KR20170041528A - The nano island solid matrix for matrix assisted laser desorption/ionization time-of-flight mass spectrometry and the method thereof - Google Patents
The nano island solid matrix for matrix assisted laser desorption/ionization time-of-flight mass spectrometry and the method thereof Download PDFInfo
- Publication number
- KR20170041528A KR20170041528A KR1020150141081A KR20150141081A KR20170041528A KR 20170041528 A KR20170041528 A KR 20170041528A KR 1020150141081 A KR1020150141081 A KR 1020150141081A KR 20150141081 A KR20150141081 A KR 20150141081A KR 20170041528 A KR20170041528 A KR 20170041528A
- Authority
- KR
- South Korea
- Prior art keywords
- thin film
- nano
- metal thin
- island structure
- island
- Prior art date
Links
Images
Classifications
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/26—Mass spectrometers or separator tubes
- H01J49/34—Dynamic spectrometers
- H01J49/40—Time-of-flight spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/55—Specular reflectivity
- G01N21/552—Attenuated total reflection
- G01N21/553—Attenuated total reflection and using surface plasmons
- G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/64—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using wave or particle radiation to ionise a gas, e.g. in an ionisation chamber
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
- H01J49/0409—Sample holders or containers
- H01J49/0418—Sample holders or containers for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates
Abstract
The present invention relates to a nano-island solid matrix applicable to a sample plate used in a Matrix Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and a method for producing the same, The solid matrix includes a conductive base material, a metal thin film formed on the conductive base material, and a nano-island structure formed at least partially on the metal thin film surface. When the laser is irradiated, the sample to be analyzed is ionized by surface plasmon resonance And the nano-island structure for producing such a surface plasmon resonance is obtained by heat treatment after being manufactured through a gold thin film, and even distribution of various samples and drying are maintained. Therefore, a mass analysis A peak can be obtained.
Description
The present invention relates to a nano-island solid matrix formed with a nano-island structure applicable to a sample plate used in a Matrix Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) will be.
In general, the MALDI-TOF MS (MALDI-TOF MS) is a device for measuring the molecular weight of ions after ionizing the molecules of the sample to be analyzed. The MALDI-TOF MS It is used for mass spectrometry of biochemicals with large molecular weights.
The mass spectrometry using the Maldistop mass spectrometer is performed as follows. When an organic matrix is mixed with a sample to be analyzed and then a laser is irradiated, the organic matrix absorbs laser energy in the ultraviolet region to ionize the sample during the activation process.
The ionization method of Maltodest mass spectrometry (MALDI-TOF MS) generally causes a simple ionization such as +1 or +2 to the sample. In the case of a polymer substance such as a protein, Is an easy method for measuring the molecular weight.
For the Maddystone mass analysis, the sample to be analyzed is mixed with the organic matrix, or the organic matrix is preliminarily dispensed on the metal plate, and the sample to be analyzed is dispensed and dried before use. Thus, the Maltodest Mass Spectrometer provides simple sample preparation, high sensitivity measurements, and wide analytical range advantages.
However, in the case of low molecular weight samples in the Maldistop mass spectrometry, the organic matrix used for ionization of the sample is ionized by the irradiation of the laser, and at the same time, the low molecular weight sample decomposes and the noise of the matrix is generated in the low molecular weight region. There is a drawback that measurement is not easy. Therefore, when the analytical peak of the low molecular weight sample overlaps with the noise of the matrix, analysis becomes impossible.
Solid-state matrices have been developed to replace organic matrices for analyzing samples in such low molecular weight regions.
As the solid matrix, those made of a semiconductor material having an energy band gap as a nano structure are the main species. As such a solid matrix, porous silicon, silicon dioxide (SiO 2 ), titanium dioxide (TiO 2 ), tin oxide Nanoparticles of semiconductor nanoparticles, nanowires, etc. have been reported.
