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Solid state nanopores for gene expression profiling
Superlattices and Microstructures 46 (2009) 59–63
Contents lists available at ScienceDirect
Superlattices and Microstructures journal homepage: www.elsevier.com/locate/superlattices
Solid state nanopores for gene expression profiling V. Mussi a,b,∗ , P. Fanzio a,b , L. Repetto a,b , G. Firpo a,b , U. Valbusa a,b , P. Scaruffi c , S. Stigliani c , G.P. Tonini c a
Nanomed Labs, Advanced Biotechnology Center, Largo Rosanna Benzi, 10 16133 Genova, Italy
b
Physics Department University of Genova, Via Dodecaneso, 33 16146 Genova, Italy
c
Translational Paediatric Oncology, National Institute for Cancer Research (IST) Largo R. Benzi, 10 Genova, 16132, Italy
article
info
Article history: Available online 10 October 2008 Keywords: Nanopore Functionalization Ionic current Biosensor Gene expression
abstract Recently, nanopore technology has been introduced for genome analysis. Here we show results related to the possibility of preparing ‘‘engineered solid state nanopores’’. The nanopores were fabricated on a suspended Si3 N4 membrane by Focused Ion Beam (FIB) drilling and chemically functionalized in order to covalently bind oligonucleotides (probes) on their surface. Our data show the stable effect of DNA attachment on the ionic current measured through the nanopore, making it possible to conceive and develop a selective biosensor for gene expression profiling. © 2008 Elsevier Ltd. All rights reserved.
The Human Genome project allowed the identification of new genes and Expressed Sequence Tags (EST) that will complete the decoding of the human genome (http://www.ornl.gov/hgmis/). In the meantime, new technologies for genome analysis have been developed, i.e. microarray technology, able to analyze ten of thousands genes in a single experiment. The microarray technology is based on the principle that single strand DNA (ssDNA) molecules attached to solid supports can hybridize to complementary sequences. Microarray devices are utilized to characterize gene expression profiles, to detect point mutations and Single Nucleotide Polymorphisms (SNPs), to identify gains or losses in chromosome regions [1]. Recently [2], nanopore technology has been introduced for genome analysis. The use of nanopores for the detection and analysis of single molecules has been inspired by the Coulter counter [3] working principle: a small channel separates two electrolyte-filled reservoirs in which target particles are dispersed. By applying a voltage over the channel the particles are drawn through it, and their passage is detected as a current drop. The magnitude of the current dip is directly correlated with
∗ Corresponding author at: Nanomed Labs, Advanced Biotechnology Center, Largo Rosanna Benzi, 10 16133 Genova, Italy. Tel.: +39 0105737382; fax: +39 0105737382. E-mail address: [email protected] (V. Mussi). 0749-6036/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.spmi.2008.09.003
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V. Mussi et al. / Superlattices and Microstructures 46 (2009) 59–63
Fig. 1. TEM images of a single solid state nanopore (A) and an array of nanopores (B) produced on a 100 nm thick Si3 N4 suspended membrane by an Ultra High Resolution Field Emission Scanning Electron Microscopy (UHR-FE-SEM) and Focused Ion Beam (FIB).
