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1 Encyclopedia of Nanotechnology Springer Science+Business Media B.V / _120 Bharat Bhushan Nanogap Biosensors J. Tanner Nevill 1 and Daniele Malleo 1 (1) Fluxion Biosciences, 384 Oyster Point Boulevard, Suite 6, South San Francisco, CA, USA J. Tanner Nevill (Corresponding author) tanner.nevill@gmail.com Daniele Malleo d.malleo@gmail.com Without Abstract Definition A nanogap biosensor is an arrangement of two electrodes separated by no more than 300 nm that is used to electrically detect biologically relevant materials, reactions, or interactions in solution. The limit of 300 nm is imposed because it represents the practical upper limit of the characteristic thickness of the electrical double layer, which forms at all charged surfaces in aqueous solutions. Overview Nanogap biosensors are made using various micro- and nano-fabrication techniques to create a nanometer-scale detection region. This region is interrogated using electrical techniques to sense and measure the presence or activity of a biomolecule. All-electrical measurements offer the promise of low cost, rapid, and label-free detection. Many arguments are made for using nanogap biosensors, including higher sensitivity, reduction of parasitic effects, overlapping double layers, and the use of molecular-sized cavities. Liang and Chou use a nanogap electrode arrangement inside of a nanofluidic channel like a pore for detecting (and eventually sequencing) single strands of DNA [1]. Im et al. exploit the fact that a molecule can occupy a significant portion of the nanogap, thereby making it very sensitive to low numbers of molecules: By functionalizing the surface of the nanogap, analyte binding significantly changes the electrical characteristic of the sensing electrodes [2]. The electrical double layer, a cloud of counterions that balances out the charge present on any surface in solution, is often seen as a parasitic component of electrical measurements: This layer shields the bulk solution from the induced electric field thus decreasing the sensitivity of alternating current detection techniques. Because nanogaps have dimensions similar or smaller than the double layer thickness, the electric potential in a nanogap distributes differently than in a macroscale gap. In fact, nanogap devices typically create environs where the volume of interest consists exclusively of double layer regions, actually eliminating the bulk solution from the measurement. Elimination of the bulk response is desirable in many situations, where 1 of 13 8/31/12 9:42 AM

2 measurement and quantification of very few or even single molecule events is of interest, making the concept of overlapping double layers in nanogaps very compelling. The sensors developed thus far can be classified by the geometry of the detection region. According to the number of dimensions along which the sensing cavity exists, there are three categories as depicted in Fig. 1: 1D nanogaps refer to point gap junctions, 2D devices consist of coplanar electrodes or fractured planes, and 3D nanogaps are nanocavities, where the electrode surfaces forming the cavity have a significant area relative to the gap length. Nanogap Biosensors, Fig. 1 Definition of the three categories of nanogap geometry. Form follows function and each geometry is typically tuned for a different function As form follows function, the measurement techniques and applications can be correlated with the geometry. 1D and 2D nanogaps are typically interrogated with DC voltages to produce a resistive measurement, while 3D nanogaps are mostly interrogated using AC measurement techniques, to measure a complex impedance response. Consequently, the discussion of nanogap biosensors is divided below into two sections. One- and Two-Dimensional Nanogap Biosensors Advantages and Disadvantages The key advantage of one- and two-dimensional nanogaps as biosensors is the opportunity for surface-bound molecules to occupy a significant portion of the interelectrode spacing, thereby increasing sensitivity, even enabling single molecule detection [3]. The main drawback of these sensors is the difficulty in ensuring specificity in the measurements. For this reason, the electrodes in the device need to have been functionalized appropriately before 2 of 13 8/31/12 9:42 AM

