CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser. No. 62/803,100 filed Feb. 8, 2019, the entire disclosure of which is hereby incorporated herein by reference.

FIELD

Embodiments of the present invention are related to systems, methods, devices, and compositions of matter for electronic sequencing of biopolymers. More specifically, the present invention includes embodiments which teach the construction of a system for detecting biopolymers electronically based on enzymatic replication.

BACKGROUND OF THE INVENTION

Collins and coworkers devised a method to monitor enzymatic process of synthesizing DNA by a Klenow fragment of DNA polymerase I attached to a single-wall carbon nanotube (SWCNT) field-effect transistor (FET)^{1, 2}In the device, when a nucleotide was incorporated into a DNA strand, a brief excursion of ΔI(t) below the mean baseline currents was recorded. The ΔI signals may be associated with the dynamics of the DNA polymerase in its conformation. Importantly, the characteristics of the signal reflected a specific nucleotide that was incorporated into DNA. It opens a way to read out DNA sequences electronically. As far as the carbon nanotube is concerned, it is a material made from just a single layer of carbon atoms locked in a hexagonal grid. Because of the rigid chemical structure, its sensing may rely on electrostatic gating motions of charged side chains of the enzyme close to the attachment site, which can be shielded by electrolytes in solution. Also, the carbon nanotube in the device had a length of 0.5-1.0 μm,³which poses a challenge to attaching a single protein molecule of a diameteress than 10 nm to a specific location in such a long wire precisely.

Huang's group reported another type of device with a DNA polymerase grabbed in an antibody bridged nanogap (FIG. 1a).⁴Electrical fluctuations were recorded as the DNA polymerase elongated a DNA chain by incorporating nucleotides. With the severe controversy, the paper was subsequently retracted (Nat. Nanotechnol. 2015, 10, 563), but it illustrates an idea of monitoring the conformational dynamics of a protein by electrical fluctuation. Besides, the transmission electron microscopy (TEM) image demonstrated that the DNA polymerase laid on the SiO₂surface out of the nanogap. This configuration would prevent the polymerase from effectively interacting with DNA due to the steric hindrance posed by the surface,

In the other prior art, the invention (WO 2017/024049) provides a nanoscale field-effect transistor (nanoFET) for DNA sequencing, where the DNA polymerase is is immobilized with its nucleotide exit region oriented toward a carbon nanotube gate.

One invention (US 2017/0044605) has claimed an electronic sensor device to sequence DNA and RNA using a polymerase immobilized on a biopolymer that bridges two separate electrodes, In another prior art (US 2018/0305727, WO 2018/208505), a single enzyme is directly wired to both positive and negative electrodes to complete a circuit such that all electrical currents must flow through the molecule. Also, the enzyme is attached to electrodes through more than two contacting points. Nonetheless, it requires a sub-10 nm nanogap, which poses a great challenge to manufacturing.

It has been demonstrated that a protein can become conductive above a bias threshold.⁵Natural Peptides lose their conductivity quickly because of their relatively flexible conformations.

The ϕ29 DNA polymerase is the only enzyme involved in the replication of the phage ϕ29 genome. Based on amino acid sequence similarities and its sensitivity to specific inhibitors, ϕ29 DNA polymerase belongs to the eukaryotic-type family B of DNA-dependent DNA polymerases (Bernad et al. 1987). As any other DNA polymerase, it accomplishes sequential template-directed addition of dNMP units onto the 3′-OH group of a growing DNA chain, showing discrimination for mismatched dNMP insertion by a factor from 10⁴to 10⁶(Esteban et al. 1993). Besides, ϕ29 DNA polymerase catalyzes 3′-5′ exonucleolysis, to release dNMP units from the 3′ end of a DNA strand (Blanco and Salas 1985), degrading preferentially a mismatched primer-terminus, and further enhancing replication fidelity by 10²-fold (Esteban et al, 1994; Garmendia et al. 1992), as it occurs in most DNA replicases.

Three factors that disrupt alfa helix formation: (a) glycine—which is the smallest amino acid; (b) proline—which is the least common amino acid in the alpha-helix which destabilizes it; (c) amino acids with like charge side chains that are close together that are incompatible with the alpha helix.

Molecular self-assembly is ideally suited to create nanostructures with dimensions ranging from 10-100 nm, a size regime suitable for most electronic materials.

