WO2004055503A1

WO2004055503A1 - A raman scattering near-field enhanced method and sample cell

Info

Publication number: WO2004055503A1
Application number: PCT/CN2003/001072
Authority: WO
Inventors: Shifa Wu; Guanying Wu
Original assignee: Shifa Wu; Guanying Wu
Priority date: 2002-12-16
Filing date: 2003-12-16
Publication date: 2004-07-01
Also published as: CN100386620C; AU2003289654A1; CN1425910A

Abstract

A supersensitive Raman scattering near-field enhanced sample cell includes two parallel substrates, both of which are transparent and have an index of refraction greater than that of the sample. The substrates' inner surface is plated by silver or gold film with the thickness of several nanometers in which silver or gold colloidal solids in the size of nanometer or nano-structures are arranged. The two boundary surfaces of said cell, between which are filled with the sample, can be clamped to a space with the distance of several ten to several hundred nanometers, to permit the laser exciting beam, having an incident angle exceeding the critical angle thereof, to produce evanescent wave, which in turn excites the sample and make it exit Raman scattering in the main form of evanescent wave. The application also relates to a Raman scattering near-field enhanced method.

Description

Raman scattering near field enhancement method and sample cell

The present invention relates to the field of spectrum analysis and testing instruments, and in particular, to a super-sensitive Raman scattering near-field enhancement (RSNE) method and a sample cell for which excitation and reception are both performed using evanescent light.

Background technique

In the spectral analysis technology, molecular Raman scattering has a very limited application because its scattering cross section is too small (approximately 10 ^'29 cm ² ); while the fluorescence analysis cross section is relatively large (approximately 1 (T ¹⁰ _cm ² ), there are already Large application areas, such as gene chip can be labeled with fluorescent molecular probes, and time-resolved immunofluorescent labeling for virus diagnosis. These methods have mature methods and instruments, but there are still shortcomings and disadvantages: (1) The analysis process is complex and requires The time is too long, and it is usually calculated in days. (2) The sample size is too large, such as blood samples, which are usually measured in milliliter. Can one finger refer to the amount of blood (such as only one microliter) and the time in minutes to complete some The diagnosis of important diseases such as various cancers, AIDS or other important infectious diseases is an issue that needs to be addressed. '

The ultra-sensitive Raman scattering near-field enhanced analysis technology will likely achieve this ideal. The advantages of this method are as follows: (1) The Raman characteristic spectrum is very narrow, so its measurement dynamic range is more than a thousand times the fluorescence with a broader characteristic spectrum. Therefore, the fluorescence detection has to be remedied by the chip method of many detection cells. A fluorescent label sample needs to be used. One chip unit, so the blood sample needs to be large enough. Raman detection can detect multiple signatures with only one drop of blood, without using a chip method; (2) As long as the Raman signatures of the diagnostic target molecule (related to the virus, etc.) can be detected, it can be compared through a database in the computer. The diagnostic results can be output immediately, and the general impurity components are less likely to interfere with the diagnosis of target molecules under the conditions of known peak positions of the characteristic spectrum; (3) The Raman characteristic spectrum is determined by the dynamic characteristics of molecular earthquakes, without any labeling, fluorescence or immune labeling There is also the question of whether they can be marked superior.

However, the key is to design an ultra-sensitive Raman scattering near-field enhancement analysis system. Over the past thirty years, it has been found that under the condition that the target molecules are adsorbed on the rough surface of noble metal such as silver, gold, copper, or the colloidal surface of this metal nanoparticle, a strong characteristic Raman spectrum signal can be obtained by using a confocal microscope Raman spectrometer. Raman spectroscopy can be used to diagnose the molecular vibration characteristics of samples. It is a new technique for ultra-micro chemical (molecular) analysis. In the past, because the conventional Raman signal was weak, it should be The use is extremely limited (only a very small number of Raman scattering cross-sections, that is, substances with strong Raman scattering activity). A few years ago, the combination of scanning near-field optical microscopy and modern Raman spectrometers was used to further discover "hot spots" in surface-enhanced Raman scattering (SERS) tests, that is, light-induced resonance excites Ag or Au particles. The local area of "Plasmon" with extremely enhanced light-excitation electromagnetic field in the "hot spot" can be thousands of times higher than the incident photoelectric magnetic field, so its Raman signal enhancement can be more than 10 times than the constant Raman signal. This value is called It is a Raman enhancement factor, and it has been experimentally estimated that it has reached 10 ¹⁴ . To date, it is generally acknowledged that this very complex augmented mechanics community has not fully figured it out, and there is great potential for development.