Particularly, in the case of titanium dioxide (TiO 2 ) nanoparticles which are widely used in industry, they are mixed with a sample and mixed with a metal plate before drying. At this time, the crystals of the dried sample and the organic matrix show an irregular shape. Therefore, the distribution of the dried titanium dioxide (TiO 2 ) particles is uneven, and the mass spectrometric peaks are different depending on the irradiation position of the laser.
In view of the foregoing, the present invention can measure a low molecular weight ion mass (m / z) range of 500 Da or less which can not be measured due to noise of the organic matrix in the past maltodext mass spectrometry, It is an object of the present invention to provide a nano-island solid matrix in which the mass spectrometry peaks appear differently depending on positions where the laser is irradiated.
In addition, the present invention, taking the above-mentioned points into consideration, can be applied to a nano-island solid matrix in which a metal thin film is deposited on a conductive base material and then heat- The present invention also provides a method for producing a nano-island solid matrix for producing a nano-island structure of a nano-island solid matrix.
In order to achieve the above object, the nano-island solid matrix of the present invention is a solid matrix usable in Matrix Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry And a nano-island structure formed at least partially on the surface of the metal thin film discontinuously.
The metal thin film may have a thickness of 10 nm or less and cause a surface plasmon resonance (SPR) phenomenon, and may include at least one selected from gold, silver, copper, and aluminum. More preferably, the thickness of the metal thin film may be 5 nm or less.
The nano-island structure is preferably a semi-spherical structure having a diameter of 160 nm or less and a thickness of 40 nm or less, and the matrix is a silicon substrate.
According to another aspect of the present invention, there is provided a method of manufacturing a nano-island solid matrix, comprising: preparing a conductive base material (S110); forming a metal thin film on the conductive base material to cause surface plasmon resonance (SPR) (S120), and forming a nano-island structure on the surface of the metal thin film by heat-treating the deposited metal thin film (S130).
The nano-island structure is formed by forming a metal thin film on the surface of the metal thin film by heat treatment at a temperature higher than the melting point of the metal thin film by 50 ° C to 100 ° C. The nano- , And a hemispherical structure having a thickness of 40 nm or less.
The metal thin film may include at least one of gold, silver, copper, and aluminum. The thickness of the metal thin film is preferably 10 nm or less, and the conductive base material may be a silicon substrate.
On the other hand, the nano-island solid matrix including the nano-island structure of the present invention is irradiated with a laser in a Maldist-M-type mass spectrometer (MALDI-FOF MS) to ionize the sample to be analyzed. surface plasmon resonance (SPR) phenomenon.
Specifically, in the surface plasmon resonance (SPR) phenomenon, a plasmon refers to an electron cloud existing on a metal surface, and a resonance phenomenon caused by a surface plasmon is a natural motion mode of a surface plasmon Is given, it indicates the property of absorbing it. That is, when the light energy corresponding to a series of wavelengths is irradiated, energy corresponding to a specific wavelength of the plasmon is absorbed. In general, surface plasmon resonance phenomenon is reported to occur strongly when the surface area is limited, such as nanoparticles (nanoparticles) and nanoislands (nano islands).
In addition, the surface plasmon resonance induced between the dielectric and the metal is determined by the dielectric constant of the metal and the dielectric, that is, when the real part of the dielectric constant of the metal is smaller than 0 and the absolute value of the dielectric constant of the metal is larger than the absolute value of the dielectric constant of the dielectric Surface plasmon complex is known to occur. It is known that the surface plasmon resonance of gold, silver, copper, aluminum and the like among the metals satisfying this relationship is large.
When the nano-island solid matrix according to the present invention is provided on a sample plate for a malduth digest mass spectrometer, the ion mass of a sample to be analyzed in a range of 500 Da or less, which can not be measured due to the noise of the organic matrix in the existing maltodext mass spectrometry, The mass-to-charge (m / z) ratio can be measured and mass analysis can be performed.