3 sample introduction, to enable the determination of presence or absence of specific molecules quickly and with off-the-shelf electrical meters: IV (current voltage) meters, electrochemical measurement stations, or patch clamp amplifiers. From a fabrication point of view, while it is in principle very easy to fabricate one-dimensional nanogap electrodes by electromigration, repeatability of this process can be an issue; conversely, fabrication via electron beam lithography allows for extreme control and repeatability but has slower throughput and higher cost. Methods of Fabrication One-dimensional nanogap structures are most commonly realized using electromigration, electron-beam lithography, or a combination of the two techniques. In some cases to shrink gaps to nanometer dimensions, methods such as surface-catalyzed metal deposition or dielectrophoretic transport of metal nanoparticles are used. The latter methods are advantageous as they enable fabrication of many nanogap electrodes at once. On the other hand, electron beam lithography offers the elegant advantage of directly writing nanoscale features in photoresist, which, incorporated with ion milling, lift-off, or unique metal deposition techniques, allows for the creation of nanogap electrodes. Electromigration is an intriguing technique, because it was an originally undesirable effect often encountered in the electronics industry. As metal lines got smaller and smaller, the relatively higher current densities would cause breaks to occur. These breaks were a result of a momentum transfer between the electrons comprising current and the atoms in the metal lattice [4]. Breaks tended to happen at grain boundaries or along defects in the lattice, and were highly dependent on temperature. Researchers interested in molectronics (a field of study whereby individual molecules are used to perform electronic functions) recognized that this technique could be used to create nanogap electrode junctions. This method offers the ability to get very small point gaps in an inexpensive manner, but there is a notable lack of control. Numerous groups have made strides in creating feedback control for electromigrated nanogap devices in order to reliably control the gap sizes in addition to offering parallel fabrication methods [5]. Another method is the chemical deposition of metal or electroplating which allows for the creation of nanogap devices using traditional fabrication methods that would not normally be capable of nanoscale dimensions. Lastly, a few groups have used either mechanical manipulation of electrodes to create nanogap devices or have mechanically broken wires to create nanoscale gaps [6]. Two-dimensional nanogap devices are similarly realized using metal deposition over a sacrificial linear feature to create a linear nanogap. Oblique metal deposition is a simple way to create a metal overhang in order to form a nanogap [7]. Similar to one-dimensional nanogap sensors, electron beam lithography is a suitable alternative for 2D nanogap fabrication as well as the use of sacrificial layers [8]. Measurement Methods Direct Current Measurements The most elementary way of acquiring data from a nanogap biosensor is to apply a DC (direct current) potential, while measuring current flow through the sensor electrodes: When the gap is open, no current flows through except for a small tunneling current. When a biomolecule, or other sample, is attached to both the electrodes or otherwise closes the circuit, a larger direct current starts to flow. Specificity in the measurement can be attained by appropriate functionalization of the electrodes surface. In the case of measurements carried out in solution a background current is continuously measured due to ion transport in the solution. Sample attachment to the electrodes is not 3 of 13 8/31/12 9:42 AM

4 necessary, and just proximity of charged species (such as DNA molecules) can be detected by measuring increases in current. Ohm s law,, is in most cases sufficient to interpret the data acquired. The tunneling current by itself can be used to characterize the device and estimate the distance between two electrodes, if the gap is too small to be detected by optical or electron microscopy. The equation describing the tunneling current between two electrodes is: (1) where,,,, A is the emission area in cm 2, s is the electrode separation or distance in Å, ϕ is the barrier height, and V is the voltage in Volts [9]. Current Voltage (IV) Measurements To determine the behavior and characteristics of the molecule under study, or the sensor itself, DC potential can be swept in incremental steps from a negative to a positive potential and the resultant current traces drawn: Asymmetries and/or nonlinearities in the measured IV traces can be used to extrapolate characteristics of the sample under study. Three-Dimensional Nanogap Biosensors Advantages and Disadvantages Three-dimensional nanogap devices are significantly different from one- and two-dimensional nanogaps in fabrication methods, measurement techniques, and in many cases, applications. 3D nanogaps contain a significant volume relative to the nanogap width. This nanocavity allows for both surface bound and free molecules to be monitored. Alternating current measurement techniques are typically used (e.g., dielectric or impedance spectroscopy), which provide data from which meaningful biological parameters can be extrapolated (e.g., binding efficiency of biomolecules, hydrodynamic radius of proteins). The key advantages of nanogap biosensors using impedance detection techniques (e.g., impedance spectroscopy) are a homogeneous interrogation electric field and the opportunity for surface-bound molecules to occupy a significant portion of the detection volume, thereby increasing sensitivity. The disadvantages of these devices include complex fabrication, difficulty in coupling to external equipment without introducing parasitic components, and the need for on-sensor reference electrodes and controls. Methods of Fabrication Three-dimensional nanogap devices are almost exclusively fabricated by provisionally incorporating a sacrificial layer of nanoscale dimensions, which is used to separate two conducting (metal or semiconductor) electrodes. When this sacrificial layer is removed or etched away, in the 4 of 13 8/31/12 9:42 AM