the volume of the particle, so that this analytical technique allows determining the concentration and size distribution of the dispersed particles. The results presented in [2] demonstrates the possibility to scale this behaviour to the nanometre regime by using α -hemolysin, a protein secreted by Staphilococcus aureous, which self-assembles into a lipid bilayer featuring a 1.4 nm transmembrane biological pore. A step forward in this direction was made in [4] to identify individual DNA strands with singlebase resolution by means of ‘‘engineered nanopores’’. This was achieved by covalently binding of DNA oligonucleotides (probes) near the entrance of the pore’s lumen, so to partially obstruct the nanopore and reduce its ion conductivity. When a short complementary oligonucleotide (target) is drawn into the lumen of the nanopore by voltage bias, it is likely to form a DNA duplex with the tethered oligomer, producing a characteristic current reduction. On the other hand, a non complementary oligonucleotide is rapidly drawn by the voltage bias into the constriction of the transmembrane region, producing a marked current reduction that signals molecular translocation without duplex formation. In this way, engineered biological nanopore devices have been used to sequence a codon in a single molecule of DNA [4], or to detect both single-base pair and single-nucleotide differences between molecules [5]. Although it has been proved that biological pores are useful for a range of applications, many groups focused the attention on the possibility to fabricate nanopores from solid-state materials which present obvious advantages, such as: (i) very high stability, (ii) control of diameter and channel length, (iii) adjustable surface properties and (iv) potential for integration into devices and arrays [6,7]. In this context, we succeeded in using an Ultra High Resolution-Field Emission-Scanning Electron Microscopy (UHR-FE-SEM) and Focused Ion Beam (FIB) to produce both single solid state nanopores (Fig. 1A) and regular arrays of nanopores (Fig. 1B) on a 100 nm thick Si3 N4 insulating membranes deposited on a Si chip. A nanopore chip can then be mounted in a measuring apparatus made of two
V. Mussi et al. / Superlattices and Microstructures 46 (2009) 59–63
61
Fig. 2. A typical I /V curve measured on a single nanopore by means of a patch clamp amplifier.
polydimethylsiloxane (PDMS) chambers filled with KCl 1 M solution buffered at pH 8 with 10 mM HEPES, placed in a double Faraday cage enclosure to reduce electrical noise. Two Ag/AgCl electrodes are used to apply a voltage bias across the membrane and to collect the ion current with picoampere sensitivity by means of a patch clamp amplifier (Axopatch 200B, Axon Instruments). Fig. 2 shows a typical I /V curve measured on a single nanopore in the range 50–250 mV with a 100 kHz sampling rate and the amplifier internal low-pass fourpole Bessel filter set at 10 kHz. A linear fit of the curve allows to obtain an estimation of the electrical resistance of the measuring apparatus, that is, by neglecting the low electrodes and cell resistance, the nanopore resistance R ∼ 12 M. The value of R can then be used to deduce information on the nanopore dimension, provided that we are able to make some hypothesis on its shape and geometry. If we approximate the nanopore as a constant diameter channel, with a fixed length l equal to the Si3 N4 membrane thickness, we can introduce an ‘‘effective diameter’’, deff , given by:
r deff = 2 ·
l
σ ·π ·R
where σ = 10 S/m is the KCl 1 M solution conductivity at 25 ◦ C. The resistance obtained by fitting the data presented in Fig. 2 thus corresponds to an effective diameter of ∼33 nm. This value can be compared with the diameter deduced from the SEM image of the pore presented in Fig. 3, which is about 32 nm. By following the approach proposed in [4] for biological nanopores, we developed a protocol to chemically functionalize the nanopore surface with specific oligonucleotide probes. This would allow a hybridization experiment with target molecules in the solution and driven towards the nanopore by the applied voltage. In principle, the duplex formation between probe molecules and complementary target at the nanopore surface can then be detected by monitoring the ionic current. The details of the functionalization protocol will be given in a further publication. Briefly, Si3 N4 membranes were activated with 3-aminopropyltriethoxysilane, then treated with the cross-linker 1,4-phenylene-diisotiocyanate and then with amino-modified 45-mer oligonucleotide (Eurofins MWG Operon, Germany). The DNA-functionalized nanopores where then mounted on the measuring apparatus to determine the modification induced by the organic layer on the ionic current. Our results show a quite large and stable enhancement of the initial pore resistance. Fig. 4 presents two typical I /V curves measured on the same chip before (black points) and after (red points) the probe bound. The comparison between the two resistance values was used to evaluate the effective diameter reduction due to the pore obstruction caused by the probe molecules. For all the measured nanopores we obtained an effective
62
V. Mussi et al. / Superlattices and Microstructures 46 (2009) 59–63
Fig. 3. SEM image of a FIB drilled nanopore on a Si3 N4 membrane. The circle highlights the pore contour to deduce an estimation of the diameter.
