KR101681951B1 - Nanofluidic fluorescence apertureless near-field microscopy - Google Patents

Nanofluidic fluorescence apertureless near-field microscopy Download PDF

Info

Publication number
KR101681951B1
KR101681951B1 KR1020100039815A KR20100039815A KR101681951B1 KR 101681951 B1 KR101681951 B1 KR 101681951B1 KR 1020100039815 A KR1020100039815 A KR 1020100039815A KR 20100039815 A KR20100039815 A KR 20100039815A KR 101681951 B1 KR101681951 B1 KR 101681951B1
Authority
KR
South Korea
Prior art keywords
fluorescence
narrow space
field microscope
nano
antenna
Prior art date
Application number
KR1020100039815A
Other languages
Korean (ko)
Other versions
KR20110120415A (en
Inventor
천홍구
Original Assignee
서울대학교산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 서울대학교산학협력단 filed Critical 서울대학교산학협력단
Priority to KR1020100039815A priority Critical patent/KR101681951B1/en
Priority to PCT/KR2011/003008 priority patent/WO2011136527A2/en
Publication of KR20110120415A publication Critical patent/KR20110120415A/en
Application granted granted Critical
Publication of KR101681951B1 publication Critical patent/KR101681951B1/en

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
    • G01Q60/00—Particular types of SPM [Scanning Probe Microscopy] or microscopes; Essential components thereof
    • G01Q60/18—SNOM [Scanning Near-Field Optical Microscopy] or apparatus therefor, e.g. SNOM probes
    • G01Q60/20—Fluorescence

Abstract

Nanofluidic-based fluorescence near-field microscopes are provided. The fluorescence near-field microscope is configured to concentrate an incident light source in a narrow space, to change a quantum yield of a fluorescent sample existing in the narrow space, and to concentrate the fluorescence signal output generated in the narrow space in a specific direction. And a nanopore or nanochannel connected to the narrow space of the nano-antenna to provide a moving path for introducing the fluorescent sample into the narrow space. As a result, it is possible to concentrate the incident light source in a specific narrow space of the nano antenna, increase the low quantum yield of the fluorescent sample, efficiently detect the fluorescence signal output in a specific direction, enable high signal to noise ratio and high resolution fluorescence detection, By introducing a fluorescent sample through a pore or a nanochannel to the nanotenna, the sample can be scanned without mechanical movement of the nanotenna. The nanofluidics-based fluorescence near-field microscope can perform DNA sequence analysis by linearly passing the fluorescence-labeled DNA through the nanopore or nanochannel and reading the fluorescence signal sequentially generated in the narrow space.

Description

[0001] Nanofluidic fluorescence apertureless near-field microscopy [0002]

The present invention relates to a fluorescent sample detection system and a method thereof, and more particularly, to a fluorescent sample detection system capable of detecting a high signal-to-noise ratio and a high-resolution fluorescence by combining a nanopore and a nanopore (or nanochannel) And a fluorescence near-field microscope based on nanofluidics.

The most important performance indicators of DNA sequencing method are DNA read length and throughput. Recently, direct DNA sequencing method using current measurement in nanopore or nanochannel has been attracting attention because of its high spatial resolution, high throughput, and theoretically unlimited read length. However, the detection sensitivity of a sample in a microchannel or a nanochannel is generally highest in fluorescence signal detection. However, due to the diffraction limit, the excitation light source can not be collected sufficiently small to read each nucleotide sequence. For example, a 488 nm laser can not be focused to a size below 244 nm. Since the distance between DNA bases is 0.33 nm, this 244 nm focused excitation light source exits at 700 or more bases at once, making deconvolution of the output signal difficult. Therefore, this diffraction limit must be overcome for high sensitivity, single-molecule DNA sequencing.