5 final steps of the fabrication protocol, a nanocavity is created. A common technique is to use a thermally grown oxide layer, which creates a precise thickness by tightly controlling growth conditions. This layer can then later be fully or partially removed with a wet etch. The materials used to form the electrodes vary, but the significant majority of nanogap devices use either gold or semiconductor electrodes. Gold has numerous advantages over semiconductors for many of these devices, including biocompatibility, ease of surface modification (through thiol-based chemistries), stability (resistance to oxidation), and off-the-shelf availability. One disadvantage of gold is the lack of compatibility with some fabrication processes. In such cases semiconductor electrodes have been used, despite their inherent shortcomings: mediocre conductivity and susceptibility to spontaneous surface oxidation [10]. Overlapping Electrical Double Layers In order to understand why 3D nanogap devices are so interesting as biosensors, the topic of overlapping electrical double layers must be covered. The electrical double layer at a single electrode surface is discussed in depth by Lyklema [11]. At a charged surface, electromigration of charged species balances with diffusion to create a diffuse layer of counterions that shields the bulk solution from the electrode s potential. Inside a nanogap, the situation is much different: The double layers can occupy the entire volume. The presence of the bulk solution, which can be measured in terms of parasitic capacitance and resistance, is virtually nonexistent. Depending on the solution s properties, the double layers can actually overlap. This term has been used to describe the situation where the Debye length is greater than the distance between the electrodes. The Debye length (λ D ) is the characteristic length scale of the double layer thickness, and is defined for a symmetric electrolyte: (2) where ε 0 is the permittivity of free space, ε is the relative permittivity of the solution, R is the gas constant, T is the temperature in Kelvin, z is the chemical valence of the electrolyte, F is the Faraday constant, and c 0 is the molar concentration of the electrolyte. Table 1 shows theoretical Debye lengths for a symmetric monovalent electrolyte at room temperature. These lengths can range from less than a nanometer to hundreds of nanometers, so for many of the nanogap devices in use and under development, overlapping double layers are a commonality. Nanogap Biosensors, Table 1 Debye lengths (λ D ) as a function of electrolyte concentration expressed as molarity (M) Debye lengths c 0 (M) λ D (nm) of 13 8/31/12 9:42 AM

6 To investigate the electric potential that results from the distribution of ions or charges inside of a nanogap, Poisson s equation is used: (3) where ϕ is the electric potential and ρ E is the charge density: (4) where i represents the ion species. If the nanogap is to be considered a closed system, mass must be conserved, so the following equation is also required: (5) where J i is the flux density in mol/cm 2 /s and R i is the production of species i, which is assumed to be zero at the voltages common for impedance spectroscopy. The flux density can be shown as: where D i is the diffusion coefficient and u i is the ion mobility. This represents diffusive flux and electric flux, as represented by the two terms in this equation, respectively. Flux across the boundaries is assumed to be zero, and the potential on the two electrode surfaces are: (6) (7) The distance between the electrodes, L, is varied. A dimensionless ratio of the distance between the electrodes and the Debye length is defined to be S n : (8) The results from this solution are shown in Fig. 2. Although this analysis is based on steady-state assumptions, it is a good approximation for most impedance spectroscopy systems, since the relaxation frequency of the electrical double layer is typically faster than the frequency of the current injected by the analyzer. 6 of 13 8/31/12 9:42 AM