Conventional Raman spectrometers and micro-Raman spectrometers combined with confocal microscopes have mature products abroad. Near-field Raman spectrometers combined with near-field optical microscopes also have many experimental research results, but near-field Raman Technology and instruments are far from mature and commercialized.

Norihiko Hayazawa et al. In Chemical Physics Letters 335 (2001) 369-374—proposed the use of a metal tip to enhance near-field Raman scattering (Near-field Raman scattering enhanced by a metallized tip), as shown in Figure 5, through NA: 1.4 The hollow cone-shaped beam of the microscope objective lens with a numerical aperture of 1 <NA <1.4 excites the Raman spectrum of the sample, and the received signal (Signal) is set within NA <1; however, the metal tip enhances the emission Raman light at the metal The sharp axial direction (within NA <1) is mainly the near-field loss of light, while within NA <1, only the far-field light that can be transmitted can be received, and the relationship between the emission and the reception is not matched, so the efficiency is very low.

M. Futamata et al. Also proposed a system in Chemical Physics Letters 341 (2001) 425-430, which uses ATR (attenuated total reflection) to stimulate samples to produce evanescent light, as shown in Figure 6, although the system uses both excitation and reception Lost light, but because the fiber tip (Probe) is used to receive the lost light, the solid angle at which the fiber tip receives the lost light is very small, only a small part of the upward emission, the downward and the other azimuth solid angles The transmitted Raman light is not received, so the reception efficiency is also low.

Summary of the Invention

Reflective micro Raman spectrometers are excited by and transmittable light (far-field light). They are a type of far-field light with low excitation and reception efficiency. Field light receiving Raman spectrometer, put liquid sample on silver or gold rough surface, or put silver or gold glue For bulk particles, there can be a degree of surface-enhanced Raman scattering (SERS). If a Raman Scattering Near Field Enhancement (RSNE) sample cell is designed to use excitation and reception on the micro Raman spectrometer, both efficient near-field evanescent light excitation and efficient near-field evacuation are used. With light reception, a more effective Raman enhancement and a larger Raman enhancement factor will be obtained.

The purpose of the present invention is to use the near-field optics and Raman near-field enhancement new concepts and new developments to invent an ultra-highly sensitive excitation and reception Raman scattering near-field enhanced sample cell with hidden light, which overcomes the above problems. There are deficiencies in the technical part that increase the Raman scattering enhancement factor.

According to an aspect of the present invention, an ultra-high-sensitivity excitation and reception method is used to enhance the sample cell with evanescent light Raman scattering near field, and the excitation and reception are used to enhance the sample cell with evanescent light Raman scattering near field. It is characterized by comprising two transparent substrates with parallel surfaces and a refractive index greater than the refractive index of the sample, the inner surface of the substrate is plated with a few nanometers of thick silver or gold film, and nanoscale silver or gold colloids are distributed between the silver or gold films. Particles or nanostructures. The two interfaces of the sample cell can clamp the sample to a distance of tens to hundreds of nanometers in order to allow the excitation laser beam with an incident angle exceeding the critical angle to generate evanescent light. Exciting the sample and emitting the main form of evanescent light. Raman scattered light, this sample cell is suitable for liquid samples, nano powder samples and thin film samples.

The sample cell also includes a light-collecting element connected to the substrate, and the evanescent light emitted by the sample Raman scattering toward the two substrates is collected in the light-collecting element in a transmittable forbidden light form. '

At least one of the above-mentioned light-collecting elements is an over-focus truncated ellipsoidal reflector or a large-aperture oil-immersed objective lens, so as to focus the forbidden light that is converted by the sample's Raman scattering and evanescent light into the interior and collect and couple it into the light. Man spectrometer.