It has been reported that a silicon nanowire field-effect transistor (FET) built on the edge of a SiN nanopore can detect DNA translocation by sensing the changes in electrical potential.⁶Also, a field-effect transistor can sense conformational changes in proximity to semiconductor channels gated conductance in physiological buffers, resulting in the highly sensitive detection of ligand and receptor interactions.⁷However, these FET devices have not exhibited a capacity to read single DNA bases in a DNA strand.

A simple nanojunction can be formed by connecting a molecular wire to two electrodes separated by a nanoscale gap. It allows electrons to flow when integrated into an electrical circuit. In general, the molecular component is covalently attached to the electrodes, and the electrical conductivity of a junction is affected by the molecular structure and molecule-metal contact.⁸However, its electronic state can be switched by stereoelectronic effect⁹and altered by external stimuli. For example, the conductance of a host-guest molecular junction can be tuned by the insertion of guest molecules.¹⁰Also, a protein transistor can be fabricated by bridging a nanogap using an antigold nanoparticle antibody.¹¹

Electron transfer (ET) can be mediated along with proteins and peptides.^{12, 13}Arguably, ET through peptides may operate through tunneling and hopping in parallel; however, their contributions change with the length of the mediating bridge. For short bridges, tunneling is dominating, whereas for long bridges, hopping becomes more pronounced,¹⁴which was demonstrated experimentally by Isied and coworkers.¹⁵The composition of the side chains, hydrogen bonds, and an α-helical secondary structure have been identified as important factors contributing to the hopping and tunneling conductivity in these peptide systems over short distances. Thus, the charge transfer properties of peptides can be modulated by manipulating their secondary structure.

Long-range electron transport in conductive pili represents a natural inspiration for the design of molecular bioelectronics and tunable synthetic platforms for molecular sensing. The proteinaceous pili of Geobacter sulfurreducens can conduct electrons over micrometer distance with metallic-like conductivity.¹⁶They are unique biological electronic materials. The conductive pili (e-pili) are composed of a single peptide monomer, PilA, which is homologous to the pilin monomer of type IV pili.¹³In Geobacter sulfurreducens, the major pilin subunit is encoded by the gene pila, which produces the protein PilA with a sequence shown in FIG. 3a. The protein PilA itself is not conductive¹⁷since it only contain a few of aromatic amino acids residues which are scattered in an a helix (FIGS. 3b & 3c). It has been determined by NMR that the Geobacter sulfurreducens PilA adopts a long, kinked α-helix with a dynamic C-terminal region (FIG. 3, A).¹⁸Thus, the conductivity of e-pili can be putatively explained by a continuous arrangement of aromatic amino acids in a G. sulfurreducens pilus (FIG. 3, B).¹⁹It has been demonstrated that aromatic amino acid residues are required for pili conductivity and long-range extracellular electron transport in Geobacter sulfurreducens.²⁰

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: a prior art of electrical detection system for polymerase fluctuations reported by Chen et al.⁴(a) schematic description where phi29 DNA polymerase (light blue) is conjugated to a secondary antibody (beige lines) and binds the Fc domain of IgG (blue lines) bearing two gold nanoparticles separately attached to two electrodes to ensure that the antibody assembly is bound into the integrated circuit; (b) Transmission electron microscopy (TEM) image of a phi29-conjugated protein transistor carrying a bound oligonucleotide template and annealed primer.

FIG. 2: A prior art of using biopolymers to connect a DNA polymerase to in electrodes.

FIG. 3: (a) Amino acid sequence of PilA protein of the conductive pilus; (b) PilA protein's α helical model; (c) helical wheel plots of PilA protein.

FIG. 4: (a) a predicted structure of G. sulfurreducens pilin monomers derived from NMR (b) a predicted structure of a Geobacter sulfurreducens pilus derived using monomer pilin based on NMR structure.

FIG. 5: (a) an amino acid sequence of the modified PilA protein; (b) helical wheel plots of the modified PilA protein. (c) the modified PilA protein's α helical model.

FIG. 6: a library of unnatural L-aromatic amino acids for the construction of conductive proteins and peptides.

FIG. 7: a library of unnatural D-aromatic amino acids for the construction of conductive proteins and peptides.

