English:
[0002] Ever increasing attention is being paid to detection and analysis of low concentrations of analytes in various biologic and organic environments. Qualitative analysis of such analytes is generally limited to the higher concentration levels, whereas quantitative analysis usually requires labeling with a radioisotope or fluorescent reagent. Such procedures are generally time consuming and inconvenient.
[0003] Solid-state sensors and particularly biosensors have received considerable attention lately due to their increasing utility in chemical, biological, and pharmaceutical research as well as disease diagnostics. In general, biosensors consist of two components: a highly specific recognition element and a transducing structure that converts the molecular recognition event into a quantifiable signal. Biosensors have been developed to detect a variety of biomolecular complexes including oligonucleotide pairs, antibody-antigen, hormone-receptor, enzyme-substrate and lectin-glycoprotein interactions. Signal transductions are generally accomplished with electrochemical, field-effect transistor, optical absorption, fluorescence or interferometric devices.
[0004] It is known that the intensity of the visible reflectivity changes of a porous silicon film can be utilized in a simple biological sensor, as disclosed in U.S. Pat. No. 6,248,539 to Ghadiri et al. As disclosed therein, the detection and measurement of wavelength shifts in the interference spectra of a porous semiconductor substrate, such as a silicon substrate, make possible detection, identification and quantification of small molecules. While such a biological sensor is certainly useful, detection of a reflectivity shift is complicated by the presence of a broad peak rather than one or more sharply defined luminescent peaks.
[0006] Raman spectroscopy or surface plasmon resonance has also been used seeking to achieve the goal of sensitive and accurate detection or identification of individual molecules from biological samples. When light passes through a medium of interest, a certain amount of the light becomes diverted from its original direction in a phenomenon known as scattering. Some of the scattered light also differs in frequency from the original excitatory light, due to the absorption of light and excitation of electrons to a higher energy state, followed by light emission at a different wavelength. The difference of the energy of the absorbed light and the energy of the emitted light matches the vibrational energy of the medium. This phenomenon is known as Raman scattering, and the method to characterize and analyze the medium or molecule of interest with the Raman scattered light is called Raman spectroscopy. The wavelengths of the Raman emission spectrum are characteristic of the chemical composition and structure of the Raman scattering molecules in a sample, while the intensity of Raman scattered light is dependent on the concentration of molecules in the sample.
[0007] It has been observed that molecules near roughened silver surfaces show enhanced Raman scattering of as much as two orders of magnitude or more. This surface enhanced Raman scattering (SERS) effect is related to the phenomenon of plasmon resonance, wherein metal nanoparticles or metal coatings exhibit a pronounced optical resonance in response to incident electromagnetic radiation, due to the collective coupling of conduction electrons in the metal. In essence, nanoparticles of gold, silver, copper and certain other metals can function to enhance the localized effects of electromagnetic radiation. Molecules located in the vicinity of such particles exhibit a much greater sensitivity for Raman spectroscopic analysis. SERS is the technique to utilize this surface enhanced Raman scattering effect to characterize and analyze biological molecules of interest.
[0008] Sodium chloride and lithium chloride have been identified as chemicals that enhance the SERS signal when applied to a metal nanoparticle or metal coated surface before or after the molecule of interest has been introduced. However, the technique of using these chemical enhancers has not proved sensitive enough to reliably detect low concentrations of analyte molecules, such as single nucleotides or proteins. Only one type of nucleotide, deoxyadenosine monophosphate, and only one type of protein, hemoglobin, have been detected at single molecule level. As a result SERS has not been viewed as suitable for analyzing the protein content of a complex biological sample, such as blood plasma.
[0009] Thus a need exists in the art for a method of biomolecule detection that provides information regarding the characteristics of the bound molecule and for reliably detecting and/or identifying individual molecules using a Raman spectroscopic analytical technique. In addition, there is also a need in the art for quick and simple means of qualitatively and quantitatively detecting biomolecules at low concentration levels.

中文:
[0002]对各种生物和有机环境中低浓度分析物的检测和分析一直受到越来越多的关注。对这样的分析物的定性分析一般限于较高的浓度水平,而定量分析通常要求用放射性同位素或荧光试剂进行标记。此类方法一般耗时而且不方便。
[0003]近来由于它们在化学、生物学和药物研究以及疾病诊断学上日益增长的应用,固态传感器,特别是生物传感器已经得到相当多的关注。一般而言,生物传感器由两个元件组成:高度特异性识别元件和将分子识别结果转化为可定量信号的转换结构。生物传感器已经被发展为检测多种生物分子复合物,包括寡核苷酸对、抗体-抗原、激素-受体、酶-底物和凝集素-糖蛋白相互作用。一般用电化学、场效应晶体管、光学吸收、荧光或干涉装置完成信号转换。
[0004]已知在简单的生物传感器中,可以利用多孔硅薄膜的可见反射率变化的强度,如在Ghadiri等的美国专利号6,248,539中所公开。如在该专利号中所公开,检测和测量多孔半导体基板例如硅基板中干涉光谱的波长位移使得对小分子的检测、鉴定和定量成为可能。尽管这样的生物传感器的确有用,然而,通过宽峰而不是一个或多个清晰界定的发光峰的存在而检测反射率变化是复杂的。
[0006]拉曼光谱法或表面等离子共振技术(surface plasmon resonance)也已经被用于寻求实现灵敏而准确地检测或鉴定生物样品中各个分子的目的。当光经过感兴趣的介质时,一定量的光以一种被称为散射的现象从其最初的方向发生转向。一些散射光也与最初的激发光的频率不同,原因在于光的吸收以及电子激发到较高的能态,随后在不同波长下的光发射。吸收光的能量和发射光的能量的差异与介质的振动能匹配。这种现象被称为拉曼散射,用拉曼散射光表征并分析感兴趣的介质或分子的方法被称为拉曼光谱法。拉曼发射光谱的波长是样品中拉曼散射分子的化学组成和结构的特征,而拉曼散射光的强度取决于样品中分子的浓度。
[0007]已经观察到,在粗糙的银表面附近的分子显示了增强的拉曼散射,多达两个或更多数量级。这种表面增强拉曼散射(SERS)效应与等离子共振的现象有关,其中金属纳米颗粒或金属涂层响应于入射电磁辐射(incident electromagnetic radiation),显示出显著的光学共振,原因在于金属中传导电子的并合耦合(collective coupling)。本质上,金、银、铜和某些其它金属的纳米颗粒可以起着增强电磁辐射的局部化效应(localized effect)作用。位于此类颗粒附近的分子对拉曼光谱分析表现出更大的灵敏度。SERS是利用这种表面增强拉曼散射效应表征和分析感兴趣生物分子的技术。
[0008]在引入感兴趣的分子之前或之后,当氯化钠和氯化锂被应用于金属纳米颗粒或金属涂敷表面时,将它们鉴定为增强SERS信号的化学品。然而,使用这些化学促进剂的技术没有证明对可靠地检测低浓度分析物分子例如单核苷酸或蛋白质是足够灵敏的。在单分子水平下,只有一种核苷酸,即脱氧腺苷一磷酸,和只有一种蛋白质,即血红蛋白被检测。因此,SERS不被认为适于分析复杂生物样品例如血浆的蛋白质含量。
[0009]因此,在本领域,需要一种提供所结合分子特征的相关信息的生物分子检测方法,并对使用拉曼光谱分析技术可靠地检测和/或鉴定单个分子存在需求。另外,在本领域,也对定性和定量检测低浓度水平下生物分子的快速而简单的方法存在需求。