CN113533294B - Time domain, space domain and spectrum domain single molecule characterization device based on nanometer gap electrode pair - Google Patents

Abstract

The invention discloses a time domain, space domain and spectrum domain single molecule characterization device based on a nanometer gap electrode pair, which comprises the following components: the illumination system is provided with a laser, a reflecting mirror, a femtosecond laser, a pulse selector, a dichroic mirror, a half-wave plate, a quarter-wave plate, a beam splitter, a scanning galvanometer system and a microscope objective which are sequentially arranged along a light path; the probe and tunneling electricity test system comprises a nanogap probe, a three-dimensional nano precision moving platform and a tunnel signal detection module, wherein the three-dimensional nano precision moving platform controls the movement of the nanogap probe, and the tunnel signal detection module is used for collecting signal light; the ultrafast optical modulation tunneling detection system mainly comprises a laser light source, a light beam polarization modulation module, a focusing scanning module and a femtosecond time synchronization module; and the single-molecule Raman detection system is used for collecting a Raman radiation image emitted by the sample.

Description

Time domain, space domain and spectral domain single molecule characterization device based on nanogap electrode pair

technical field

The invention relates to the field of optoelectronic technology, in particular to a single-molecule characterization device in time domain, space domain and spectrum domain based on a pair of nano-gap electrodes.

Background technique

High temporal and spatial resolution characterization at the single-molecule scale is the key to the development of molecular optoelectronics, solving biological, chemical and medical challenges, and is also considered to be one of the keys to developing precision medicine and solving major medical challenges.

At present, it mainly relies on high-energy probes (such as transmission electron microscope, X-ray diffraction and scanning electron microscope), scanning probes (such as atomic force microscope, scanning tunneling microscope, spectral fingerprint probe, etc.), fluorescent probes and nanopore technology to achieve . High-energy probe technology has been maturely applied to the characteristics of surface properties, generally has a good spatial resolution, but the ability to analyze material properties is weak, especially requires special conditions such as vacuum conduction, and has great limitations in practical applications. In particular, the characterization and analysis of dynamic organisms and single-molecule change processes has not been realized so far.

Optical imaging technology has developed rapidly in recent years. The spatial resolution of fluorescently labeled samples can reach tens of nanometers. It does not require vacuum and ultra-low temperature conditions, and the sample observation conditions are friendly. However, single-molecule real-time, There are still great difficulties in dynamic tracking and in situ detection with non-fluorescent labels at the nanometer to subnanometer scale.

The nanopore single-molecule detection technology uses the nano-confined channel as a unit component, and externally applies an excitation signal (such as a constant potential) to measure the signal change (such as ion current) when the target passes through the nanopore, so as to realize the analysis of single molecules, with high Sensitive, label-free, single-molecule detection and other performances, but this technology generally can only provide quasi-steady-state single information, and the time resolution is mostly at the second level, which greatly limits the understanding of molecular system dynamics information.

In order to obtain temporal and spatial resolution information at the same time, a feasible solution is to introduce the pump-probe method into a spatially resolved characterization technique. At present, the pump-probe technology combined with electron microscope or scanning probe has shown its function. For time-resolved transmission electron microscopy combined with electron microscopy, the special environmental requirements limit its application in the fields of biological and chemical engineering. The time-resolved scanning tunneling microscope (STM) technology combined with the scanning probe uses ultrafast laser pulses to induce electrons in the sample to be in a non-equilibrium state, and combined with high spatial resolution imaging of tunneling electrons, the ultrafast dynamics of electronic excited state changes can be obtained. academic characteristics. However, this technology also has obvious defects. One is the lack of stable and reliable electrical probes, and optical excitation causes the instability of STM probes; The STM often causes the signal to be submerged in noise, so improving the tunneling current change caused by photoexcitation is a very critical issue in the experiment.