FIG. 8: (a) a three-arm linker for connecting two helical peptides; (b) a coiled coil peptide dimer through one three-arm linker; (c) a peptide timer capped by two three-arm linkers.

FIG. 9: (a) a nanojunction composed of a peptide nanostructure bridging a nanogap; (b) a DNA polymerase immobilized on the nanojunction for DNA sequencing.

FIG. 10: Chemical structures of unnatural amino acids for attachments of peptide nanostructures as well as immobilization of proteins and peptides.

SUMMARY OF THE INVENTION

One embodiment of this invention provides conductive peptides by modifying the PilA sequence with aromatic amino acids. First, the Pila sequence is rearranged like repeats of a heptad pattern (abcdefg)_nwhere n is the number of in repeats. The aromatic amino acid (F) is substituted for the amino acids at the position a and d of the heptad. As a result, a modified peptide is created with a sequence, as shown in FIG. 5a. The modified peptide can take a helical structure with an aromatic amino acid-rich region as shown in the helical wheel plot (FIG. 5b). The modified peptide has a train of aromatic moieties exposed on its surface is with distances between the aromatic rings smaller than <6 Å (FIG. 5c), which allow electrons to flow through tunneling or hopping, functioning as a molecule wire.

This invention provides also unnatural aromatic amino acids (UAAA) for the construction of conductive proteins and peptides. In one embodiment, it provides a library of UAAAs with an L-configuration (FIG. 6), and in another embodiment, it provides a library of UAAAs with a D-configuration (FIG. 7). UAAAs are incorporated into proteins and peptides by means of bioengineering and/or chemical methods.

In one embodiment, the invention provides a three-arm linker for the formation of a peptide nanostructure and its attachment to electrodes (FIG. 8a). It also provides a method to make coiled-coil conductive peptides using the three-arm linker (FIG. 8b) and a peptide dimer with its two ends capped by the three-arm linker (FIG. 8c). The peptide nanostructure forms an aromatic tunnel for the electron flow, functioning as a metal-like wire.

The invention also provides methods to attach the peptide nanostructures to a nanogap composed of electrodes to form a nanojunction for bio- and chemo-sensing (FIG. 9a). In one embodiment, the invention provides a method to immobilize a DNA polymerase to the junction (FIG. 9b) for DNA sequencing.

In one embodiment, the invention provides unnatural amino acids for attachment of peptides to electrodes and immobilization of proteins on nanojunctions composed of peptides or peptide nanostructures (FIG. 10) using conjugation chemistry, including but not limited to click chemistry and photochemistry.

Furthermore, this invention discloses the following nanostructures and methods for constructing these nanostructures for electronic sensing, sequencing and/or identification of biomolecules or biopolymers, including but not limited to DNA, RNA, oligos, proteins, peptides, polysaccharides, etc. either natural or modified or synthesized:

1. A system for electronic identification and sequencing of a biopolymer in a is nanogap comprising a first electrode and the second electrode in proximity to said first electrode, which is bridged by a peptide nanostructure bonding to both electrodes through chemical bonds to form nanojunction that does not break over the time course of a measurement process,

2. The said nanojunction in item 1 is functionalized by attaching an enzyme, protein, receptor, nucleic acid probe, antibody and its variants, aptamer, supramolecular host to the nanostructure for the detection of chemical and biochemical reactions as well as molecular interactions.

3. Under a bias applied between the first and second electrodes, the device records current fluctuations resulting from the said nanostructure's distortions caused by the conformation changes of the enzyme attached to the nanostructure while carrying out biochemical reactions. A bias is chosen between the two electrodes so that a steady DC current is observed, and current fluctuations arise when biochemical reactions take place between the said electrodes. In a polymerization reaction, a train of electrical spikes is recorded for the determination of the polymeric sequences.

