Nanodevices

The greatest utility of the biomarker detection nanodevices discussed below is the ability to have real-time, selective, multiplexed, and tag-free detection of biomolecules in small sample volumes. The nanoscale of these devices also increases the portability and reduces costs associated with larger reagent volumes used in conventional assays.

Nanowires

Nanowires are nanoscale sensing wires that can be functionalized to bind proteins of interest and have great multiplexing potential [64] . When a bio-molecule of interest binds a nanowire, there is a drop in its conductance. The ability of nanowires to act like nanoscale field-effect biotransistors allows their use as real-time sensors to multiple binding events, as illustrated in Figure 4. Also shown in Figure 4 is a scanning electron image of a single silicone nanow-ire between two electrodes.

Nanowires made from silicon are advantageous due to their ability to be easily tuned and surface modified for specific biomolecule detection. Hahm and Lieber [65] utilized silicon nanowires for the specific detection of a DNA mutation site for cystic fibrosis transmembrane receptor. The nanowire construct allowed real-time detection and was ultrasensitive, detecting the mutation in fentomole concentrations. Hahm and Lieber also found high device - to- device reproducibility.

Simultaneous multiplexed detection of four cancer biomarkers in fentomo-lar concentrations from undiluted serum samples was achieved by Zheng et al. [66]. Real-time detection results for a multiplexed silicon nanowire

Figure 4 Nanowire detection of a single-target biomolecule using conductive-based measurements. A biomolecule is immobilized selectively by antibody interaction, causing a change in conductance, recorded on the right. Inset: SEM image of a single silicon nanowire (scale bar = 500nm). (From refs. 100 and 67, with permission.) (See insert for color reproduction of the figure. )

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Figure 4 Nanowire detection of a single-target biomolecule using conductive-based measurements. A biomolecule is immobilized selectively by antibody interaction, causing a change in conductance, recorded on the right. Inset: SEM image of a single silicon nanowire (scale bar = 500nm). (From refs. 100 and 67, with permission.) (See insert for color reproduction of the figure. )

device designed to detect three biomarkers is shown in Figure 5. The sensitivity of the nanowire device was demonstrated further by the same group when they detected binding, activity, and inhibition of telomerase from unamplified extracts of as few as 10 cells. Detection is as sensitive as current telomere repeat application protocol (TRAP) assays but does not require the use of polymerase chain reaction (PCR) amplification and labeling.

Silicon nanowires were also used by Wang et al. [67] to measure the ATP binding (and nonbinding) to an immobilized leukemia biomarker, Abl. Although this work also demonstrates the ability of nanowires for biomarker detection, the group went a step further in utilizing the device to measure the biomarker response to known inhibitors, lending potential to drug discovery

Figure 5 Multiplexed detection of cancer marker proteins. (a) Multiplexed protein detection by three silicon-nanowire devices in an array. Devices 1, 2, and 3 are fabricated from similar nanowires and then differentiated with distinct mouse antibody receptors specific to three different cancer markers. (b) Conductance versus time data recorded for the simultaneous detection of PSA, CEA, and mucin-1 on p-type silicon-nanowire array in which NW1, NW2, and NW3 were functionalized with mouse antibodies for PSA, CEA, and mucin-1, respectively. The solutions were delivered to the nanowire array sequentially as follows: (1) 0.9ng/mL PSA, (2) 1.4pg/mL PSA, (3) 0.2ng/mL CEA, (4) 2pg/mL CEA, (5) 0.5ng/mL mucin-1, (6) 5pg/mL mucin-1. Buffer solutions were injected following each protein solution at points indicated by black arrows. (From ref. 66, with permission.) (See insert for color reproduction of the figure. )

Figure 5 Multiplexed detection of cancer marker proteins. (a) Multiplexed protein detection by three silicon-nanowire devices in an array. Devices 1, 2, and 3 are fabricated from similar nanowires and then differentiated with distinct mouse antibody receptors specific to three different cancer markers. (b) Conductance versus time data recorded for the simultaneous detection of PSA, CEA, and mucin-1 on p-type silicon-nanowire array in which NW1, NW2, and NW3 were functionalized with mouse antibodies for PSA, CEA, and mucin-1, respectively. The solutions were delivered to the nanowire array sequentially as follows: (1) 0.9ng/mL PSA, (2) 1.4pg/mL PSA, (3) 0.2ng/mL CEA, (4) 2pg/mL CEA, (5) 0.5ng/mL mucin-1, (6) 5pg/mL mucin-1. Buffer solutions were injected following each protein solution at points indicated by black arrows. (From ref. 66, with permission.) (See insert for color reproduction of the figure. )

and drug efficacy research. Detection in the nanomolar range and in a concentration-dependent manner (allowing quantification) make this sensitive method tenable for drug discovery and efficacy studies for any of the tyrosine kinases responsible for many cancers and diseases. Silicon nanowires have been developed for a number of other applications not discussed here, including pH sensing [64] and virus detection [68].