In addition, nanosensor devices have also received sufficient attention in recent years, including single-molecule detection technologies based on nanopores, tunneling sensing, and field-effect transistors, which have gained widespread attention and development. Among them, the measurement mode of tunneling sensing uses the sub-5nm gap electrode device as the analysis element to measure the change of tunneling current caused by applying a constant potential to measure the target, and is applied to DNA sequencing, single-molecule detection and single-molecule chemical reaction. Mechanism research is one of the important frontier fields of current world development. However, the tunneling sensing measurement method in the constant potential mode also has inevitable technical limitations, such as single signal, poor sensitivity, low selectivity, and inability to provide analyte dynamics information, which greatly limits quantum tunneling sensing. sense application.

SUMMARY OF THE INVENTION

The present invention provides a single molecule characterization method and device in the time domain, space domain and spectral domain based on the nano-gap electrode pair, using the multi-functional integrated nano-tunnel probe of the nano-gap to replace the metal tip of the traditional STM, and introducing femtosecond excitation The single-molecule tunneling technology uses a unique probe structure to realize the combination of plasmonic localized enhanced Raman probes and fluorescent probes, and then realizes the precise and multifunctional nanoscale spatiotemporal comprehensive characterization of single molecules.

The time domain, space domain and spectrum domain single molecule characterization device system based on the nano-gap electrode pair disclosed in the present invention mainly consists of a probe and tunneling electrical test system (subsystem one), an ultrafast light modulation tunneling detection system ( Subsystem 2) and single-molecule Raman detection system (subsystem 3) are composed of three detection subsystems. There are corresponding control processors between the three detection subsystems. All the control processors and computers together form the system control system. system (subsystem four).

1) Probe and tunneling electrical test system: The system consists of a nano-gap probe, a three-dimensional nano-precision mobile stage and a tunnel signal detection module.

Preferably, the nanogap probe can realize the analysis of single molecules at a smaller nanometer scale, and observe the change of tunneling current of single molecules passing through the probe electrode gap by applying a constant potential, which is capable of realizing DNA sequencing and single molecule detection. and the ability to study the mechanism of single-molecule chemical reactions. At the same time, combining with ultrafast light modulation can further improve the time resolution; the three-dimensional nano-precision mobile stage can precisely move the nano-probe to the required position to detect the nano-scale of the sample. Scanning and high-precision sub-nanometer positioning; the high-precision tunneling signal detection module can realize the time delay and precise synchronization of the pulsed laser associated with the control system and the femtosecond laser.

2) Ultrafast optical modulation tunneling detection system: mainly includes laser light source and beam polarization modulation module, confocal scanning module and femtosecond laser pumping-detecting module.

This subsystem can combine the tunneling electrical detection platform with the femtosecond laser pump-detection system, which can provide multi-parameter signal output (such as tunneling current, fluorescence spectrum, fluorescence imaging, etc.) Single-molecule analysis at high resolution.

Preferably, the light source control module includes a titanium sapphire laser femtosecond pulse high repetition frequency tunable laser, a collimation and beam expansion unit, a wavefront control unit, a two-dimensional high-precision scanning unit and a confocal focusing unit. After the femtosecond laser beam is expanded and collimated, a pulse selector is used to select the number of pulses, and a spatial light modulator or polarizer is used to regulate the beam, so as to generate different pump photon states and form a stronger excitation signal code. At the same time, the pulsed laser passes through the confocal scanning module and focuses on the needle tip of the sample through the high numerical aperture objective lens, which can induce the local fluorescence effect and the change of the tunneling junction barrier. The change of each pulse indicates the ability of the system to detect the excitation and transition of carriers, and the frequency of the pulse reflects the continuous change of this state. The femtosecond laser pulse synchronization signal will be output to control the single-molecule photoelectric integrated detection system for tunneling sensing to realize nanoprobe-tunneling detection and time gating using femtosecond pulse waves.

3) Single-molecule Raman detection module: This invention uses the metal nanostructure of the tunnel junction to construct a special needle-tip surface plasmon wave local field enhancement characteristic, which can be combined with fluorescence and Raman spectrum detection technology to obtain the fingerprint spectrum of molecules in the tunnel junction information.