4. The enzyme in item 1 and item 3 includes but not limited to DNA polymerase, RNA polymerase, DNA helicase, DNA ligase, DNA exonuclease, reverse transcriptase, RNA primase, ribosome, sucrase, lactase, etc., either natural, mutated or synthesized, wherein the DNA polymerase is selected among the group of ϕ29 DNA polymerase, T7 DNA polymerase, Tag polymerase, DNA polymerase Y, DNA Polymerase Pol I, Pol II, Pol III, Pol IV and Pol V, Pol α (alpha), Pol β (beta), Pol σ (sigma), Pol λ (lambda), Pol δ (delta), Pol ε (epsilon), Pol μ (mu), Pol ι (iota), Pol κ (kappa), pol η (eta), terminal deoxynucleotidyl transferase, telomerase, etc., either natural, mutated or synthesized;

5. The said electrodes in item 1 are composed of:

a) metal electrodes that can be functionalized on their surfaces by self-assembling monolayers that can react with anchoring molecules by forming covalent bonds.

b) metal oxide electrodes that can be functionalized with silanes that can react with anchoring molecules to form covalent bonds.

c) carbon electrodes that can be functionalized with organic reagents that can react with anchoring molecules to form covalent bonds.

Wherein metal electrodes include but not limited to Au, Pd, Pt, Cu, Ag, Ti, TiN, or other transition metals.

6. The said nanogap in item 1:

(a) has a length of 3 to 10,000 nm, preferably 5 to 100 nm, and most preferably 5 to 50 nm; a width of 3 to 1000 nm, preferably 10 to 50 nm; and a depth of 2 to 1000 nm, preferably 5 to 50 nm.

(b) is fabricated on substrates including but not limited to glass, silicon and silicon oxide, and polymer films.

7. The said nanostructure in item 1:

(a) is a single peptide chain with helical structure, constructed using a modified bacterial PilA sequence with aromatic amino acid arrangement depicted in FIG. 5 or similar amino acid composition and arrangement;

(b) is a single peptide chain with helical structure, constructed using unnatural aromatic amino acids with either an L-configuration (FIG. 6) or a D-configuration (FIG. 7), or a combination thereof;

(c) is a single peptide/DNA/RNA mixed helical chain constructed using either s natural or modified or synthesized aromatic amino acids and nucleic acids with distance between any two adjacent aromatic rings smaller than 0.6 nm (6 Å), preferably less than 0.35 nm;

(d) is a single peptide coupled with conductive organic conjugates and/or conductive polymers;

(e) is a dual peptide chain consisting of two helical peptide chains either the same composition and arrangement or different composition and arrangement, and with each peptide chain attaching to the electrodes individually or two peptide chain forming a peptide dimer and attaching to the electrodes through a three-arm linker, such as that shown in FIG. 8;

(f) is a peptide chain and a nucleic acid chain forming a dual linear chain structure, helical or non-helical, wherein the peptide chain is made of aromatic amino acids, either natural or synthesized, and the aromatic rings of the amino acids and nucleic acids interacting with each other with distance between any two adjacent rings, either from the peptide chain or from the nucleotide chain, less than 0.6 nm, preferably less than 0.35 nm.

(g) is a multiple peptide chain or a multiple peptide/DNA/RNA mixed chain bundled together forming a two-dimensional nanostructure, or a three-dimensional nanostructure including a bundle of columns, a stack of two-dimensional structures or a folded chain structure such as coiled coils, with a length that can bridge the said two electrodes.

Wherein all the nanostructures mentioned above have a length equivalent to the said nanogap size and able to bridge the two electrodes, and contain functional groups for attachment to electrodes and functional groups for the immobilization of enzymes.

8. The said functional groups for attachment in item 7 include but not limited to:

(a) those thiols on the sugar rings of nucleosides and amino acids.

(b) those thiols and selenols on the nucleobases of nucleosides.

(c) those aliphatic amines on nucleosides.

(d) Those catechols on nucleoside.

(e) azide, alkyne and alkene on unnatural amino acids.

(f) Photoactive groups such as benzophenone

9. The said anchoring molecules in item 5 are

(a) those that can interact with the metal surface through multivalent bonds.

(b) a tripod structure that can interact with the metal surface through trivalent bonds.

(c) Those that are composed of a tetraphenylmethane core of which three phenyl rings are functionalized with —CH₂SH and —CH₂SeH and the last phenyl ring is functionalized with azide, carboxylic acid, boronic acid, and organic groups that can react with those functional groups incorporated into peptide, DNA and RNA nanostructures.

10. The said anchoring molecules in item 5 are

(a) N-heterocyclic carbenes (NHC);

(b) N-heterocyclic carbenes (NHC) that are selectively deposited to cathode electrodes by electrochemical methods with their metal complexes in solutions.

(c) N-heterocyclic carbenes (NHC) that are deposited to both metal electrodes in organic and aqueous solutions.

(d) N-heterocyclic carbenes (NHC) containing functional groups including amines, carboxylic acids, thiol, boronic acids, or other organic groups for attachment.

11. The said NHC metal complexes in item 10 include but not limited to those composed of Au, Pd, Pt, Cu, Ag, Ti, TiN, or other transition metals or a combination thereof.

12. The said nanogap in item 6 is functionalized with chemical reagents on its bottom.

13. The said chemical reagent in item 12 is:

(a) Silanes that can react with oxide surfaces;

(b) Silatranes that can react with oxide surfaces;

(c) A multi-arm linker that contains silatranes and functional groups;

(d) A four-arm linker that is composed of an adamantane core;

(e) A four-arm linker that contains two silatranes and two biotin moieties.

(f) A four-arm linker that is composed of adamantane core and silatranes and biotin

14. The said chemical reagents in item 12 are used to immobilize proteins in the nanogap, which include antibodies, receptors, streptavidin, avidin.

15. The said streptavidin in item 14 is used to immobilized the said nanostructures.

16. The said nanostructure in item1 is functionalized with biotins.

17. The system of item 1 can contain a single nanogap or a plurality of nanogaps, each with a pair of electrodes, an enzyme, a peptide nanostructure and all other features associated with a single nanogap. Furthermore, the system can consist of an array of nanogaps between 100 to 100 million, preferably between 10,000 to 1 million.

18. The said nanostructure in item 1 is in general conductive by itself. However, in some special cases, it can be made non-conductive by itself but conductive when combines with the enzyme or at least during a portion of the chemical reaction process of the enzyme.

19. Features of nanostructure, nanogap, enzyme and electrode, their composition and construction as well as other associated features and methods mentioned in our provisional application Ser. No. 62/794,096 that are relevant to this invention and can apply to the said nanostructure in this invention are included here in its entirety

General Remarks

All publications, patents and other documents mentioned herein are incorporated by reference in their entirety.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs. While the present invention has been illustrated by a description of various embodiments and while these embodiments have been described in considerable detail, it is not the intention of the applicants to restrict or in any way limit the scope of the applications. Additional advantages and modifications will readily appear to those skilled in the art. The invention in its broader aspects is therefore not limited to the specific details, representative device, apparatus and method, and illustrative example shown and described. Accordingly, departures may be made from such details without departing from the spirit of applicant's general inventive concept. Finally, the use of singular articles such as “a,” “the,” “said,” etc. should be read to recite one or more of the indicated elements unless a claim recites an explicit limitation to the contrary.

REFERENCES

1. Olsen, T. J.; Choi, Y.; Sims, P. C.; Gul, O. T.; Corso, B. L.; Dong, C.; Brown, W. A.; Collins, P. G.; Weiss, G. A., Electronic measurements of single-molecule processing by DNA polymerase I (Klenow fragment). J Am Chem Soc 2013, 135 (21), 7855-60.

2. Pugliese, K. M.; Gul, O. T.; Choi, Y.; Olsen, T. J.; Sims, P. C.; Collins, P. G.; Weiss, G. A., Processive Incorporation of Deoxynucleoside Triphosphate Analogs by Single-Molecule DNA Polymerase (Klenow Fragment) Nanocircuits. J Am Chem Soc 2015, 137 (30), 9587-94.

3. Choi, Y.; Olsen, T. J.; Sims, P. C.; Moody, I. S.; Corso, B. L; Dang, M. N.; Weiss, G. A.; Collins, P. G., Dissecting Single-Molecule Signal Transduction in Carbon Nanotube Circuits with Protein Engineering. Nano Lett. 2013, 13, 625-631.

4. Chen, Y. S.; Lee, C. H.; Hung, M. Y.; Pan, H. A.; Chiou, J. C.; Huang, G. S., DNA sequencing using electrical conductance measurements of a DNA polymerase. Nat Nanotechnol 2013, 8 (6), 452-458.

5. Zhang, B.; Song, W.; Pang, P.; Zhao, Y.; Zhang, P.; Csabai, I.; Vattay, G.; Lindsay, S., Observation of Giant Conductance Fluctuations in a Protein. Nano Futures 2017, 1 (3), 035002.