It is also possible to produce a solid matrix comprising a nano-island structure in which the surface plasmon resonance is maximized for optimal maldistone mass analysis.
Further, even when the sample to be analyzed is dried on a sample plate containing the nano-island solid matrix according to the present invention, the nano-island structure is uniformly distributed, so that mass spectrometry with high reproducibility can be performed.
1 is a flow chart of a method of manufacturing a nano-island solid matrix according to a preferred embodiment of the present invention.
2 is a schematic diagram showing a method of manufacturing a nano-island solid matrix according to a preferred embodiment of the present invention.
FIG. 3 is a result of an atomic force microscopy (AFM) measurement of a nanoisland structure according to a preferred embodiment of the present invention.
4 is a view showing a shape of a nano island structure according to a preferred embodiment of the present invention.
FIG. 5 illustrates a surface plasmon resonance phenomenon by a nano-island structure according to a preferred embodiment of the present invention.
FIG. 6 is a mass analysis result of a low molecular weight sample on a gold thin film before a heat treatment process for forming a nano-island solid matrix according to a preferred embodiment of the present invention.
FIG. 7 is a mass analysis result of a low molecular weight sample using a nano-island solid matrix according to a preferred embodiment of the present invention.
8 is a mass analysis result of a low molecular weight sample in a mixture state using a nano-island solid matrix according to a preferred embodiment of the present invention.
Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention may be embodied in many different forms without departing from the scope of the present invention. The present invention is not limited to the embodiment described.
Figures 1 and 2 illustrate a method of making a nano-island solid matrix according to one embodiment of the present invention.
As shown in FIG. 1, a method of manufacturing a nano-island solid matrix according to the present invention includes the steps of preparing a conductive base material (S110), forming a metal thin film on the prepared conductive base material to cause surface plasmon resonance (SPR) (S120); and forming a nano-island structure on the surface of the metal film through heat treatment (S130).
In detail, in the step of preparing the conductive base material (S110), the conductive base material may be a silicon substrate doped with a P-type semiconductor for the conductive adhesion to a sample plate in a Maldøt mass spectrometer, but the present invention is not limited thereto, , Copper, and the like, so that the present invention is not limited to the above materials and methods.
In the step of depositing the metal thin film (S120), a gold thin film was deposited to a thickness of 10 nm or less as a metal thin film to form a nano-island structure, and in the examples, 1 nm, 5 nm, Is deposited by thermal evaporation.
In the step of forming the nano-island structure (S130), the heat treatment is preferably performed at a temperature higher than the melting point of the metal thin film by 50 ° C to 100 ° C. As another example, in the case of the gold thin film, The heat treatment can be performed at 500 ° C for 30 minutes.
As described above, the nano-island structure used in the present embodiment is formed using a gold thin film, but a metal thin film that causes surface plasmon resonance (SPR) phenomenon such as silver, copper, and aluminum may be used. And can be manufactured through heat treatment at a temperature near the melting point of each metal.
2 is a view showing a method of manufacturing a nano-island solid matrix according to a preferred embodiment of the present invention. As shown in FIG. 1, a nano-island solid matrix in which a nano-island structure is formed on the surface of a metal thin film can be manufactured through heat treatment after depositing a metal thin film.
FIG. 3 is a result of an atomic force microscope measurement showing the shape of a nano-island structure manufactured according to a preferred embodiment of the present invention.
As shown in FIG. 3, each atomic force microscope photograph shows the shape of a nano-island structure obtained by heat treatment after depositing a gold thin film with thicknesses of 1 nm, 5 nm, and 10 nm, respectively, . As shown in the figure, each of the 1 nm, 5 nm, and 10 nm gold thin films was annealed to form nano-sized structures with diameters of 40 nm, 60 nm, and 160 nm, respectively. As a result, The nano-island structure can be determined depending on the thickness of the metal thin film.
