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Silicon Nanopore Arrays: Fabrication and Applications for DNA Sensing
KTH, School of Engineering Sciences (SCI), Applied Physics, Materials and Nanophysics. (Nano-Silicon group)ORCID iD: 0000-0002-8962-1844
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Nanopore biomolecule sensing and sequencing has emerged as a simple but powerful tool for single molecule studies over the past two decades. By elec- trophoretically driving single molecules through a nanometer-sized pore, often sitting in an insulating membrane that separates two buffer solutions, ionic current blockades can be detected to reveal rich information of the molecules, such as DNA length, protein size and conformation, even nucleic acid se- quence. Biological protein pores, as well as solid-state nanopores have been used, but both suffer from relatively low throughput due to the lack of abil- ity to scale up to a large array. In this thesis, we tackled the throughput issue from the fabrication aspect as well as from the detection aspect, aim- ing at a parallel optical single molecule sensing on an array of well-separated nanopores.

From the fabrication aspect, several lithography-based self-regulating meth- ods were tested to obtain nanopore arrays in silicon membranes, including anisotropic KOH etching, thermal oxidation-induced pore shrinkage, metal- assisted etching and electrochemical etching. Among those, the most success- ful method was the electrochemical etching of silicon. By electron-beam or photo lithography, the positions of the pores were defined on a silicon mem- brane. Followed by anisotropic KOH etching, inverted pyramids were formed as etching pits. The nanopores were then formed by anodic etching of silicon in HF. Using this concept, the size of the pores does not depend on the lithog- raphy step; only the positions of pores were defined by lithography. In this way, an array of ∼ 900 pores with an average entrance diameter of 18 ± 4 nm was fabricated on a 120 μm × 120 μm membrane.

From the detection aspect, parallel readout of fluorescence signals from the labelled DNA molecules while translocating through an array of nanopores was performed using a wide-field microscope with a relatively fast CMOS camera recording at 1 KHz frame rate. Statistics of duration and frequency of the translocation events were extracted and studied. It was found that the event duration decreases with rising excitation laser power. This can be attributed to a laser-induced heating effect. Simulation suggested that a sig- nificant thermal gradient was generated at the pore vicinity by the excitation laser due to photon absorption by the silicon membrane. Such temperature rise affects all mass transport in a solution via a viscosity change. The ther- mal effect has also been proven by that conductance of an array of nanopores scales with the laser power. The thermal effect on the translocation frequency has been studied systematically as well. Due to thermophoresis of DNA in a thermal gradient, the thermophoretic force serves as a repulsion force, op- posing the electrophoretic force at the pore vicinity, depleting molecules away from the pore. Because of the molecule-size-dependent thermal depletion, a size-dependent translocation frequency was observed. This can be potentially used for a high throughput molecule sorting by adjusting the balance between the thermophoretic force and the electrophoretic force.

Abstract [sv]

Detektion av biomolekyler med hjälp av nanoporer har under de senaste två decennierna vuxit fram som ett enkelt men kraftfullt verktyg för studi- er av enstaka molekyler. Genom att driva molekyerna elektroforetiskt (med elektriskt fält) genom en nanometer-stor por, som ofta sitter i ett isoleran- de membran som separerar två buffertlösningar, kan molekylerna detekteras genom att jonströmmen genom membranet delvis blockeras. Detta kan ge detaljerad information om de detekterade molekylerna, såsom t ex ett pro- teins storlek, längden på en DNA-sekvens, och även sekvensen på ingående nukleinsyror. Naturliga, biologiska protein-baserade porer, liksom nanoporer gjorda i fasta material har använts, men båda lider av relativt låg effektivitet på grund av svårigheten att skala upp tekniken till stora matriser av nano- porer. I denna avhandling, tacklas denna fråga från både tillverknings-sidan såväl som från detektions-sidan genom att använda en matris av nanoporer för parallell optisk detektering av enskilda molekyler vilket resulterar i en hög ge- nomströmningshastighet. Detta möjliggörs genom att nanoporerna separeras optiskt, dvs med några mikrometers mellanrum.

För att tillverka matriser av nanoporer i kisel-membran har flera olika självreglerande metoder undersökts experimentellt, bland annat anisotrop KOH etsning för att minska porstorleken, termisk oxidation för att krym- pa porer, metall-inducerad etsning samt elektrokemisk etsning. Bland dessa befanns den elektrokemiska metoden ge bäst resultat. För att bestämma posi- tionen av porerna på membranet användes optisk litografi eller elektronstråle- litografi varefter KOH etsning resulterade i pyramid-formade gropar. Dessa initierade sedan etsningen av nanoporer genom membranet i den efterföljan- de elektrokemiska etsprocessen. Genom detta förfarande bestäms inte stor- leken (diametern) på porerna av litografi-processen utan endast deras lägen på membranet. På detta sätt kunde 900 porer med en medel-diameter på 18 ± 4 nm tillverkas på ett membran av storleken 120 μm × 120μm.