At least one of the above-mentioned light collecting elements is an over-focus truncated parabolic reflector (replaces the above-over-focus truncated ellipsoidal mirror) or a large-aperture oil immersion objective with infinite back focal length (replaces the conventional back-focus large-aperture oil immersion objective) Objective lens) is used to make the forbidden light converted into the interior of the sample by the Raman scattering and evanescent light coupled into the Raman spectrometer in a parallel beam.

One of the above-mentioned light collecting elements is an over-focus truncated hemispherical reflector or a hemispherical reflector or a reflector thickened and modified by the silver or gold film of the substrate, and is used to collect toward one of the substrates of the sample cell. The emitted Raman scattered light is reflected by the sample cell and connected by another substrate Connected to the light collecting element.

One of the above-mentioned light-collecting elements is provided with a small hole in the side reflection layer for introducing a fine laser beam with an incident angle exceeding a critical angle, or a large numerical aperture oil immersion objective lens is used to introduce a laser beam with an incident angle exceeding the critical angle.

According to another aspect of the present invention, there is provided a Raman scattering near-field enhancement method in which both Raman scattering excitation and reception use evanescent light, including the following steps.

Two transparent substrates with parallel surfaces and a refractive index greater than the refractive index of the sample are provided to form a sample cell. The inner surface of the substrate is plated with a few nanometers of silver or gold thin film, and nanoscale silver or gold colloid particles are distributed between the silver or gold films. Or structure

A liquid sample or a nano powder sample or a thin film sample is placed between the two interfaces of the sample cell, and the sample is clamped to a distance of tens to hundreds of nanometers.

Wherein, each substrate is provided with a light collecting element connected to the substrate, the outer wall of the light collecting element is plated with a metal reflective layer and a small hole is introduced in the side reflective layer of one of the light collecting elements to introduce a thin laser beam or A laser beam is introduced into the aperture oil immersion objective lens, and the incident angle of the introduced laser beam exceeds a critical angle in order to generate a hidden light excitation sample in the sample cell.

Wherein, one of the above-mentioned light collecting elements is an over-focus truncated hemispherical reflector or a hemispherical reflector or a reflector thickened and changed by the silver or gold film of the substrate, and the direction of one of the substrates of the sample cell is Raman scattered light is collected by a light collecting element that is reflected to the other substrate direction of the sample cell.

Among them, at least one of the two light collecting elements of the sample cell is an over-focus truncated ellipsoidal mirror or an over-focus truncated parabolic object mirror or a large-aperture oil immersion objective, and is used to The Raman scattered light of the sample in the 4 IT solid angle is collected in all directions from the substrate and coupled into the Raman spectrometer.

According to the effective volume of the excitation sample of the present invention, when collecting light with a very large numerical aperture oil immersion objective lens, it is a micrometer diameter of the focal spot of the microscope, and when collecting light with an over-focus truncated ellipsoidal reflector, the diameter can be as large as hundreds of microns. Scanning near-field optical microscope-Raman spectrometer with a thickness of several tens to hundreds of nanometers, according to which the number of excited Raman hot spots and the number of excited sample molecules has only one metal tip (only one hot spot, in pursuit of high spatial resolution) will be 3-4 orders of magnitude higher, Raman light's excitation efficiency and reception efficiency Compared with the above Raman spectrometer, the rate is expected to be two orders of magnitude higher. Therefore, the detection of Raman scattered light using the RSNE sample cell of the present invention can reach an ultra-high level of sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a Raman scattering near-field enhancement (RSNE) sample cell that uses evanescent light for both excitation and reception according to the present invention;

FIG. 2 is one of the preferred embodiments of the Raman scattering near field enhanced sample cell according to the present invention; FIG. 3 is the second of the preferred embodiments of the Raman scattering near field enhanced sample cell according to the present invention; FIG. 4 is according to the present invention The third preferred embodiment of the Raman scattering near-field enhanced sample cell; FIG. 5 is a drawing of a paper by Norihiko Hayazawa and others in the prior art;

Figure 6 is a drawing of a paper by M. Futamata and others in the prior art.