7 Nanogap Biosensors, Fig. 2 Numerical solution of overlapping double layers. The electrode surfaces are represented by the left and right plot borders. The y-axis is the potential, and the x-axis is the distance between the electrodes. The potential distribution is plotted as the dimensionless number, S n It is clear from the numerical solution shown in Fig. 2 that when the double layers overlap, the potential drop across the electrodes approaches a linear regime. This is significantly different from a macroscopic gap, where virtually all of the potential drops across the double layers and the bulk solution in a macro-gap typically exhibits a null electric field. This is an important point for biosensors, since the entire solution should ideally be subjected to the same conditions. For macroscale electrode sensors, molecules in the middle of the gap will behave much differently from molecules found on or near the electrode surfaces. The uniform field inside a nanogap is a significant advantage for these devices, since all molecules inside of the gap will see the same electrical conditions. The bulk solution is essentially removed, thereby providing, at least in theory, a more homogenous response. Figure 3 depicts a simple illustration of the electrical double layers in a macroscale gap and a nanogap with overlapping double layers. 7 of 13 8/31/12 9:42 AM

8 Nanogap Biosensors, Fig. 3 Electrical double layers and overlapping electrical double layers. Schematic of ion distribution inside macroscale gaps and nanogap devices, and resultant potential distribution. λ D represents the Debye length Measurement Methods The detection method is typically achieved by measuring the complex impedance response to either a single excitation frequency or a full range of frequencies, or spectrum: The impedance spectrum of a system is measured by applying a frequency-dependent excitation voltage and measuring the resulting current. From the impedance spectrum and the geometrical parameters of the system, the complex permittivity or dielectric spectrum is derived. Kaatze and Feldman have comprehensively reviewed the basic principles of dielectric spectroscopy and the methods in use to measure the dielectric properties of liquid samples over the frequency range from about Hz [12]. Conventionally, a small AC (alternating current) potential, V * (jω), swept over a range of frequencies is applied. The electrical current response, I * (jω), is measured and the complex impedance Z * (jω), of the system is: (9) Here Z * RE(jω) and Z * IM(jω) are the real and imaginary parts of the complex impedance, respectively. The magnitude and phase angle of the complex impedance are 8 of 13 8/31/12 9:42 AM

9 (10) and (11) respectively. Knowing the geometrical and physical parameters of the biosensor, it is possible to model the physical mechanisms of conductivity and polarizability with equivalent circuit analysis. Using networks of resistors and capacitors, the response of the system under controlled conditions can be predicted, and consequently the detection of biological events that cause deviations from the predicted response. Determination of an appropriate equivalent model requires some a priori knowledge of the chemistry and physics of the system. Because multiple models can be found to fit any acquired set of impedance data, the choice of the most appropriate model is often the result of a complex balance between complexity, in terms of raw number of circuit elements employed, and efficiency, in terms of which chemical and physical phenomena of the system need to be characterized and with what level of sensitivity. In general, it is of the utmost importance to design a nanogap sensor system where the number of parasitic circuit elements (which tend to increase the complexity of the resultant equivalent circuit model) is minimized, while the impedance response of the system is maximally affected by specific biological events taking place in the nanogap. This is usually achieved by a combination of clever design, appropriate choice of materials, and a reasoned choice of range of frequencies to be measured. To maximize sensitivity, it is advantageous to have large active areas and small interelectrode distances, maximizing device capacitance. In the particular case where biomolecules are sensed as they bind to the surface and modify the double layer, it is also convenient to minimize the contribution of the bulk solution to the overall impedance, which leads to a sensitivity response that is inversely proportional to the interelectrode distance. Defining ξ as the quality factor, the two dependencies can be formulated as follows: For a generic parallel-plate capacitive sensor, the measured quantity is capacitance, while the sensed parameter is the permittivity ε. The two are related by the well-known expression: (12) where A is the area of the electrodes, d the distance separating them, and ε the permittivity of the dielectric between the two plates. To maximize the measured capacitance given a fixed permittivity it is necessary to maximize the area-to-interelectrode distance ratio. Therefore, the first quality factor can be defined as (13) For the specific case of sensors sensitive to surface binding events where bulk contributions (due 9 of 13 8/31/12 9:42 AM