Preferably, the present invention adopts a DC laser. After the laser beam emitted by the DC laser is expanded and collimated, it passes through a high numerical aperture objective lens, converges and irradiates the sample and the electrode probe to generate localized plasmon waves, and induces the pull of the sample. Mann radiation. After the Raman radiation is collected, the Raman monochromatic system forms a spectral image, and the area array EMCCD camera is used to detect the spectral signal to realize real-time high-resolution imaging and spectral analysis.

Based on the plasmon enhancement effect, its electromagnetic field intensity distribution and Raman enhancement exponential attenuation characteristics have near-field enhancement effect, which can greatly improve the Raman molecular signal and reduce the interference of background light noise. When the molecule passes through the tunnel junction, the molecular probe with Raman activity can locate and track the molecule to be detected in real time. Using methods such as adding Raman or fluorescence on the surface, the interaction between the perforated molecule and the tunnel junction can be reflected through the changes in the Raman fingerprint information, light intensity, wavelength, and lifetime of the molecule, so as to achieve the purpose of spectroscopic monitoring of the molecule passing through the tunnel junction, and the interaction with the DC tunnel junction. Wear sensing analysis techniques complement each other.

4) System control system: It mainly includes four parts: light source controller, light spot modulation controller, scanning controller, and signal detection controller, all of which are connected to the computer.

Preferably, the high-precision synchronization and time delay of the control system in time, control the sub-nanometer movement and alignment of the moving and scanning system, control the detection of spectrum, image detection, and tunneling current signal detection.

The opto-electrical comprehensive characterization method for single molecules based on nano-gap electrode pairs disclosed in the present invention uses nanoprobes to detect tunneling currents to achieve high-precision detection of single molecules, and utilizes ultrafast optical modulation tunneling detection systems to detect tunneling currents. The combination of the electrical detection platform and the femtosecond laser pump-detection system can provide multi-parameter signal output (such as tunneling current, fluorescence spectrum, fluorescence imaging, etc.) for the measurement process, and realize single-molecule analysis with higher temporal and optical resolution; Utilizing the surface plasmon wave local field enhancement characteristics of the probe tip, combined with fluorescence and Raman spectroscopy detection technology, the fingerprint spectrum information of molecules in the tunnel junction can be obtained, so as to realize the characterization of single molecules in the time domain, space domain and spectral domain.

Description of drawings

Fig. 1 is a device system diagram of the present invention;

2 is a schematic diagram of the structure of a single-molecule detection device with a pair of nano-gap electrodes;

Fig. 3 is a conductance-time variation graph of the tunneling electrode in the presence of single-stranded DNA molecules.

Detailed ways

In the following description, many specific details are set forth in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described here, therefore, the present invention is not limited to the specific embodiments disclosed below limit.

The following describes in detail the embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals refer to the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the figures are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.

As shown in Figure 1, the multifunctional single molecule characterization device system device based on the sub-5 nanometer gap electrode pair in this embodiment includes: a laser 1 with a wavelength of 632nm, a single-mode polarization-maintaining fiber 2a and a single-mode polarization-maintaining fiber 2b, Collimator beam expander 3a and collimator beam expander 3b, reflector 4a, reflector 4b and reflector 4c, femtosecond laser 5, pulse picker 6, dichroic mirror 7a and dichroic mirror 7b, one-half Wave plate 8, quarter wave plate 9, beam splitter 10, scanning galvanometer system 11, lens 12a, lens 12b and lens 12c, field lens 13, microscope objective lens 14, sample stage 15, 20-60 times collection Objective lens 16, CCD 17, nano-gap electrode tunneling probe 18, optical filter 19, multimode optical fiber 20a and multimode optical fiber 20b, detector avalanche photodiode APD21, Raman spectrometer 22, three-dimensional nanometer precision mobile stage 23, tunnel junction A single molecule analyzer 24, a synchronous control FPGA (Field Programmable Gate Array) board 25, a signal acquisition FPGA board 26, and a computer 27.

Among them, the pulse selector 6 is used to obtain femtosecond laser pulses with different delay times, and can obtain continuous time delays in the range of femtoseconds to microseconds, so as to obtain dynamic information of the excited state of the sample and improve the time resolution to femtoseconds.