detailed description

As shown in FIG. 1, the sample cell of the present invention is composed of two upper and lower substrates whose surfaces are substantially parallel. The substrate is made of a transparent material with a refractive index greater than that of the sample and is plated with a thin, approximately It is a thin silver or gold film 2 with a thickness of about 3-5 nanometers for enhancing the loss of light field strength. The silver or gold film on the lower substrate is provided with nano-scale silver or gold particles 4, and the particle size and its difference do not exceed The optimized control value given by numerical simulation. The very large particles are used to support the control of the sample cell spacing and are controlled to a small number and uniform distribution to ensure that the most uniformly distributed silver or gold particles exist on the interface of the sample cell. The controlled spacing of a few nanometers leaves room for the extreme value of the electric field to enhance the so-called "hot spots". .

The upper and lower interfaces of the sample cell clamp the liquid sample 5. The diameter of the sample cell is selected in millimeters or sub-millimeters. The upper and lower interface substrates of the sample cell require ultra-high-precision planar optical polishing.

The P-polarized total internal reflection fine laser beam 1 of the excitation sample is incident on the lower substrate silver or gold film through the aperture of the light-blocking layer, and the incident angle exceeds the critical angle of total internal reflection, and is then led out by another aperture of the light-blocking layer to improve Signal-to-noise ratio of Raman scattered light. Of course, it is also possible to use a large-aperture oil immersion objective to introduce a laser beam with an incident angle exceeding the critical angle of total internal reflection.

·· The total internal reflection laser beam generates plasmon and evanescent waves on the silver or gold film of the lower substrate. The schematic diagram of the evanescent light is shown in reference numeral 3 in the right illustration, where the amplitude of the electric field (abscissa) and the longitudinal distance (ordinate) of the lower interface are approximately exponentially attenuated. Under the interaction of nano-scale silver or gold particles, the evanescent field will generate so-called "hot spots" with enhanced electric field extremes in the vicinity of the particles. With the enhancement of the "hot spot" electric field, the electric field gradient will also increase. The Raman scattering of 5 molecules in the liquid sample at the "hot spot" is extremely enhanced. This enhanced Raman scattering caused by the evanescent field is called near-field enhancement. The Raman scattering induced by the "hot spot" stimulates the molecules of the sample to exhibit a dipole form. Emission 6, the emission of Raman scattering of the molecule to the upper and lower directions of the sample cell is evanescent light, and the inset 7 on the left in Figure 1 is a schematic diagram of the exponential attenuation of the emanating light emitted from the Raman scattering in the two directions.

According to the present invention, since the evanescent light will decay exponentially with distance, the designed sample cell should be very thin, and the Raman scattering evanescent light is converted into forbidden Hght 8 in the substrate, which is transmissive light It has a large aperture angle and an approximately hollow core cone form. Receive the forbidden light with two large aperture angles near the hollow conical shape, and introduce it into the Raman spectrometer with the light collecting element to obtain the near-field enhanced Raman spectrum of the sample.

Figures 2, 3 and 4 respectively show three preferred embodiments of the sample cell according to the present invention for receiving forbidden light in two directions, up and down. The same reference numerals as in FIG. 1 will not be repeated.

As shown in FIG. 2, the near-field excitation and reception according to the present invention employs a lost-light Raman scattering near-field enhancement (RSNE) sample cell composed of two over-focus truncated ellipsoidal mirrors 9 and 10. The spheres are also transparent solids with a higher refractive index than the sample, such as optical glass, and they are plated with two parallel interfaces that pass through the focal point and are coated with a very thin silver or gold film to form a sample cell. The Raman scattering forbidden light 11 and 12 with a large aperture angle are emitted near the focal point, and the internal reflection through the metal-plated reflective layer on the outer wall of the ellipsoid converges near the other focal point of the ellipsoid, and is output as a small aperture angle beam. Multi-mode fiber 13 is coupled at this focal point. The two output fibers coupled to the upper and lower over-focus truncated ellipsoids are combined with the Raman spectrometer inlet 14 after being bundled. One of the ellipsoids has a small hole incident on the side, and the total reflection beam is drawn out through a small hole in a symmetrical place.