Near-field scanning optical microscopy (NSOM) allows local excitation and detection beyond the diffraction limit using nanometer-sized antennas smaller than the wavelength of the incident light source, resulting in a resolution of approximately 10 nm . The biggest advantage of a nano-antenna is that it can enhance the optical field by concentrating the incident light source in a specific narrow space like a lens. In the meantime, the nano antenna has two other advantages than the lens. First, the low quantum yield of the fluorescent sample increases in the narrow space of the nano antenna. Second, the output of the fluorescence signal generated in the narrow space is directional So that the fluorescence signal can be efficiently detected in a specific direction.

These three effects - high signal-to-noise ratio and high-resolution fluorescence detection of the fluorescent near-field microscope are possible by the incident light concentration and amplification, the increase in the yield of the fluorescent sample quantum yield, and the directionality of the fluorescence signal output.

To perform DNA sequencing using a fluorescence near-field microscope, the fluorescence-labeled DNA must be linearly stretched over the sample stage and fixed, and then a two-dimensional image acquired. It is a complicated process to linearly expand and fix the DNA on the sample stage without overlapping. Mechanical scanning of the nano antenna for 2D image acquisition is complex and takes a long time. Therefore, it is necessary to develop a fluorescence near-field microscope that does not require mechanical scanning and DNA spreading and fixation process of the nano-antenna and can increase the image acquisition speed.

Since DNA is a linear polymer, image acquisition for sequencing does not need to be a two-dimensional image through mechanical scanning. When a nanopore (or nanochannel) is placed on a nano-antenna and DNA is linearly passed through it, a one-dimensional image is obtained. This nano-antenna-nanopore (or nanochannel) integrated structure can precisely position the fluorescent sample on the nano-antenna, eliminating elements such as a two-dimensional mechanical scanning stage, position feedback control, and incident light source focus alignment in a conventional fluorescence near- can do.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method of detecting a high signal-to-noise ratio and a high-resolution fluorescence by combining a nano-antenna and a nanopore (or a nanochannel) Scanning microscope based on nanofluidics, and a method therefor.

In order to achieve the above object, a nanofluidics-based fluorescence near-field microscope according to the present invention is characterized by focusing an incident light source in a narrow space, changing a quantum yield of a fluorescent sample existing in the narrow space, A nanoantenna configured to concentrate the fluorescence signal output in a specific direction; And a nanopore or nanochannel connected to the narrow space of the nano-antenna to provide a moving path for introducing the fluorescent sample into the narrow space.

The nano-antenna may be composed of two or more adjacent conductors.

In addition, the nano-antennas may have one or more concentric circular structures.

In addition, the nano-antenna may be a dipole antenna composed of a yttrium silicide (YSi 2 ) nanowire.

The nano-antenna may be a dipole antenna composed of carbon nanotubes.

In addition, the fluorescence near-field microscope can analyze the DNA base sequence by reading fluorescent signals sequentially generated in the narrow space while linearly passing the fluorescence-labeled DNA through the nanopore or nano channel.

A method of detecting a fluorescent sample using a fluorescence near-field microscope according to the present invention is characterized in that the fluorescence near-field microscope focuses the incident light source in a narrow space, changes the quantum yield of the fluorescent sample existing in the narrow space, A nanoantenna configured to focus the fluorescence signal generated in the narrow space in a specific direction; And a nanopore or nanochannel connected to the narrow space of the nano-antenna to provide a moving path for introducing the fluorescent sample into the narrow space.

Meanwhile, the method of analyzing the DNA base sequence using the fluorescence near-field microscope according to the present invention is characterized in that the fluorescence near-field microscope focuses the incident light source in a narrow space, changes the quantum yield of the fluorescent sample existing in the narrow space A nano-antenna configured to focus the fluorescence signal generated in the narrow space in a specific direction; And a nanopore or nanochannel connected to the narrow space of the nano-antenna to provide a moving path for introducing the fluorescent sample into the narrow space.

As described above, according to the present invention, a nanofluidic-based fluorescent near-field microscope capable of detecting a high signal-to-noise ratio and high-resolution fluorescence by combining a nano-antenna and a nanopore (or a nanochannel) Configuration is possible. In addition, it can be applied to DNA sequencing because it does not require mechanical scanning and DNA spreading and fixing process of nano antenna, and can increase 1-dimensional image acquisition speed.