Fig. 4. Typical I /V curves measured on the same nanopore chip before (squares) and after (circles) the DNA covalent attachment which produces a reduction of the pore effective diameter, i.e. an enhancement of its electrical resistance.
radius reduction between 10 and 18 nm (18 nm for the chip of Fig. 4). These data are in quite good agreement with the expected ones for a DNA probe molecule of 45 bases, that is a total length of 15 nm, even if we also noted a surface charge induced effect on the ionic current. Finally, data of Fig. 5 demonstrate the stability of the diameter reduction as measured for few days after the functionalization. In conclusion our results demonstrate that solid state nanopores can be stably functionalized by DNA molecules making it possible to conceive and develop a selective biosensor for gene expression profiling. We foresee developing robust solid-state nanopores, as a high throughput platform for routine molecular diagnosis of tumors and genetic diseases.
V. Mussi et al. / Superlattices and Microstructures 46 (2009) 59–63
63
Fig. 5. The stability of the diameter reduction as measured for few days after the functionalization procedure, indicated by the arrow.
Acknowledgment This work has been supported by Ministero dell’Università e della Ricerca (MIUR), Italy. References [1] M. Schena, Microarray Biochip Technology, Eaton Publishing, 2000. [2] J.J. Kasianowicz, E. Brandin, D. Branton, D.W. Deamer, Proceedings National Academy of Sciences United States of America 93 (1996) 13770–13773. [3] W.H. Coulter, Proceedings of the National Electronics Conference, 1957, pp. 1034–1042. [4] S. Howorka, S. Cheley, H. Bayley, Nature Biotechnology 19 (2001) 636–639. [5] S. Winters-Hilt, M. Landry, M. Akeson, M. Tanase, I. Amin, A. Coombs, E. Morales, J. Millet, C. Baribault, S. Sendamangalam, Cheminformatics BMC Bioinformatics 7 (2006) S22. [6] J. Li, D. Stein, C. McMullan, D. Branton, M.J. Aziz, J.A. Golovchenko, Nature 412 (2001) 166–169. [7] C.J. Lo, T. Aref, A. Bezryadin, Nanotechnology 17 (2006) 3264–3267.
Contents lists available at ScienceDirect
Superlattices and Microstructures journal homepage: www.elsevier.com/locate/superlattices
Solid state nanopores for gene expression profiling V. Mussi a,b,∗ , P. Fanzio a,b , L. Repetto a,b , G. Firpo a,b , U. Valbusa a,b , P. Scaruffi c , S. Stigliani c , G.P. Tonini c a
Nanomed Labs, Advanced Biotechnology Center, Largo Rosanna Benzi, 10 16133 Genova, Italy
b
Physics Department University of Genova, Via Dodecaneso, 33 16146 Genova, Italy
c
Translational Paediatric Oncology, National Institute for Cancer Research (IST) Largo R. Benzi, 10 Genova, 16132, Italy
article
info
Article history: Available online 10 October 2008 Keywords: Nanopore Functionalization Ionic current Biosensor Gene expression
abstract Recently, nanopore technology has been introduced for genome analysis. Here we show results related to the possibility of preparing ‘‘engineered solid state nanopores’’. The nanopores were fabricated on a suspended Si3 N4 membrane by Focused Ion Beam (FIB) drilling and chemically functionalized in order to covalently bind oligonucleotides (probes) on their surface. Our data show the stable effect of DNA attachment on the ionic current measured through the nanopore, making it possible to conceive and develop a selective biosensor for gene expression profiling. © 2008 Elsevier Ltd. All rights reserved.