FIG. ₃ shows an RSNE sample cell composed of a hemispherical evanescent mirror 15 and an off-focus truncated hemispherical mirror 10 (or with a large-aperture oil immersion objective 19 in FIG. 4). The refractive index of the focal truncated ellipsoid 10 is also greater than the refractive index of the sample. The RSNE sample cell is composed of the interface between the two and the outer surface is plated with a metal reflective layer, one of which (15 or 10) is on the side. (2) Two small holes for introducing and exporting a total internal reflection P-polarized fine laser beam 1 for exciting a sample. In FIG. 3, a hole is opened in the side of the off-focus truncated ellipsoid 10. The hemispherical mirror 15 converts the evanescent light that is sampled downward by Raman scattering into forbidden light 12 and returns after reflection in the hemisphere, and then converts it into the evanescent light 16 that is upward in the sample pool and upwards that are reflected by the hemisphere. Both the lost light 16 and the lost light in the original direction are collected by the over-focus truncated whole ellipsoid 10 in the form of forbidden light, namely 17, 11 and collected near the other focus of the ellipsoid, and then passed through the multimode fiber or lens optics. The system is coupled into a Raman spectrometer.

FIG. 4 is a schematic structural diagram of an RSNE sample cell composed of an over-focus truncated hemispherical 18 reflector and a large-aperture oil immersion objective 19 cover sheet. Here, the over-focus truncated hemispherical reflector 18 and the hemispherical reflection in FIG. 2 Mirror 15 is similar. After the downward Raman scattering evanescent light is converted into forbidden light, the incident light angle is kept smaller as the incident angle is smaller than the critical angle. Therefore, the form of transmittable light has been passed through the sample after secondary reflection in the focal truncated semi-ellipsoid 18. Since the angle of incidence is smaller than the critical angle, the light can be transmitted through the sample cell and then received by the large-aperture oil immersion objective 19. The cover lens of the oil immersion objective lens 19 and the off-focus truncated semi-ellipsoid 18 both have a refractive index greater than that of the sample and are plated with a thin silver or gold film 2. The upward Raman scattering evanescent light is converted to forbidden light by the cover sheet. The large numerical aperture oil immersion objective lens 19 is coupled to the Raman spectrometer 14, and all Raman scattered light is reflected by the mirror 20 and coupled to the Raman spectrometer 11. Similarly, the excitation laser beam 1 is introduced through an objective lens or a small hole plated with a metal reflective layer on the side of the truncated hemi-spheroid.

It is conceivable that at least one of the above-mentioned light-collecting elements may be an over-focus truncated parabolic object reflection. A mirror or a large-aperture oil-immersed objective lens with an infinite back focal length, so that the forbidden light converted into the interior by the sample Raman scattering and evanescent light is entered Coupling into a Raman spectrometer with parallel light beams.

When the light-collecting element is an over-focus truncated ellipsoidal reflector or an over-focus truncated parabolic reflector or a large-aperture oil-immersed objective, the Raman samples in a 4 π solid angle can be collected in all directions toward the two substrates of the sample cell. Scattered light.

In addition, the above-focus truncated hemispherical reflector or hemispherical reflector is used to collect the Raman scattered light emitted in the direction of one substrate of the sample cell and reflect the collected light connected to the other substrate through the sample cell. Component collection. If the gain is allowed to be affected to some extent, a mirror made by thickening the silver or gold film of the substrate may be used instead, which is also used for reflecting Raman scattering. Light is collected through a sample cell by a light collecting element connected to another substrate.

Of course, the sample cell of the present invention can also be used to analyze solid samples. In this case, a nano-scale powder or film is used for the sample.

By using the Raman scattering near field enhancement (RSNE) sample cell of the present invention, the Raman enhancement factor can be improved, and the sensitivity of Raman analysis can be greatly improved. Under the condition that the calibration of the Raman spectrum of some important diseases has been obtained, a drop of finger is used for blood The ideal of diagnosing early major illnesses is possible. On-line Raman testing and molecular trace analysis technology in many fields such as physicochemical, biological, medical, geological, and materials will provide a new method for catalyzing reactions.