Detektering av de fluorescens-inmärkta DNA-molekylerna utfördes paral- lellt genom avläsning av fluorescenssignaler när de passerade genom matrisen av nanoporor med hjälp av ett mikroskop med en relativt snabb CMOS- kamera med video-sekvenser på upp till 1 KHz. Statistiken över tiden för passage genom membranet av enskilda molekyler och frekvens av händelser- na extraherades och studerades. Det visade sig att tiden för passage mins- kade med ökande laser excitation. Detta kan hänföras till en laser-inducerad uppvärmnings-effekt. Uppskattning av denna effekt genom dator-simulering visade att en termisk gradient bildas i närheten av porerna på grund av upp- värmning av kisel-membranet genom absorption av laser-strålen. En sådan temperaturhöjning påverkar all mass-transport i en lösning via ändringar av viskositeten. Den termiska effekten bevisas också av att konduktansen genom matrisen av nanoporer ökar linjärt med lasereffekten. Den termiska effekten på frekvensen av molekyl-passager har också studerats systematiskt. På grund av termofores av DNA i en termisk gradient, verkar den termoforetiska kraf- ten som en repulserande kraft som motsätter sig den elektroforetiska kraften i porområdet. På grund av att utarmningen av molekyler vid porerna beror på storleken observerades en storleks-beroende passage-frekvens. Detta skulle potentiellt kunna användas för molekylsortering med hög genomströmnings- hastighet genom att justera balansen mellan den termoforetiska kraften och den elektroforetiska kraften.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2018. , p. 87
Series
TRITA-SCI-FOU ; 2018:18
Keywords [en]
nanopore, array, electrochemical etching, DNA, optics, thermophoresis
National Category
Nano Technology
Identifiers
URN: urn:nbn:se:kth:diva-228469ISBN: 978-91-7729-804-5 (print)OAI: oai:DiVA.org:kth-228469DiVA, id: diva2:1209929
Public defence
2018-06-15, Sal C, Electrum, Kistagången 16, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20180525

Available from: 2018-05-25 Created: 2018-05-24 Last updated: 2019-08-20Bibliographically approved
List of papers
1. Oxidation of nanopores in a silicon membrane: self-limiting formation of sub-10nm circular openings
Open this publication in new window or tab >>Oxidation of nanopores in a silicon membrane: self-limiting formation of sub-10nm circular openings
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2014 (English)In: Nanotechnology, ISSN 0957-4484, E-ISSN 1361-6528, Vol. 25, no 35, p. 355302-Article in journal (Refereed) Published
Abstract [en]

We describe a simple but reliable approach to shrink silicon nanopores with nanometer precision for potential high throughput biomolecular sensing and parallel DNA sequencing. Here, nanopore arrays on silicon membranes were fabricated by a self-limiting shrinkage of inverted pyramidal pores using dry thermal oxidation at 850 degrees C. The shrinkage rate of the pores with various initial sizes saturated after 4 h of oxidation. In the saturation regime, the shrinkage rate is within +/- 2 nm h(-1). Oxidized pores with an average diameter of 32 nm were obtained with perfect circular shape. By careful design of the initial pore size, nanopores with diameters as small as 8 nm have been observed. Statistics of the pore width show that the shrinkage process did not broaden the pore size distribution; in most cases the distribution even decreased slightly. The progression of the oxidation and the deformation of the oxide around the pores were characterized by focused ion beam and electron microscopy. Cross-sectional imaging of the pores suggests that the initial inverted pyramidal geometry is most likely the determining factor for the self-limiting shrinkage.