10 to the total volume enclosed by two parallel electrodes) are to be minimized, a second quality factor can be defined as the area-to-volume ratio, i.e., (14) The combination of these two factors yields: (15) This dimensionless parameter can be used to quantitatively compare the efficiency of different nanogap sensors, as well as to optimize the design of new sensors. Although alternating current methods are the most common methods used with 3D nanogap biosensors, changes in direct currents (and similarly, shifts in IV curves) have been used [13]. Key Research Findings Some key pieces of literature are briefly listed below. This list is not meant to be exhaustive, but rather illustrative of the most significant research findings achieved using nanogap sensors, and a starting point for further exploration. One- and Two-Dimensional Nanogap Biosensors One- and two-dimensional nanogap devices have been used mostly in proof-of-concept studies, to determine the ability to detect and to a limited extent, characterize nucleic acids (DNA, RNA) binding events, and presence of other biologically relevant biomolecules such as proteins and antibodies. Chen et al. detected specific target DNA concentrations of as low as 10 picomolar identifying single-base-pair mismatches [14]. Liang et al. observed electrical signals caused by 1.1 kilobase-pair (kbp) double-stranded (ds)-dna passing through the gap in the nanogap detectors with a gap equal to or less than 13 nm [15]. More recently, Cingolani et al. demonstrated that hybridization events are detected by the discrete increase in conductivity when the target AuNC DNA conjugates bridge the electrode gap [16]. The electrical detection of other biomolecular interactions has also been demonstrated. For example, Haguet et al. have detected the capture of antibodies as well as the specific interaction between the biotin and the streptavidin molecules [17]. Subsequently, the same group has shown that the same method can be extended to the detection of immunoglobulin G (IgGs) in serum using protein probes [18]. Three-Dimensional Nanogap Biosensors Three-dimensional nanogap resistive sensors with gaps of 5, 10, and 15 nm have been used to detect biotin streptavidin binding events by measuring peak increases in direct current [13]. Single-frequency AC impedance measurements have been used successfully with threedimensional nanogap sensors adopting metal electrodes and integrated reference sensing on-chip, for the detection of thrombin, a blood clotting factor, and to investigate its binding properties to a specific antibody and RNA-aptamer. A similar device was used to study the interaction of the Rev peptide with a corresponding RNA anti-rev aptamer in concentration spanning the range of of 13 8/31/12 9:42 AM

11 nm 2 μm. The parasitic effect of the double layer was significantly reduced by using measurement frequencies of 800 1,280 MHz [19]. AC impedance spectroscopy has also been used to specifically detect the presence of thrombin in solution by looking at impedance spectra recorded using an 20mV AC signal swept over a frequency range of 10 Hz 100 khz [20]. Similarly, hybridization of poly-a nucleotides to immobilized poly-t between nanogap electrodes has been shown to result in a measurable change in capacitance over a frequency range of 100 Hz 10 khz [21]. Future Directions At this point in time, the development of nanogap biosensors is still in the research phase, i.e., there are no commercially available nanogap biosensors. However, the promise of sensitive, label-free devices with small footprints is alluring. Great strides have been made in terms of nanogap manufacturing and detection. Future work is needed in scale up of manufacturing, as few groups have achieved repeatable large-scale fabrication. This will be essential in developing sensors in quantities at low cost. For applications relating to nucleic acid determination and sequencing, increases in sensitivity and specificity are necessary for these biosensors to compete with existing industry standard sensors which offer fluorescence-based readouts. Specific label-free, all-electrical nucleic acid detection can still be valuable for diagnostic point of care devices, although additional work is needed to enable multiplexed detection which will allow the analysis of a panel of biomarker sequences, rather than a single short target sequence. Similarly useful will be nanogap devices that enable the detection of specific proteins in serum or blood, forfeiting the cost and complexity associated with traditional antigen antibody assays such as ELISAs. Devices aimed at point-of-care testing are particularly exciting because this application capitalizes on the inherent advantages of a nanogap: small volumes, small footprint, low power consumption, and label-free, all-electrical detection. The future will likely see many point-of-care biosensors using electrical-based detection, and perhaps nanogap technology can play an important role. Cross-References Biosensors Dry Etching SU-8 Photoresist Wet Etching References 1. Liang, X., Chou, S.Y.: Nanogap detector inside nanofluidic channel for fast real-time label-free DNA analysis. Nano. Lett. 8, (2008) 11 of 13 8/31/12 9:42 AM