Nanocantilevers

Nanocantilevers are a promising new approach to multimolecular sensing. Nanocantilever arrays, illustrated in Figure 6, consist of many nano- diving boardlike beams that can have antibodies attached covalently to their surface. When a biomolecule of interest binds, the beam bends and the deflection can be detected by either laser light observation or changes in resonant-vibration r

Tumor biomarker proteins »

Tumor biomarker proteins »

Bent cantilever

Figure 6 Nanocantilever array. The biomarker proteins are affinity bound to the cantilevers and cause them to deflect. The deflections can be observed directly with lasers. Alternatively, the shit in resonant frequencies caused by the binding can be detected electronically. (From ref. 1, with permission.) (See insert for color reproduction of the figure. )

Bent cantilever

Figure 6 Nanocantilever array. The biomarker proteins are affinity bound to the cantilevers and cause them to deflect. The deflections can be observed directly with lasers. Alternatively, the shit in resonant frequencies caused by the binding can be detected electronically. (From ref. 1, with permission.) (See insert for color reproduction of the figure. )

frequency [69-71] , The utility of cantilever detection has been demonstrated by multiplexed DNA assays to detect BRCA1 mutations, indicative of early breast cancer onset [72]. Additionally, nanocantilevers were able to detect and quantitate prostate-specific antigen in clinically significant concentrations [70] .

A nanomechanical cantilever array was used by Arntz et al. [ 2] for the multiplexed detection of the cardiac biomarkers creatin kinase and myoglo-bin. Both biomarkers could be detected at micromolar concentrations from plasma samples. Further development of this system is being investigated for the early and rapid diagnosis of myocardial infarction.

Nanomechanical cantilever detection systems are not limited to the detection of biomarkers due to hybridization and antibody interactions. Environmentally sensitive polymers coated onto nanocantilevers can also produce detectable cantilever stresses in response to sample chemistries. For instance, pH-sensitive poly(methacrylic acid) coated onto nanocantilevers were used as highly sensitive, small-volume pH sensors [73]. As new environmentally responsive polymers are developed, this application may grow rapidly. Similarly, analyte vapors in a gas phase resulted in a changed surface stress of nanocantilevers in a device termed an artificial nose [74].

Nanoarrays

Currently, microarrays are highly utilized in research and the clinic for molecular diagnostics, genotyping, and biomarker-guided therapy. Development of nanoarrays will advantageously advance current uses by allowing higher degrees of multiplexing, higher specificity, more portability, and reduced cost associated with sample and reagent volumes. Advanced methods have already

Figure 7 SEM images of arrays of multiwalled carbon nanotubes at (a) Ultraviolet lithography and (b) e-beam patterned Ni spots. Panels are 45 ° perspective views with scale bars of (a) 2 and (b) 5 |im. (From ref. 79, with permission.)

been developed for the fabrication of nanoarrays for proteomic profiles and diagnostics [75-78].

Carbon nanotube electrode arrays were used by Li et al. [79] for ultrasensitive DNA detection. Scanning electron images of the arrays (Figure 7) demonstrate the precision and versatility in the fabrication of carbon nanotube electrode arrays. Nanoelectrodes are highly desirable, due to the fact that electrode performace in terms of speed and spatial resolution scale inversely with electrode radius. Oligonucleotides covalently bound to the terminus of the carbon nanotubes were able to detect target DNA sequence hybridization at concentrations of only a few attamoles. The specificity and versatility of carbon nanotube electrode arrays allow statistically significant, multiplexed detection of biomolecules in clinically relevant concentrations.

A carbon nanotube-based array sensor was employed to detect glucose by binding glucose oxidase to the terminus of the carbon nanotubes [80]. Direct electron transfer from the enzyme through the carbon nanotube and to a platinum transducer allowed real-time sensitive detection of the reaction and glucose presence. This technology may be expanded for the detection of many other biomarkers with electron-producing enzymatic reactions.

Gold nanoarrays with immobilized anti- Escherichia coli antibodies have been developed for the early detection of bacteria during human kidney infections [81]. A novel aspect of this work was that the antibodies were all arranged in a post configuration as opposed to a random configuration used in most studies. This allowed for the detection of bacteria in concentrations two orders of magnitude below the detection limits of current methods. Additionally, the rapid detection method eliminated the need to run bacterial cultures overnight, as is a common practice in the clinic.

Other Novel Nanodevices for Biomarker Detection

Although the majority of research into nanodevices for biomarker detection fall into the three categories discussed above (nanowires, nanocantilevers, and nanoarrays), a plethora of other novel detection devices have been investigated, as discussed here briefly. Additionally, nanotechnology methods contribute to biomarker detection in a general sense, such as the ability to make precise nanoscale pores and channels for utilization of picoliter volumes [82-84].

In addition to their application to nanowires and nanoarrays, carbon nano-tubes can be utilized as nano-biomarker probes for the precise detection of specific genes following a DNA hybridization assay [85] . The use of carbon nanotubes as probes has great potential, due to their intense Raman scattering (making it possible to detect a single nanotube) [86,87] and near-infrared fluorescence [88-91].

Nanotube-based sensors were first developed for the detection of NO2 and NH3 gas [ 92] but have since become more sophisticated to allow sensitive detection of biomarkers. Surface-modified carbon nanofiber electrodes have been developed for small-volume detection of glucose [93-95] and are being developed for glutamate and lactate [93]. Carbon nanoelectrodes coupled with oxidases may also be utilized for sensitive detection of cholesterol, alcohol, lactate, acetylcholine, choline, hypoxanthine, and xanthine from a variety of biological fluids [95]. Nanotubes have also been developed as sensors for autoimmune disease [96] , single-nucleotide polymorphisms (SNPs) [97] , and for reversible, small-volume pH detection [94] .

Nanogap actuators have been proposed for the use of protein detection [ 98]. Immobilized antibodies in a nanogap actuator enable protein- binding specificity. Once bound, the target protein can be detected based on rigidity measurements yielding information on protein presence, concentration, and size.

The diverse range of nanodevices discussed above will address key issues in biomarker research, including multiplexing ability, specificity, real - time detection, and tag-free detection. As a result, they will greatly aid diagnostics, treatment monitoring, and drug discovery.

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