As shown in FIG. 4, the shape of the nano-island structure fabricated by heat treatment of the gold thin film having a thickness of 10 nm using an atomic force microscope was measured. As a result, It can be seen that the island structure is formed.
FIG. 5 is a graph showing a surface plasmon resonance peak by scanning a wavelength range of 400 to 800 nm with a UV-VIS spectrophotometer using a nano-island solid matrix prepared according to a preferred embodiment of the present invention.
Specifically, as shown in FIG. 5, in the case of a gold thin film for fabricating a nano-island structure, a peak indicating absorption of a specific wavelength showing surface plasmon resonance only in a gold thin film having a thickness of 1 nm was observed at a wavelength of 549.5 nm .
After the heat treatment of the gold thin film, the absorption peak due to the surface plasmon resonance of each nano-island structure was confirmed, and the degree of the surface plasmon resonance according to the absorption peak size was measured using a gold thin film of 1 nm and 5 nm Especially in island structures. In the annealed 1 nm gold film, the absorption peak was observed at the wavelength of 523 nm and at the wavelength of 547 nm in the heat treated gold thin film of 5 nm.
FIG. 6 shows the results of mass spectrometry using a gold thin film before heat treatment used for the production of the nano-island structure of the present invention.
As shown in FIG. 6 (a), it is known that CHCA (a-Cyano-4-hydroxycinnamic acid), which is an organic matrix used in the prior art, generates a matrix noise with a high density at an ion mass of 500 m / have.
6 (b) shows a case where the gold thin film before the heat treatment was measured without a sample, and the matrix noise observed in the organic matrix CHCA (a-Cyano-4-hydroxycinnamic acid) Respectively.
6 (c) and 6 (d) show that arginine and phenylalanine, low in molecular weight, were prepared at a concentration of 400 μg / ml on a gold thin film deposited to a thickness of 1 nm before the heat treatment, As a result of the ditto mass analysis, the ion peak of arginine and phenylalanine, which are the two amino acids, was not observed in the gold thin film having a thickness of 1 nm before the heat treatment. These results show that the gold thin film before the simple heat treatment is not suitable for the ionization of the low molecular weight sample.
FIG. 7 is a graph showing the results of mass spectrometry of arginine and phenylalanine as a low molecular weight sample using a nano-sized solid matrix prepared by heat-treating a gold thin film deposited to a thickness of 1 nm according to a preferred embodiment of the present invention. In the case of the a-Cyano-4-hydroxycinnamic acid (CHCA) which is an organic matrix used in the above-described FIG. 6, a matrix noise with a high density is generated at an ion mass below 500 m / z, , Arginine and phenylalanine were prepared at a concentration of 400 μg / ml in a nano-island solid matrix of the present invention, in which the nano-islands structure was formed, and 0.5 μl of each of them was separately spray dried and then dried to obtain a low- Ion peak was observed.
These results show that the nano-island structure can perform the dynamics as a solid matrix in the maldistop mass analysis, and it can be applied to mass spectrometry, especially by ionizing low molecular weight samples.
FIG. 8 is a graph showing mass spectrometry results of an amino acid mixture as a low-molecular-weight sample using a nano-island structure prepared according to a preferred embodiment of the present invention. As shown, the sample of the mixture contains 8 kinds of total (8) kinds of glycine (1), serine (2), proline (3), aspartic acid (4), glutamate (5), histidine Was prepared as a solution at a concentration of 1 mg / ml, each solution was taken in a pipet by 50 쨉 l, and all the solutions taken were mixed to prepare a mixture sample.
Here, the amino acid of the sample of the mixture is selected from amino acids having chemical functional groups having polar, non-polar, acidic, and basic properties.
As shown in FIG. 7, the result of mass spectrometry of the thus-prepared mixture sample on a nano-island solid matrix revealed that eight ion pits of the mixed amino acid samples were observed.