Wherein, the microscope objective lens 14 is a high numerical aperture objective lens, and the numerical aperture NA is 1.4; the collection objective lens 16 and the CCD 17 are used to obtain wide-field imaging information of the sample.

Among them, nanogap generally refers to the holes, channels or passages formed or provided in other ways in the material, and then connected to the electrode to access the sensing area, and can be used to measure the change of tunneling current caused by single molecule (as shown in Figure 2) . At the same time, the nano-metal gap probe can generate localized plasmon excitations and induce enhanced Raman radiation; the three-dimensional nano-precision mobile stage 23 is used to precisely move the nano-probe to the required position for nano-scanning of the detection sample and high-precision sub-nanometer positioning.

Among them, the Raman spectrometer is used to receive the Raman radiation signal enhanced by the localized plasmon wave of the probe to realize Raman spectrum detection.

Wherein, realize the synchronous control to laser 1, femtosecond laser 5, pulse picker 6, scanning mirror system 11, three-dimensional nano-precision mobile platform 23 and signal acquisition FPGA board 26 by computer control synchronous control FPGA board 25, and signal acquisition The FPGA board 26 can synchronously collect the wide-field signal of the CCD17, the electrical signal of the tunnel junction single molecule analyzer 24, the fluorescent signal of the confocal excitation detection of the detector avalanche photodiode APD21, the spectral signal of the Raman spectrometer 22, and related information The data is transmitted to the computer 27 and communicated to the user.

Using the device shown in Figure 1 to obtain time-domain information for single-molecule characterization is as follows:

The device includes a tunneling current detection module that can use the traditional DC constant potential mode to record the tunneling current. By using the ion current detection technology, the tunneling single molecule can provide molecular intrinsic property information, including molecular substructure and energy level structure information . The tunneling detection module is simplified and expressed in Fig. 1, which is composed of a nanoprobe 18 and a tunnel junction single molecule analyzer 24, while the single molecule detection embodiment expressed in Fig. 2 shows in detail the device of the present invention to obtain a single molecule pointer in the time domain Information, the process of detecting tunneling current.

2 is a schematic diagram of the structure of a single-molecule detection device with nano-gap electrode pairs. The probe is composed of sub-nano-gap electrode pairs 28a-28b and dielectrics 29a-29b. Terminal A is near the sample and terminal B is the current detection terminal. The single molecule analyzer is composed of a measuring power supply 30 , electrophoretic electrode pairs 31 a - 31 b , an electrophoretic power supply 32 , a current amplification module 35 , an ammeter 36 and a control unit 33 . The control unit 33 can control the measurement power supply 30, the electrophoresis power supply 32 and the current amplification module 35, and is responsible for controlling the bias voltage of the tunnel probe and the amplification and detection of the current signal, so as to realize the detection of tunneling electrical information, and transfer the amplified current to the The signal is collected in the FPGA board 26.

In the embodiment of DNA single-strand detection, as shown in FIG. 2( b ), the electrophoretic power supply 32 is used to set the voltage of the appropriate pair of electrophoretic electrodes 31a-31b, so as to control the movement of single molecules through the tunnel junction. The measuring power supply 30 can apply a voltage to the electrodes of the nanogap electrode pairs 28a-28b. When the single molecule 34 passes through the tunnel junction, the nanogap electrode pairs 28a-28b generate a tunneling current, and the current flows from A to A in the nanogap electrode pairs 28a-28b. Conducted to the B terminal, so as to facilitate the measurement of the current at the B terminal. Terminal B is connected to the current amplification module 35 , and the ammeter 36 measures the amplified tunneling current and sends the current to the signal acquisition FPGA board 26 . When the analyte passes through the tunneling junction (nano-electrode gap) under the action of the driving force, the tunneling current changes instantaneously, which causes the conductance to change, and its size and duration reflect the information of the analyte. Statistical analysis enables the detection of analytes. Figure 3 is the conductance-time diagram generated when the single-stranded DNA molecule continues to pass through the tunneling electrode with a gap of about 1.1 nanometers. When the DNA molecule is in the middle of the electrode gap, a spiked pulse signal (tunneling current) can be detected , so the change of current (conductance) reflects the information corresponding to each single molecule passing through the electrode pair.