1 is a block diagram of a nano-antenna-nanofoam integrated system according to an embodiment of the present invention;
FIG. 2 is a view illustrating a manufacturing process of a nano-antenna-nanochannel integration system,
FIG. 3 is a view provided to explain various nano-antenna structures of a nano-antenna-nanofoam integrated system,
FIG. 4 is a diagram provided for explaining a DNA sequencing method using a nanofluidics-based fluorescence near-field microscope.

Hereinafter, the present invention will be described in detail with reference to the drawings.

1 is a block diagram of a nano-antenna-nanofoam integrated system according to an embodiment of the present invention. The nano-antenna integrated system shown in FIG. 1 includes a nano-antenna and a nanopore that provides a moving path for introducing a fluorescent sample into the nano-antenna, and focuses the incident light source on a specific narrow space of the nano- It is possible to efficiently detect the fluorescence signal output in a specific direction by increasing the low quantum yield of the fluorescent sample and to realize high signal-to-noise ratio and high-resolution fluorescence detection, and by introducing the fluorescent sample into the nanotube through the nanoforer, It is a system that enables the scanning of samples without mechanical movement.

As shown in the side view 10a and the bottom view 10b of FIG. 1, the nano-antenna integration system according to the present embodiment includes a nanomembrane support layer 11, a nanomembrane 12, a nanopore 14 And a nano antenna 13a.

The nanomembrane support layer 11 is a layer for fixing the nanomembrane 12 in which the nanopore 14 is open.

The nanomembrane 12 is a thin film where the nanophoton 13a and the nanopore 14 are located.

The nano antenna 13a is a structure configured to concentrate an incident light source in a narrow space, change the quantum yield of a fluorescent sample existing in the narrow space, and concentrate the fluorescence signal generated in the narrow space in a specific direction. The nano antenna 13a according to the present embodiment is a dipole antenna.

The nanopore 14 is a path for introducing a fluorescent sample into the narrow space of the nano-antenna.

FIG. 2 is a view illustrating a manufacturing process of a nano-antenna-nanochannel integration system according to an embodiment of the present invention. The nano-antenna integrated system shown in FIG. 2 includes a nano-antenna and a nanochannel that provides a moving path for introducing a fluorescent sample into the nano-antenna, and focuses the incident light on a specific narrow space of the nano-antenna It is possible to efficiently detect the fluorescence signal output in a specific direction by increasing the low quantum yield of the fluorescent sample and to provide a high signal-to-noise ratio and high-resolution fluorescence detection. By introducing the fluorescent sample into the nanotenna through the nanotube, It is a system that enables the scanning of samples without mechanical movement.

As shown in the side views 20a, 20c, 20e and 20g and the top views 20b, 20d, 20f and 20h of FIG. 2, the nano antenna- A nanoanode cover layer 24, a nanochannel 25, a nanochannel sample inlet 26 and a nanochannel sample outlet 27. The nanochannel cover layer 24,

The first stage side view 20a and the top view 20b show a state in which the nano antenna 13a is flat on the nano antenna support layer 21. [

The second-stage side view 20c and the top view 20d show the step of covering the processed nano-antenna 13a with the nano-antenna cover thin-film layer 23.

The third-stage side view 20e and the top view 20f are formed on the nano-antenna cover layer 23, the nano-antenna 13a, the nano-channel support layer 21, the nanochannel 25, And the step of processing the channel sample outlet portion 27 is shown. If necessary, this step can be carried out without the nanotube antenna cover thin film layer 23 process. It is preferable that focused ion beam (FIB) milling is used for the process of the nanochannel 25, the nanochannel sample inlet part 26 and the nanochannel sample outlet part 27 in this step. The length of the nanochannel 25 is preferably longer than the diffraction limit of the incident light source, and the nanochannel sample introduction part 26 and the nanochannel sample exit part 27 are preferably microchannels. After the nanochannel 25, the nanochannel sample introduction part 26 and the nanochannel sample exit part 27 are completed, the nano antenna cover layer 24 is bonded.