The Human Genome project allowed the identification of new genes and Expressed Sequence Tags (EST) that will complete the decoding of the human genome (http://www.ornl.gov/hgmis/). In the meantime, new technologies for genome analysis have been developed, i.e. microarray technology, able to analyze ten of thousands genes in a single experiment. The microarray technology is based on the principle that single strand DNA (ssDNA) molecules attached to solid supports can hybridize to complementary sequences. Microarray devices are utilized to characterize gene expression profiles, to detect point mutations and Single Nucleotide Polymorphisms (SNPs), to identify gains or losses in chromosome regions [1]. Recently [2], nanopore technology has been introduced for genome analysis. The use of nanopores for the detection and analysis of single molecules has been inspired by the Coulter counter [3] working principle: a small channel separates two electrolyte-filled reservoirs in which target particles are dispersed. By applying a voltage over the channel the particles are drawn through it, and their passage is detected as a current drop. The magnitude of the current dip is directly correlated with
∗ Corresponding author at: Nanomed Labs, Advanced Biotechnology Center, Largo Rosanna Benzi, 10 16133 Genova, Italy. Tel.: +39 0105737382; fax: +39 0105737382. E-mail address: [email protected] (V. Mussi). 0749-6036/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.spmi.2008.09.003
60
V. Mussi et al. / Superlattices and Microstructures 46 (2009) 59–63
Fig. 1. TEM images of a single solid state nanopore (A) and an array of nanopores (B) produced on a 100 nm thick Si3 N4 suspended membrane by an Ultra High Resolution Field Emission Scanning Electron Microscopy (UHR-FE-SEM) and Focused Ion Beam (FIB).
the volume of the particle, so that this analytical technique allows determining the concentration and size distribution of the dispersed particles. The results presented in [2] demonstrates the possibility to scale this behaviour to the nanometre regime by using α -hemolysin, a protein secreted by Staphilococcus aureous, which self-assembles into a lipid bilayer featuring a 1.4 nm transmembrane biological pore. A step forward in this direction was made in [4] to identify individual DNA strands with singlebase resolution by means of ‘‘engineered nanopores’’. This was achieved by covalently binding of DNA oligonucleotides (probes) near the entrance of the pore’s lumen, so to partially obstruct the nanopore and reduce its ion conductivity. When a short complementary oligonucleotide (target) is drawn into the lumen of the nanopore by voltage bias, it is likely to form a DNA duplex with the tethered oligomer, producing a characteristic current reduction. On the other hand, a non complementary oligonucleotide is rapidly drawn by the voltage bias into the constriction of the transmembrane region, producing a marked current reduction that signals molecular translocation without duplex formation. In this way, engineered biological nanopore devices have been used to sequence a codon in a single molecule of DNA [4], or to detect both single-base pair and single-nucleotide differences between molecules [5]. Although it has been proved that biological pores are useful for a range of applications, many groups focused the attention on the possibility to fabricate nanopores from solid-state materials which present obvious advantages, such as: (i) very high stability, (ii) control of diameter and channel length, (iii) adjustable surface properties and (iv) potential for integration into devices and arrays [6,7]. In this context, we succeeded in using an Ultra High Resolution-Field Emission-Scanning Electron Microscopy (UHR-FE-SEM) and Focused Ion Beam (FIB) to produce both single solid state nanopores (Fig. 1A) and regular arrays of nanopores (Fig. 1B) on a 100 nm thick Si3 N4 insulating membranes deposited on a Si chip. A nanopore chip can then be mounted in a measuring apparatus made of two
V. Mussi et al. / Superlattices and Microstructures 46 (2009) 59–63
61
Fig. 2. A typical I /V curve measured on a single nanopore by means of a patch clamp amplifier.
