and Dr Gunter Kletetschka (NASA and Catholic University) to develop silicon nanowire (SiNW) transistors. Both operate very similar to silicon based metal oxide field effect transistors (MOSFETS). Notably, it has been shown that CNTs have a higher intrinsic mobility than silicon, leading to much higher charge sensitivity when used as a transistor. The geometry of these nanomaterials (cylinders) can better focus electric field than the planar (planks) geometry used in conventional MOSFETs, adding to the sensitivity of the nanodevice. The CNT system, however, presents challenges such as inter-nanotube variation in electronic properties and high temperature processing, which can be mitigated using SiNWs.