The fourth stage side view 20g and the top view 20h show a state in which the nano antenna cover layer 24 is bonded. By bonding the nano antenna cover layer 24, the nano channel 25 is completed.

An advantage of the nano-antenna integrated system of the nano antenna-nanochannel integration system according to the present embodiment is that high signal-to-noise ratio fluorescence detection is possible. In the case of the nanofoil integrated system, since the incident light source is irradiated with the nano antenna through the space containing the sample, a high background fluorescence signal output may occur from the fluorescent sample existing in the space. If the length of the nanochannel 25 of the nano-antenna-nanochannel integrated system is made longer than the diffraction limit of the incident light source, the incident light source can be collected so as to be confined to the nanochannel 25, The channel sample introduction section 26 and the nanochannel sample outlet section 27 can be prevented from being irradiated with the incident light source. Therefore, the background fluorescence signal output from the nanochannel sample introducing portion 26 and the nanochannel sample outlet portion 27 can be largely lowered, resulting in high signal-to-noise ratio fluorescence detection.

FIG. 3 is a view illustrating a structure of various nano antennas of a nano-antenna-nanofoam integration system according to an embodiment of the present invention. The overall structure is the same as illustrated in Fig.

2 is different from the example of FIG. 1 in that the nano antenna 13a of FIG. 1 is composed of a bowtie-shaped nano antenna 13b and a concentric nano- And the nano-antenna 13c.

As shown in the side view 10c and the bottom view 10d of the nano-antenna integration system using the Bowtie nano antenna 13b, the incident light source is concentrated by the bowtie nano antenna 13b, A nanopore 14 for introducing the sample is located in a specific narrow region that enhances the quantum yield and directs the fluorescence signal output. The dipole antenna type 13a illustrated in FIG. 1 and the bowtie nano antenna 13b illustrated in FIG. 2 are examples in which a nano antenna is implemented by two or more adjacent conductors.

As shown in the side view 10e and the bottom view 10f of the nano-antenna-integrated nanopore integrated system using the concentric nano antenna 13c, the incident light source is concentrated by the concentric nano-antenna 13c, A nanopore 14 for introducing the sample is located in a specific narrow region that enhances the quantum yield and directs the fluorescence signal output.

FIG. 4 is a view for explaining a DNA sequencing method using a nanoparticle-based fluorescence near-field microscope according to an embodiment of the present invention.

The DNA sequencing method using the nanofluidics-based fluorescence microscopy based on the nanofluidics-based microscope shown in FIG. 4 is a specific narrow region in which the incident light source is concentrated by the nano-antenna, the low quantum yield of the fluorescent sample is increased, , A method of analyzing the DNA sequence by reading the fluorescent signal sequentially generated while linearly passing the fluorescently labeled DNA through the nanopore (or nanochannel).

As shown in FIG. 4, the DNA base sequence analysis method using the fluorescence near-field microscope based on the nanofluidic dynamic microscope according to the present embodiment includes a nanomembrane support layer 11, a nanomembrane 12, a nanopore 14, The fluorescent lamps 34a, 34g, 34t, and 34c, the incident light source 35i, the fluorescent signal output 35e, and the detector 36 are connected to the light source 13, the electrode 31, the power supply 32, the DNA 33, need.

The electrode 31 is for causing the output voltage or current of the power supply 32 to be applied across the nanofoer 14 to allow the DNA 33 to pass through the nanopore 14 by electrophoresis.

The power supply 32 is a system for applying voltage or current to control electrophoresis of the DNA 33.

The incident light source 35i is used to excite the fluorescent labels 34a, 34g, 34t, and 34c.

The fluorescent signal output 35e is a fluorescent signal of the fluorescent labels 34a, 34g, 34t, and 34c generated in a narrow space where the incident light source 35i is collected by the nano-antenna 13.