In order to improve the time resolution and spatial resolution of tunnel current detection, the device can also realize optically coupled tunnel current detection through femtosecond laser pump-probe. The device uses a confocal system for excitation to achieve improved spatial resolution. The femtosecond laser 5 outputs the femtosecond laser through the single-mode polarization-maintaining fiber 2b and enters the pulse selector 6 after passing through the collimating beam expander 3b. The pulse selector can control the pulse delay of the femtosecond laser through different delay settings. After being modulated by the pulse picker, the excitation light enters the scanning galvanometer system 11 after being reflected by the dichroic mirror 7a and the dichroic mirror 7b to realize the scanning of the excitation light on the sample surface, and is scanned by the half-wave plate 8 and The quarter-wave plate 9 is modulated into circularly polarized light (or other required polarized light). After passing through the 4f system composed of the lens 12 a and the field lens 13 , the femtosecond excitation light is focused onto the tip of the nanoprobe 18 by the microscope objective lens 14 . Femtosecond laser excitation produces changes in the potential barrier of the tunneling junction, forming a rapid change, and then under the control of the tunneling junction single molecule analyzer 24, the fast and accurate electrical signal detection of the tunneling electrode realizes the time resolution of the tunneling current detection significant increase in rate.

Using the device shown in Figure 1 to obtain single-molecule characterization spatial information is as follows:

The present invention can obtain fluorescence information of single molecule characterization based on the confocal system of the device itself. After the excitation light passes through the above-mentioned confocal system, it can be focused by the microscope objective lens 14 onto the sample fixed on the sample stage 15. The sample The generated fluorescence can first be collected by the collection objective lens 16 and then received by the CCD17 and collected into the signal acquisition FPGA board 26, and can be collected by the microscope objective lens 14 at the same time, after passing through the 4f system of the field lens 13 and the lens 12a again, and then passed through the scanning vibration Mirror system solution scan. Then the fluorescent signal passes through the dichromatic mirror 7b, and after the stray light is filtered out by the filter sheet 19, it is collected by the multimode optical fiber 20a and sent to the detector APD21, and finally the fluorescent information is collected by the signal acquisition FPGA board. Under the excitation detection, the resolution of the spatial domain is improved.

In addition, the present invention can also use the plasma excitation of the nanoprobe to obtain the spatial information of the single molecule. Under appropriate voltage control or femtosecond excitation light excitation, the nanoprobe 18 and the sample realize localized plasma excitation in the near field, and the excited optical signal passes through the field lens 13 and the mirror after being collected by the microscope objective lens 14 4b, lens 12a, scanning galvanometer system 11, dichroic mirror 7b and optical filter 19, collected by multimode optical fiber 20a and detector APD21 after being converged by lens 12b, and three-dimensional nano-precision mobile stage 23 is controlled by synchronously controlling FPGA board Realize the high-precision movement of the probe, so as to realize the scanning of the sample and obtain the ultra-high resolution spatial information of the sample.

The method for obtaining single-molecule characterization spectral domain information using the device shown in Figure 1 is as follows:

The laser 1 with a wavelength of 632nm outputs the excitation light through the single-mode polarization-maintaining fiber 2a. After being collimated and expanded by the collimator beam expander 3a and reflected by the mirror 4a, it passes through the dichromatic mirror 7a and merges with the femtosecond excitation light optical path, and enters the to the confocal system. After passing through the above-mentioned optical path, the microscopic objective lens is focused between the nanoprobe and the sample to generate localized plasmon waves, which induce the Raman radiation of the sample, and the Raman radiation signal is collected by the microscopic objective lens 14 again. , through the field lens 13, the mirror 4b, the lens 12a, the scanning galvanometer system 11, after being reflected by the beam splitter 10, enters the Raman spectrum detection optical path, is reflected by the mirror 4c, and the lens 12c is focused into the multimode optical fiber 20b for transmission Collect and analyze in the Raman spectrometer 22, and the final spectral information is collected by the signal acquisition FPGA board 26.

The above descriptions are only examples of the preferred implementation of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention within.