Claims

Rights request

What is claimed is: 1. An ultra-high sensitivity near-field enhanced sample cell using stray light Raman scattering for excitation and reception, comprising two transparent substrates with parallel surfaces and a refractive index greater than the refractive index of the sample. The surface is coated with a few nanometers of thick silver or gold film, and nanoscale silver or gold colloidal particles or nanostructures are distributed between the silver or gold films. The two interfaces of the sample cell can clamp the sample to a distance of tens to hundreds of nanometers. In order to allow the excitation laser beam with an incident angle exceeding the critical angle to generate evanescent light to excite the sample and emit Raman scattered light with evanescent light as the main form.

2. The Raman scattering near-field enhanced sample cell according to claim 1, further comprising a light collecting element connected to each of the substrates, and the sample Raman scattering is emitted in the direction of two substrates. Lost light is collected in a light-collecting element in the form of a transmissive forbidden light.

3. The Raman scattering near field enhanced sample cell according to claim 2, characterized in that at least one of the light collecting elements is an over-focus truncated ellipsoidal reflector or a large-aperture oil-immersed objective lens, so that After entering the interior, the forbidden light converted by the sample Raman scattering and evanescent light is focused and collected and coupled into the Raman spectrometer.

4. The Raman scattering near-field enhanced sample cell according to claim 2, wherein at least one of the light collecting elements is an over-focus truncated parabolic reflector or a large-aperture oil-immersed objective lens with an infinite back focal length It is used to make the forbidden light converted into the interior of the sample by the Raman scattering and evanescent light coupled into the Raman spectrometer as parallel beams.

5. The Raman scattering near-field enhanced sample cell according to claim 2, wherein one of the light-collecting elements is an off-focus truncated hemispherical mirror or a hemispherical mirror, or the substrate silver Or a thickened gold mirror is used to collect the Raman scattered light emitted toward one of the substrates of the sample cell, and after it is reflected, it is collected by the light collecting element connected to the other substrate through the sample cell.

6. The Raman scattering near-field enhanced sample cell according to claim 2, wherein a small reflective hole is provided on a side reflection layer of one of the light-collecting elements to introduce a fine laser having an incident angle exceeding a critical angle. Beam, or a laser beam with an incident angle exceeding a critical angle introduced by a large numerical aperture oil immersion objective.

7. A Raman scattering near-field enhancement method in which both Raman scattering excitation and reception use evanescent light, including the following steps,

Two transparent substrates with parallel surfaces and a refractive index greater than the refractive index of the sample are provided to form a sample cell. The inner surface of the substrate is plated with a few nanometers of thick silver or gold film, and nanoscale silver or gold colloids are distributed between the silver or gold films. Particles or structure

8. The Raman scattering near-field enhancement method for both Raman scattering excitation and reception according to claim 7, wherein each substrate is provided with a light collecting element connected to the substrate, and the light collecting element The outer wall is plated with a metal reflective layer, and a small hole is introduced in the side reflective layer of one of the light collecting elements to introduce a fine laser beam or a laser beam into a large-aperture oil immersion objective lens. The incident angle of the introduced laser beam exceeds a threshold Angle in order to create a stray light excitation sample in the sample cell.

9. The Raman scattering near-field enhancement method using both stray light for excitation and reception of Raman scattering according to claim 8, wherein one of the light collecting elements is an off-focus truncated hemispherical reflector or a hemisphere A body reflector or a reflector made by thickening the silver or gold film of the substrate, collects Raman scattered light in the direction of one substrate of the sample cell through a light collecting element that is reflected to the direction of the other substrate of the sample cell.

10. The Raman scattering near-field enhancement method using both stray light for excitation and reception of Raman scattering according to claim 8, wherein at least one of the two light collecting elements of the sample cell is a light collecting element. Focus truncated ellipsoidal reflector or over-focus truncated parabolic reflector or large-aperture oil-immersed objective, used to collect the Raman scattered light of the sample within 4 π solid angle in all directions towards the two substrates of the sample cell, and coupled into Ra Man spectrometer.