The detector 36 detects the fluorescent signal output 35e.

The fluorescent labels 34a, 34g, 34t and 34c are fluorescent substances attached to DNA for emitting fluorescence signal output 35e corresponding to A, G, T and C, respectively, and preferably have different output wavelengths. Each base interval of DNA (33) is 0.33 nm, and it is difficult to attach all bases fluorescence. Even if all bases can be attached with fluorescence, the close distance between each fluorescent label causes a fluorescence resonance energy transfer, which makes the analysis of the fluorescence signal output complicated. Therefore, it is preferable that the fluorescent labels are located at specific intervals. Or target DNA is augmented using Designed DNA polymer technology, and a fluorescent label is attached to a code corresponding to each base so that fluorescent labels (34a, 34g, 34t, 34c) corresponding to each base are spaced apart from each other desirable.

13: Nano antenna 14: Nano-pore
25: Nano channel 33: DNA
34: fluorescent label 36: detector

Claims (8)

As a nanofluidic-based fluorescence near-field microscope,
A nanoantenna configured to concentrate an incident light source in a narrow space, to change a quantum yield of a fluorescent sample existing in the narrow space, and to concentrate a fluorescence signal output generated in the narrow space in a specific direction;
A nanopore or nanochannel connected to the narrow space of the nano antenna to provide a moving path for introducing the fluorescent sample into the narrow space; And
And an electrode for holding an output voltage or current of the power supply at both ends of the nanopore or the nanochannel.
The fluorescence near-field microscope according to claim 1, wherein the nanotube comprises two or more adjacent conductors.
The fluorescence near-field microscope according to claim 1, wherein the nanotubes have one or more concentric circular structures.
The fluorescence near-field microscope according to claim 1, wherein the nanotube is a dipole antenna composed of a yttrium silicide (YSi 2 ) nanowire.
The fluorescence near-field microscope according to claim 1, wherein the nanotenna is a dipole antenna composed of carbon nanotubes.
The fluorescence near-field microscope according to claim 1, wherein the fluorescence-labeled DNA is linearly passed through the nanopore or nanochannel, and fluorescence signals sequentially generated in the narrow space are read to analyze the DNA base sequence.
A method for detecting a fluorescent sample using a fluorescence near-field microscope, wherein the fluorescence near-field microscope is a fluorescence near-field microscope according to claim 1.
A method for analyzing a DNA base sequence using a fluorescence near-field microscope, wherein the fluorescence near-field microscope is the fluorescence near-field microscope according to claim 1.

KR1020100039815A 2010-04-29 2010-04-29 Nanofluidic fluorescence apertureless near-field microscopy KR101681951B1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
KR1020100039815A KR101681951B1 (en) 2010-04-29 2010-04-29 Nanofluidic fluorescence apertureless near-field microscopy
PCT/KR2011/003008 WO2011136527A2 (en) 2010-04-29 2011-04-26 Nanofluidic fluorescence apertureless near-field scanning optical miscroscope

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020100039815A KR101681951B1 (en) 2010-04-29 2010-04-29 Nanofluidic fluorescence apertureless near-field microscopy

Publications (2)

Publication Number Publication Date
KR20110120415A KR20110120415A (en) 2011-11-04
KR101681951B1 true KR101681951B1 (en) 2016-12-05

Family

ID=44862027

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020100039815A KR101681951B1 (en) 2010-04-29 2010-04-29 Nanofluidic fluorescence apertureless near-field microscopy

Country Status (2)

Country Link
KR (1) KR101681951B1 (en)
WO (1) WO2011136527A2 (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013112698A1 (en) 2012-01-24 2013-08-01 Src, Inc. Methods and systems for long distance tagging, tracking, and locating using wavelength upconversion
US9718668B2 (en) 2012-02-16 2017-08-01 Board Of Trustees Of The University Of Arkansas Method of fabricating a nanochannel system for DNA sequencing and nanoparticle characterization
CN109261230B (en) * 2018-09-30 2020-05-08 东南大学 Monomolecular controllable output device of light-operated nanopore and use method thereof
CN114113017B (en) * 2021-11-29 2024-02-23 中国科学院重庆绿色智能技术研究院 Solid-state nanopore-based functional protein photoelectric combined detection method

