Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Nanopore arrays in a silicon membrane for parallel single-molecule detection: DNA translocation
KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics, Material Physics, MF.ORCID iD: 0000-0002-8962-1844
KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics, Material Physics, MF.
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-2219-0197
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
Show others and affiliations
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.

Place, publisher, year, edition, pages
2015. Vol. 26, no 31, article id 314002
Keyword [en]
nanopore, parallel, single-molecule, optical
National Category
Nano Technology
Identifiers
URN: urn:nbn:se:kth:diva-172676DOI: 10.1088/0957-4484/26/31/314002ISI: 000358676300004PubMedID: 26180050Scopus ID: 2-s2.0-84937774164OAI: oai:DiVA.org:kth-172676DiVA, id: diva2:850310
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
In thesis
1. Silicon Nanopore Arrays: Fabrication and Applications for DNA Sensing
Open this publication in new window or tab >>Silicon Nanopore Arrays: Fabrication and Applications for DNA Sensing
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
Keyword
nanopore, array, electrochemical etching, DNA, optics, thermophoresis
National Category
Nano Technology
Identifiers
urn:nbn:se:kth:diva-228469 (URN)978-91-7729-804-5 (ISBN)
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: 2018-05-25Bibliographically approved

Open Access in DiVA

No full text in DiVA

Other links

Publisher's full textPubMedScopus

Authority records BETA

Sychugov, IlyaLinnros, Jan

Search in DiVA

By author/editor
Zhang, MiaoSchmidt, TorstenJemt, AndersSahlén, PelinSychugov, IlyaLundeberg, JoakimLinnros, Jan
By organisation
Material Physics, MFGene TechnologyScience for Life Laboratory, SciLifeLab
In the same journal
Nanotechnology
Nano Technology

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 682 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf