Applications and Mechanisms for Biological Sensing 31 Single CellSingle Molecule Probes

The most direct application of CNTs/NWs for biological sensing is to use them as single probes, which gives great spatial resolution. With the small size, such probes can be inserted into a single cell for in situ measurements with minimum disturbance as well as ultrahigh sensitivity. Vo-Dinh et al. (74) demonstrated antibody-based nanobiosensors for the detection of benzo[a] pyrene tetrol (BPT), a biomarker for human exposure to the known carcinogen benzo[a] pyrene, by simply pulling an optical fiber to nanometer sizes at the tip. The distal end was covalently coated with anti-BPT antibodies through silane linkers. The nanobiosensors were inserted into individual cells, incubated for 5 min to allow antigen-antibody binding, and then removed for detection. The small size of the tips of these optical fiber nanosensors gives them several advantages over normal probes, including decreased response time and increased sensitivity. Such a nanobiosensor, based on fluorescence spectroscopy, shows a sensitivity down to approx 1.0 x 1010 M for BPT (74,102) and an absolute limit of detection for BPT of approx 300 zeptomol (10-21 mol) (103).

With the recent development in CNTs and NWs, such wirelike nanomaterials can be adapted for similar techniques. In particular, ZnO NWs have been found with nanolasing properties along the axial direction (40,104), which could be explored for optical-biomolecular detection. CNTs or metal NWs, on the other hand, can be used as nanoelectrodes (75) to measure the electrochemical reaction in a single cell or the reactivity of a single molecule. The electrical signal has the potential to reach single molecular sensitivity, by using a nano-electrode (87).

Single CNTs attached to a scanning probe microscope tip have also attracted intensive interest owing to their small diameter, high aspect ratio, large Young's modulus, and mechanical robustness (24). They can be used as physical probes to obtain the highest resolution in studying the structure of macro-molecules or cell surfaces. Li et al. (26) demonstrated the method for implementing and characterizing CNTs attached to a scanning probe microscope (SPM) tip for in situ imaging of DNA molecules on a mica surface within a buffer solution. They used a magnetically driven oscillating probe atomic force microscope with a silicon nitride cantilever (spring constant of k = 0.1) driven at a frequency of approximately kHz. A bundle of MWCNTs was attached to the pyramidal tip with an acrylic adhesive. The diameter of the bundles ranged from tens to hundreds of nanometers. Typically, a single MWCNT extended out and was used as a probe for SPM imaging. X DNA molecules were spontaneously bound to the surface from 2 ^g/mL solutions with the presence of approx 1 mM MgCl2 for enhancing the DNA/mica interaction. Figure 4 shows a SPM image of DNA molecules in a 2.3 x 2.3 ^m2 area. Single DNA molecules were clearly resolved. The resolution of DNA images is very uniform and consistent with the diameter of the CNT tip (approx 5 nm). SWCNT tips could provide even higher resolution (25,91). The double-helix structure of DNA may be resolved in the future by SPM techniques using SWCNT tips. The tip can also be functionalized to provide additional information about chemical force (25,91).

Woolley et al. (105) reported the direct haplotyping of kilobase-size DNA using SWCNT SPM probes. The haplotype of a subject—the specific alleles associated with each chromosome homolog—is a critical element in single-nucleotide polymorphism (SNP) mapping that leads to a greater comprehension of the genetic contribution to risk of common diseases such as cancer and heart disease. However, current methods for determining haplotypes have significant limitations that have prevented their use in large-scale genetic screening. For example, molecular techniques for determining haplotypes, such as allele-specific or single-molecule polymerase chain reaction (PCR) amplification, are hampered by the need to optimize stringent reaction conditions and the potential for significant error rates. Using SPM with high-resolution SWCNT probes, multiple polymorphic sites can be directly visualized by hybridizing specifically labeled oligonucleotides with the template DNA frag-

Fig. 4. SPM image of DNA molecules on 2.3 x 2.3 |im2 mica surface submerged under 1 mM MgCl2 buffer solution obtained with a single MWCNT tip.

ments of approx 100 to 10,000 bases. The positions of streptavidin and IRD800 labels at two sequences in M13mp18 were demonstrated. The SWCNT tips, with tip radii <3 nm and resolution of approx 10 base, made it possible for high-resolution multiplex detection to differentiate between labels such as streptavidin and the fluorophore IRD800 based on their size. This concept has been further applied to the determination of haplotypes on the UGT1A7 gene (105), which is under study for its role in cancer epidemiology. Direct haplotyping using SWCNT SPM probes represents a significant advance over conventional approaches and could facilitate the use of SNPs for association and linkage studies of inherited diseases and genetic risk (106).

The electronic properties of biomolecules such as DNAs have been extensively studied in the past few years owing to their potential for single molecular sensing. However, DNA molecules were found to be not very conductive even in the duplex form. There are also difficulties in assembling them into reliable devices. Williams et al. (92) reported on a study to couple SWCNTs covalently with PNA (an uncharged DNA analog). Hybridization of DNA fragments to the PNA sequence was measured with an atomic force microscope under ambient conditions and indicated that the functionalization is specific to the open ends of SWCNTs with rare attachment to the sidewall. PNA was chosen owing to its chemical and biological stability. The uncharged PNA backbone gives rise to PNA-DNA duplexes that are more thermally stable than their DNA-DNA counterparts because there is no electrostatic repulsion. This method unites the unique properties of SWCNTs with the specific molecular recognition features of DNA, which may provide new means to incorporate SWCNTs into larger electronic devices by recognition-based assembly as well as biological sensing.

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