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003016781A2 (en) * 2001-08-14 2003-02-27 The President And Fellows Of Harvard College Surface plasmon enhanced illumination system
US20070138376A1 (en) * 2005-08-24 2007-06-21 The Trustees Of Boston College Nanoscale optical microscope
JP2010503868A (en) * 2006-09-18 2010-02-04 アプライド バイオシステムズ， エルエルシー Method, system and apparatus for light concentrating mechanism

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7057151B2 (en) * 2001-08-31 2006-06-06 Universite Louis Pasteur Optical transmission apparatus with directionality and divergence control
EP2133688A1 (en) * 2008-06-11 2009-12-16 Koninklijke Philips Electronics N.V. Nanoantenna and uses thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003016781A2 (en) * 2001-08-14 2003-02-27 The President And Fellows Of Harvard College Surface plasmon enhanced illumination system
US20070138376A1 (en) * 2005-08-24 2007-06-21 The Trustees Of Boston College Nanoscale optical microscope
JP2010503868A (en) * 2006-09-18 2010-02-04 アプライド バイオシステムズ， エルエルシー Method, system and apparatus for light concentrating mechanism

Also Published As

Publication number Publication date
WO2011136527A2 (en) 2011-11-03
WO2011136527A9 (en) 2011-12-29
KR20110120415A (en) 2011-11-04
WO2011136527A3 (en) 2012-04-19

Similar Documents

Publication Publication Date Title
KR101233663B1 (en) Method and apparatus for enhanced nano-spectroscopic scanning
Hartschuh Tip‐enhanced near‐field optical microscopy
EP2356430B1 (en) Single molecule optical spectroscopy in solid-state nanopores in a transmission-based approach
Domke et al. Studying surface chemistry beyond the diffraction limit: 10 years of TERS
US8618508B2 (en) Detection system and method
WO2012165400A1 (en) Method and device for optical analysis of biopolymer
Toprak et al. New fluorescent tools for watching nanometer-scale conformational changes of single molecules
KR101681951B1 (en) Nanofluidic fluorescence apertureless near-field microscopy
AU2010214699A1 (en) Method and apparatus for enhanced nano-spectroscopic scanning
Meyer et al. Latest instrumental developments and bioanalytical applications in tip-enhanced Raman spectroscopy
Cang et al. Progress in single-molecule tracking spectroscopy
US7286224B2 (en) Time-multiplexed scanning light source for multi-probe, multi-laser fluorescence detection systems
Lu et al. Electro‐Optical Detection of Single Molecules Based on Solid‐State Nanopores
Oheim et al. Supercritical angle fluorescence microscopy and spectroscopy
WO2019176705A1 (en) Infrared analysis apparatus, infrared analysis chip, and infrared imaging device
Martiradonna et al. Spectral tagging by integrated photonic crystal resonators for highly sensitive and parallel detection in biochips
JP4887475B2 (en) System and method for using multiple detection channels to eliminate autofluorescence
Li et al. A versatile optical microscope for time-dependent single-molecule and single-particle spectroscopy
Dong et al. Spectroscopic analysis beyond the diffraction limit
Wenger Fluorescence spectroscopy enhancement on photonic nanoantennas
CN104880453A (en) Synchronous light-electricity sensing method for dark-field imaging-based solid nano channel
JP2009519464A (en) Sample analysis element
Umakoshi Near-field optical microscopy toward its applications for biological studies
Marcet et al. High spatial resolution confocal microscope with independent excitation and detection scanning capabilities
CHAIGNEAU et al. Tip-Enhanced Raman Spectroscopy

Legal Events

Date Code Title Description
A201 Request for examination
N231 Notification of change of applicant
E902 Notification of reason for refusal
E701 Decision to grant or registration of patent right