Place, publisher, year, edition, pages
Institute of Physics (IOP), 2014
Keywords
Solid-state nanopores, silicon, thermal oxidation
National Category
Nano Technology
Identifiers
urn:nbn:se:kth:diva-153850 (URN)10.1088/0957-4484/25/35/355302 (DOI)000341807600006 ()2-s2.0-84906085347 (Scopus ID)
Funder
Swedish Foundation for Strategic Research
Note

QC 20141013

Available from: 2014-10-13 Created: 2014-10-09 Last updated: 2018-05-24Bibliographically approved
2. Fabrication of ultra-high aspect ratio silicon nanopores by electrochemical etching
Open this publication in new window or tab >>Fabrication of ultra-high aspect ratio silicon nanopores by electrochemical etching
2014 (English)In: Applied Physics Letters, ISSN 0003-6951, E-ISSN 1077-3118, Vol. 105, no 12, p. 123111-Article in journal (Refereed) Published
Abstract [en]

We report on the formation of ultra-high aspect ratio nanopores in silicon bulk material using photo-assisted electrochemical etching. Here, n-type silicon is used as anode in contact with hydrofluoric acid. Based on the local dissolution of surface atoms in pre-defined etching pits, pore growth and pore diameter are, respectively, driven and controlled by the supply of minority charge carriers generated by backside illumination. Thus, arrays with sub-100 nm wide pores were fabricated. Similar to macropore etching, it was found that the pore diameter is proportional to the etching current, i.e., smaller etching currents result in smaller pore diameters. To find the limits under which nanopores with controllable diameter still can be obtained, etching was performed at very low current densities (several mu A cm(-2)). By local etching, straight nanopores with aspect ratios above 1000 (similar to 19 mu m deep and similar to 15 nm pore tip diameter) were achieved. However, inherent to the formation of such narrow pores is a radius of curvature of a few nanometers at the pore tip, which favors electrical breakdown resulting in rough pore wall morphologies. Lowering the applied bias is adequate to reduce spiking pores but in most cases also causes etch stop. Our findings on bulk silicon provide a realistic chance towards sub-10 nm pore arrays on silicon membranes, which are of great interest for molecular filtering and possibly DNA sequencing.

Keywords
N-Type Silicon, Formation Mechanism, Macroporous Silicon, Array Fabrication, Porous Silicon, Pore Arrays, Limits
National Category
Other Physics Topics
Identifiers
urn:nbn:se:kth:diva-155801 (URN)10.1063/1.4896524 (DOI)000343004400073 ()2-s2.0-84908300823 (Scopus ID)
Funder
Swedish Foundation for Strategic Research , RMA08-0090
Note

QC 20141113

Available from: 2014-11-13 Created: 2014-11-13 Last updated: 2018-05-24Bibliographically approved
3. Nanopore arrays in a silicon membrane for parallel single-molecule detection: fabrication
Open this publication in new window or tab >>Nanopore arrays in a silicon membrane for parallel single-molecule detection: fabrication
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2015 (English)In: Nanotechnology, ISSN 0957-4484, E-ISSN 1361-6528, Vol. 26, no 31, article id 314001Article in journal (Refereed) Published
Abstract [en]

Solid state nanopores enable translocation and detection of single bio-molecules such as DNA in buffer solutions. Here, sub-10 nm nanopore arrays in silicon membranes were fabricated by using electron-beam lithography to define etch pits and by using a subsequent electrochemical etching step. This approach effectively decouples positioning of the pores and the control of their size, where the pore size essentially results from the anodizing current and time in the etching cell. Nanopores with diameters as small as 7 nm, fully penetrating 300 nm thick membranes, were obtained. The presented fabrication scheme to form large arrays of nanopores is attractive for parallel bio-molecule sensing and DNA sequencing using optical techniques. In particular the signal-to-noise ratio is improved compared to other alternatives such as nitride membranes suffering from a high-luminescence background.

Keywords
nanopore, parallel, single-molecule
National Category
Nano Technology
Identifiers
urn:nbn:se:kth:diva-172675 (URN)10.1088/0957-4484/26/31/314001 (DOI)000358676300003 ()26180043 (PubMedID)2-s2.0-84937797128 (Scopus ID)
Funder
Swedish Foundation for Strategic Research , RMA08-0090
Note

QC 20150901

Available from: 2015-09-01 Created: 2015-08-27 Last updated: 2018-05-24Bibliographically approved
4. Nanopore arrays in a silicon membrane for parallel single-molecule detection: DNA translocation
Open this publication in new window or tab >>Nanopore arrays in a silicon membrane for parallel single-molecule detection: DNA translocation
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2015 (English)In: Nanotechnology, ISSN 0957-4484, E-ISSN 1361-6528, Vol. 26, no 31, article id 314002Article in journal (Refereed) Published
Abstract [en]

Optical nanopore sensing offers great potential in single-molecule detection, genotyping, or DNA sequencing for high-throughput applications. However, one of the bottle-necks for fluorophore-based biomolecule sensing is the lack of an optically optimized membrane with a large array of nanopores, which has large pore-to-pore distance, small variation in pore size and low background photoluminescence (PL). Here, we demonstrate parallel detection of single-fluorophore-labeled DNA strands (450 bps) translocating through an array of silicon nanopores that fulfills the above-mentioned requirements for optical sensing. The nanopore array was fabricated using electron beam lithography and anisotropic etching followed by electrochemical etching resulting in pore diameters down to similar to 7 nm. The DNA translocation measurements were performed in a conventional wide-field microscope tailored for effective background PL control. The individual nanopore diameter was found to have a substantial effect on the translocation velocity, where smaller openings slow the translocation enough for the event to be clearly detectable in the fluorescence. Our results demonstrate that a uniform silicon nanopore array combined with wide-field optical detection is a promising alternative with which to realize massively-parallel single-molecule detection.