Claims (6)

1. A time domain, space domain and spectral domain single molecule characterization device based on the nanogap electrode pair, characterized in that it comprises: Probe and tunneling electrical test system, including nano-gap probe, three-dimensional nano-precision mobile stage and tunnel junction single-molecule analyzer; the movement of nano-gap probe is controlled by the three-dimensional nano-precision mobile stage; under appropriate voltage control or femtosecond excitation Under optical excitation, the nanogap probe and the sample realize local plasmon excitation in the near field, and the excited optical signal is collected by the microscope objective lens and then passed through the field mirror, mirror, lens, scanning galvanometer system, and dichroic mirror. And the filter is collected by the detector, and the high-precision movement of the nano-gap probe is realized by controlling the three-dimensional nano-precision mobile stage, and the ultra-high-resolution spatial information of the sample is obtained by scanning the sample; The ultrafast optical modulation tunneling detection system realizes optically coupled tunneling current detection through femtosecond laser pump-detection. The femtosecond laser output from the femtosecond laser enters the pulse picker after passing through the collimating beam expander. The pulse picker The pulse delay of the femtosecond laser is controlled by different delay settings; after being modulated by the pulse picker, the excitation light enters the scanning galvanometer system to scan the excitation light on the sample surface, and is scanned by the half-wave plate and The quarter-wave plate is modulated into circularly polarized light, which is then focused on the tip of the nanoprobe by the microscope objective lens; the excitation stimulation of the femtosecond laser produces a change in the barrier of the tunnel junction, forming a rapid change, and then passing through the tunnel junction Under the control of the molecular analyzer, the fast and accurate electrical signal detection of the tunneling electrode realizes a significant improvement in the time resolution of the tunneling current detection; The single-molecule Raman detection system is used to collect the Raman radiation images emitted by the sample; the laser is collimated and expanded by the collimator beam expander, passes through the dichromatic mirror and merges with the femtosecond excitation light path, and enters the confocal system; The microscopic objective lens is focused between the nanogap probe and the sample to generate localized plasmon waves, which induce the Raman radiation of the sample. The Raman radiation signal is collected by the microscopic objective lens again, and passed through the field mirror, reflector, and lens. , The scanning galvanometer system, after being reflected by the beam splitter, enters the Raman spectrum detection optical path, and then transmits it to the Raman spectrometer for collection and analysis. 2. The time domain, space domain and spectral domain single molecule characterization device based on the nanogap electrode pair according to claim 1, characterized in that, after the femtosecond laser beam is collimated through beam expansion, a pulse selector is used to select the number of pulses , spatial light modulators or polarizers for beam modulation, in order to generate different pump photon states, forming stronger excitation signal encoding. 3. The time domain, space domain and spectral domain single molecule characterization device based on the nanogap electrode pair according to claim 2, wherein the pulsed laser passes through a confocal system and is focused to the nanogap probe through a high numerical aperture objective lens On the tip of the needle, the localized fluorescence effect and the change of the tunnel junction barrier are induced; The change of each pulse indicates the ability of the system to detect the excitation and transition of carriers, and the frequency of the pulse reflects the continuous change of this state. 4. The time domain, space domain and spectral domain single molecule characterization device based on the nanogap electrode pair according to claim 1, characterized in that, in the single molecule Raman detection system, a DC laser is used, and after beam expansion quasi- After straightening, the laser passes through the high numerical aperture objective lens, converges and irradiates the sample and the electrode probe, generates local plasmon waves, and induces the Raman radiation of the sample; the Raman radiation is collected and passed through the Raman monochromatic system to form a spectral image , using an area array EMCCD camera to detect spectral signals. 5. The time domain, space domain and spectral domain single molecule characterization device based on the nanogap electrode pair according to claim 1, further comprising a control system, specifically comprising a light source controller, a spot modulation controller, a scan control The four parts of the device and the signal detection controller are all connected with a computer. 6. The time-domain, space-domain and spectral-domain single-molecule characterization device based on the nanogap electrode pair according to claim 5, characterized in that, the high-precision synchronization and time delay of the control system in time control the detection of the spectrum, Image detection, tunneling current signal detection.

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