6. Xie, P.; Xiong, Q.; Fang, Y.; Qing, Q.; Lieber, C. M., Local electrical potential detection of DNA by nanowire-nanopore sensors. Nature Nanotechnology 2012, 7, 119-125.

7. Nakatsuka, N.; Yang, K.-A.; Abendroth, J. M.; Cheung, K. M.; Xu, X.; Yang, H.; Zhao, C.; Zhu, B.; Rim, Y. S.; Yang, Y.; Weiss, P. S.; Stojanović, M. N.; Andrews, A. M., Aptamer-field-effect transistors overcome Debye length limitations for small-molecule sensing. Science 2018, 362, 319-324.

8. Wang, G.; Kim, T.-W.; Jang, Y. H.; Lee, T., Effects of Metal-Molecule Contact and Molecular Structure on Molecular Electronic Conduction in Nonresonant Tunneling Regime: Alkyl versus Conjugated Molecules. J. Phys. Chem, C 2008, 112, 13010-13016,

9. Xin, N.; Wang, J.; Jia, C.; Liu, Z.; Zhang, X.; Yu, C.; Li, M.; Wang, S.; Gong, Y.; Sun, H.; Zhang, G.; Liu, Z.; Zhang, G.; Liao, J.; Zhang, D.; Guo, X., Stereoelectronic Effect-Induced Conductance Switching in Aromatic Chain Single-Molecule Junctions. Nano Lett. 2017, 17, 856-861.

10. Fujii, S.; Tada, T.; Komoto, Y.; Osuga, t; Murase, T.; Fujita, M.; Kiguchi, M., Rectifying Electron-Transport Properties through Stacks of Aromatic Molecules Inserted into a Self-Assembled Cage. J Am Chem Soc 2015, 137 (18), 5939-47.

11. Chen, Y.-S.; Hong, M.-Y.; Huang, G. S., A protein transistor made of an antibody molecule and two gold nanoparticles. Nature Nanotechnology 2012, 7, 197-203.

12. Amdursky, N., Electron Transfer across Helical Peptides. ChemPlusChem 2015, 80(7), 1075-1095.

13. Reguera, G.; McCarthy, K. D.; Mehta, T.; Nicoll, J. S.; Tuominen, M. T.; Lovley, D. R., Extracellular electron transfer via microbial nanowires. Nature 2005, 435 (7045), 1098-101.

14. Petrov, E. G.; Shevchenko, Y. V.; Teslenko, V. I.; May, V., Nonadiabatic donor-acceptor electron transfer mediated by a molecular bridge: A unified theoretical description of the superexchange and hopping mechanism. The Journal of chemical physics 2001, 115 (15), 7107-7122.

15. Malak, R. A.; Gao, Z.; Wishart, J. F.; !sled, S. S., Long-Range Electron Transfer Across Peptide Bridges: The Transition from Electron Superexchange to Hopping. J. AM. CHEM. SOC. 2004, 126, 13888-13889

16. Lovley, D, R., e-Biologics: Fabrication of Sustainable Electronics with “Green” Biological Materials. mBio 2017, 8 (3).

17. Lebedev, N.; Mahmud, S.; Griva, I.; Blom, A.; Tender, L. M., On the Electron Transfer through Geobacter sulfurreducens PilA Protein. JOURNAL OF POLYMER SCIENCE, PART B: POLYMER PHYSICS 2015, 53, 1706-1717.

18. Reardon, P. N.; Mueller, K. T., Structure of the type IVa major pilin from the electrically conductive bacterial nanowires of Geobacter sulfurreducens. J Biol Chem 2013, 288 (41), 29260-6.

19. Malvankar, N. S.; Vargas, M.; Nevin, K.; Tremblay, P. L.; Evans-Lutterodt, K.; Nykypanchuk, D.; Martz, E.; Tuominen, M. T.; Lovley, D. R., Structural basis for metallic-like conductivity in microbial nanowires. mBio 2015, 6 (2), e00084.

20. Vargas, M.; Malvankar, N. S.; Tremblay, P. L,; Leang, C.; Smith, J. A.; Patel, P.; Synoeyenbos-West, O.; Nevin, K. P.; Lovley, D. R., Aromatic Amino Acids Required for Pili Conductivity and Long-Range Extracellular Electron Transport in Geobacter sulfurreducens. mBio 2013, 4 (2).