Keywords
nanopore, parallel, single-molecule, optical
National Category
Nano Technology
Identifiers
urn:nbn:se:kth:diva-172676 (URN)10.1088/0957-4484/26/31/314002 (DOI)000358676300004 ()26180050 (PubMedID)2-s2.0-84937774164 (Scopus ID)
Funder
Swedish Foundation for Strategic Research , RMA08-0090Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20150901

Available from: 2015-09-01 Created: 2015-08-27 Last updated: 2018-05-24Bibliographically approved
5. Optical detection of two-color-fluorophore barcode for nanopore DNA sensing
Open this publication in new window or tab >>Optical detection of two-color-fluorophore barcode for nanopore DNA sensing
2015 (English)Conference paper, Published paper (Refereed)
Abstract [en]

A simple schematic on parallel optical detection of two-fluorophore barcode for single-molecule nanopore sensing is presented. The chosen two fluorophores, ATTO-532 and DY-521-XL, emitting in well-separated spectrum range can be excited at the same wavelength. A beam splitter was employed to separate signals from the two fluorophores and guide them to the same CCD camera. Based on a conventional microscope, sources of background in the nanopore sensing system, including membranes, compounds in buffer solution, and a detection cell was characterized. By photoluminescence excitation measurements, it turned out that silicon membrane has a negligible photoluminescence under the examined excitation from 440 nm to 560 nm, in comparison with a silicon nitrite membrane. Further, background signals from the detection cell were suppressed. Brownian motion of 450 bps DNA labelled with single ATTO-532 or DY-521-XL was successfully recorded by our optical system.

Series
Proceedings of SPIE, ISSN 0277-786X
National Category
Nano Technology Other Physics Topics
Identifiers
urn:nbn:se:kth:diva-171918 (URN)10.1117/12.2179626 (DOI)000357932000004 ()978-1-62841-642-8 (ISBN)
Conference
Conference on Nanotechnology VII, MAY 04-06, 2015, Barcelona, SPAIN
Note

QC 20150811

Available from: 2015-08-11 Created: 2015-08-10 Last updated: 2018-05-24Bibliographically approved
6. Thermophoresis-Controlled Size-Dependent DNA Translocation through an Array of Nanopores
Open this publication in new window or tab >>Thermophoresis-Controlled Size-Dependent DNA Translocation through an Array of Nanopores
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2018 (English)In: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 12, no 5, p. 4574-4582Article in journal (Refereed) Published
Abstract [en]

Large arrays of nanopores can be used for high-throughput biomolecule translocation with applications toward size discrimination and sorting at the single-molecule level. In this paper, we propose to discriminate DNA length by the capture rate of the molecules to an array of relatively large nanopores (50–130 nm) by introducing a thermal gradient by laser illumination in front of the pores balancing the force from an external electric field. Nanopore arrays defined by photolithography were batch processed using standard silicon technology in combination with electrochemical etching. Parallel translocation of single, fluorophore-labeled dsDNA strands is recorded by imaging the array with a fast CMOS camera. The experimental data show that the capture rates of DNA molecules decrease with increasing DNA length due to the thermophoretic effect of the molecules. It is shown that the translocation can be completely turned off for the longer molecule using an appropriate bias, thus allowing a size discrimination of the DNA translocation through the nanopores. A derived analytical model correctly predicts the observed capture rate. Our results demonstrate that by combining a thermal and a potential gradient at the nanopores, such large nanopore arrays can potentially be used as a low-cost, high-throughput platform for molecule sensing and sorting.

Keywords
array; capture rate; electrochemical etching; nanopore; silicon; sorting; thermophoresis
National Category
Nano Technology
Identifiers
urn:nbn:se:kth:diva-228463 (URN)10.1021/acsnano.8b00961 (DOI)000433404500054 ()2-s2.0-85047380512 (Scopus ID)
Note

QC 20180525

Available from: 2018-05-24 Created: 2018-05-24 Last updated: 2018-06-19Bibliographically approved

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