12 2. Im, H., et al.: A dielectric-modulated field-effect transistor for biosensing. Nat. Nanotech 2(7), (2007) 3. Gu, B., et al.: Nanogap field-effect transistor biosensors for electrical detection of avian influenza. Small 5(21), (2009) 4. Chiras, S., Clarke, D.R.: Dielectric cracking produced by electromigration in microelectronic interconnects. J. Appl. Phys. 88, (2000) 5. Fernandez-Martinez, I., Gonzalez, Y., Briones, F.: Parallel nanogap fabrication with nanometer size control using III-V semiconductor epitaxial technology. Nanotechnology 19(27), (2008) 6. Higuchi, Y., et al.: Application of simple mechanical polishing to fabrication of nanogap flat electrodes. Jpn. J. Appl. Phys. 45, L145 L147 (2006) 7. Li, T., Hu, W., Zhu, D.: Nanogap electrodes. Adv. Mater. 22(2), (2009) 8. Lazzarino, M., et al.: Twin cantilevers with a nanogap for single molecule experimentation. Microelectron. Eng. 83(4 9), (2006) 9. Stroscio, J.A., Kaiser, W.J.: Scanning tunneling microscopy. In: Celotta, R., Lucatorto, T. (eds.) Methods of experimental phsics, vol. 27. Academic, San Diego (1993) 10. Ionescu-Zanetti, C., et al.: Nanogap capacitors: sensitivity to sample permittivity changes. J. Appl. Phys. 99(2), (2006) 11. Lyklema, J.: Fundamentals of Interface and Colloid Science, p Academic, London (1995) 12. Kaatze, U., Feldman, Y.: Broadband dielectric spectrometry of liquids and biosystems. Meas. Sci. Technol. 17(2), R17 (2006) 13. Jang, D.Y., et al.: Sublithographic vertical gold nanogap for label-free electrical detection of protein-ligand binding. J. Vac. Sci. Technol. B 25(2), (2007) 14. Chen, F., et al.: Electrochemical approach for fabricating nanogap electrodes with well controllable separation. Appl. Phys. Lett. 86(12), (2005) 15. Liang, X., Chou, S.Y.: Nanogap detector inside nanofluidic channel for fast real-time label-free DNA analysis. Nano. Lett. 8(5), (2008) 16. Maruccio, G., et al.: A nanobiosensor to detect single hybridization events. Analyst 134(12), (2009) 12 of 13 8/31/12 9:42 AM

13 17. Haguet, V., et al.: Combined nanogap nanoparticles nanosensor for electrical detection of biomolecular interactions between polypeptides. Appl. Phys. Lett. 84(7), 1213 (2004) 18. Marcon, L., Stievenard, D., Melnyk, O.: Characterization of nanogap chemical reactivity using peptide-capped gold nanoparticles and electrical detection. Bioconjug. Chem. 19(4), (2008) 19. Schlecht, U., et al.: Detection of Rev peptides with impedance-sensors Comparison of device-geometries. Biosens. Bioelectron. 22(9 10), (2007) 20. Mannoor, M.S., et al.: Nanogap dielectric spectroscopy for aptamer-based protein detection. Biophys J. 98(4), (2010) 21. Choi, Y.K., et al.: Sublithographic nanofabrication technology for nanocatalysts and DNA chips. J. Vac. Sci. Technol. B 21(6), (2003) 13 of 13 8/31/12 9:42 AM