polydimethylsiloxane (PDMS) chambers filled with KCl 1 M solution buffered at pH 8 with 10 mM HEPES, placed in a double Faraday cage enclosure to reduce electrical noise. Two Ag/AgCl electrodes are used to apply a voltage bias across the membrane and to collect the ion current with picoampere sensitivity by means of a patch clamp amplifier (Axopatch 200B, Axon Instruments). Fig. 2 shows a typical I /V curve measured on a single nanopore in the range 50–250 mV with a 100 kHz sampling rate and the amplifier internal low-pass fourpole Bessel filter set at 10 kHz. A linear fit of the curve allows to obtain an estimation of the electrical resistance of the measuring apparatus, that is, by neglecting the low electrodes and cell resistance, the nanopore resistance R ∼ 12 M. The value of R can then be used to deduce information on the nanopore dimension, provided that we are able to make some hypothesis on its shape and geometry. If we approximate the nanopore as a constant diameter channel, with a fixed length l equal to the Si3 N4 membrane thickness, we can introduce an ‘‘effective diameter’’, deff , given by:
r deff = 2 ·
l
σ ·π ·R
where σ = 10 S/m is the KCl 1 M solution conductivity at 25 ◦ C. The resistance obtained by fitting the data presented in Fig. 2 thus corresponds to an effective diameter of ∼33 nm. This value can be compared with the diameter deduced from the SEM image of the pore presented in Fig. 3, which is about 32 nm. By following the approach proposed in [4] for biological nanopores, we developed a protocol to chemically functionalize the nanopore surface with specific oligonucleotide probes. This would allow a hybridization experiment with target molecules in the solution and driven towards the nanopore by the applied voltage. In principle, the duplex formation between probe molecules and complementary target at the nanopore surface can then be detected by monitoring the ionic current. The details of the functionalization protocol will be given in a further publication. Briefly, Si3 N4 membranes were activated with 3-aminopropyltriethoxysilane, then treated with the cross-linker 1,4-phenylene-diisotiocyanate and then with amino-modified 45-mer oligonucleotide (Eurofins MWG Operon, Germany). The DNA-functionalized nanopores where then mounted on the measuring apparatus to determine the modification induced by the organic layer on the ionic current. Our results show a quite large and stable enhancement of the initial pore resistance. Fig. 4 presents two typical I /V curves measured on the same chip before (black points) and after (red points) the probe bound. The comparison between the two resistance values was used to evaluate the effective diameter reduction due to the pore obstruction caused by the probe molecules. For all the measured nanopores we obtained an effective
62
V. Mussi et al. / Superlattices and Microstructures 46 (2009) 59–63
Fig. 3. SEM image of a FIB drilled nanopore on a Si3 N4 membrane. The circle highlights the pore contour to deduce an estimation of the diameter.
Fig. 4. Typical I /V curves measured on the same nanopore chip before (squares) and after (circles) the DNA covalent attachment which produces a reduction of the pore effective diameter, i.e. an enhancement of its electrical resistance.
radius reduction between 10 and 18 nm (18 nm for the chip of Fig. 4). These data are in quite good agreement with the expected ones for a DNA probe molecule of 45 bases, that is a total length of 15 nm, even if we also noted a surface charge induced effect on the ionic current. Finally, data of Fig. 5 demonstrate the stability of the diameter reduction as measured for few days after the functionalization. In conclusion our results demonstrate that solid state nanopores can be stably functionalized by DNA molecules making it possible to conceive and develop a selective biosensor for gene expression profiling. We foresee developing robust solid-state nanopores, as a high throughput platform for routine molecular diagnosis of tumors and genetic diseases.
V. Mussi et al. / Superlattices and Microstructures 46 (2009) 59–63
63
Fig. 5. The stability of the diameter reduction as measured for few days after the functionalization procedure, indicated by the arrow.
Acknowledgment This work has been supported by Ministero dell’Università e della Ricerca (MIUR), Italy. References [1] M. Schena, Microarray Biochip Technology, Eaton Publishing, 2000. [2] J.J. Kasianowicz, E. Brandin, D. Branton, D.W. Deamer, Proceedings National Academy of Sciences United States of America 93 (1996) 13770–13773. [3] W.H. Coulter, Proceedings of the National Electronics Conference, 1957, pp. 1034–1042. [4] S. Howorka, S. Cheley, H. Bayley, Nature Biotechnology 19 (2001) 636–639. [5] S. Winters-Hilt, M. Landry, M. Akeson, M. Tanase, I. Amin, A. Coombs, E. Morales, J. Millet, C. Baribault, S. Sendamangalam, Cheminformatics BMC Bioinformatics 7 (2006) S22. [6] J. Li, D. Stein, C. McMullan, D. Branton, M.J. Aziz, J.A. Golovchenko, Nature 412 (2001) 166–169. [7] C.J. Lo, T. Aref, A. Bezryadin, Nanotechnology 17 (2006) 3264–3267.