These results indicate that the nano-island solid matrix of the present invention, in which the nano-islands structure is formed, serves as a solid matrix when used for the mass spectrometry of maltodis and ionizes low-molecular samples of various chemical properties such as polarity, non-polarity, acidity or basicity Mass spectrometry can be applied.
The nano-island solid matrix according to the preferred embodiment of the present invention and its manufacturing method have been described in detail with reference to the accompanying drawings. However, it will be understood by those skilled in the art that various modifications and variations can be made in the present invention. Accordingly, the scope of the present invention is defined by the claims that follow.
Claims (11)
Conductive base metal;
A metal thin film formed on the conductive base material; And
And a nano-island structure formed at least partially on the surface of the metal thin film discontinuously.
Wherein the metal thin film is a metal causing a surface plasmon resonance (SPR) phenomenon, and comprises at least one of gold, silver, copper, and aluminum.
Wherein the metal thin film has a thickness of 10 nm or less.
Wherein the nano-island structure is a hemispherical structure having a diameter of 160 nm or less and a thickness of 40 nm or less.
Wherein the base material is a silicon substrate.
Depositing a metal thin film on the conductive base material to cause a surface plasmon resonance (SPR) phenomenon; And
And thermally treating the deposited metal thin film to form a nano-island structure on the surface of the metal thin film.
Wherein the nano-island structure is formed by heat-treating the metal thin film at a temperature higher than the melting point of the metal thin film by 50 ° C to 100 ° C to form the nano-island structure on the surface of the metal thin film.
Wherein the nano-island structure is a hemispherical structure whose diameter is determined according to the thickness of the deposited metal thin film, and the thickness of the metal thin film is 10 nm or less.
Wherein the metal thin film comprises at least one of gold, silver, copper, and aluminum.
Wherein the metal thin film has a thickness of 10 nm or less.
Wherein the conductive base material is a silicon substrate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150141081A KR20170041528A (en) | 2015-10-07 | 2015-10-07 | The nano island solid matrix for matrix assisted laser desorption/ionization time-of-flight mass spectrometry and the method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150141081A KR20170041528A (en) | 2015-10-07 | 2015-10-07 | The nano island solid matrix for matrix assisted laser desorption/ionization time-of-flight mass spectrometry and the method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20170041528A true KR20170041528A (en) | 2017-04-17 |
Family
ID=58703051
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020150141081A KR20170041528A (en) | 2015-10-07 | 2015-10-07 | The nano island solid matrix for matrix assisted laser desorption/ionization time-of-flight mass spectrometry and the method thereof |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR20170041528A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111426743A (en) * | 2020-04-21 | 2020-07-17 | 四川大学 | Application of silver nanosphere material in MA L DI-TOF MS (Mass Spectrometry) detection of small molecule metabolites |
| KR20200114753A (en) * | 2019-03-29 | 2020-10-07 | 연세대학교 산학협력단 | Sample plate and method of fabricating the same |
| CN113686947A (en) * | 2021-09-15 | 2021-11-23 | 华东理工大学 | Preparation method and application of mass spectrum matrix material |
-
2015
- 2015-10-07 KR KR1020150141081A patent/KR20170041528A/en active Search and Examination
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20200114753A (en) * | 2019-03-29 | 2020-10-07 | 연세대학교 산학협력단 | Sample plate and method of fabricating the same |
| CN111426743A (en) * | 2020-04-21 | 2020-07-17 | 四川大学 | Application of silver nanosphere material in MA L DI-TOF MS (Mass Spectrometry) detection of small molecule metabolites |
| CN113686947A (en) * | 2021-09-15 | 2021-11-23 | 华东理工大学 | Preparation method and application of mass spectrum matrix material |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Stolee et al. | Laser–nanostructure interactions for ion production | |
| TWI434314B (en) | Substrate for mass spectrometry, mass spectrometry, and mass spectrometer | |
| Coffinier et al. | Diamond nanowires for highly sensitive matrix-free mass spectrometry analysis of small molecules | |
| KR101752656B1 (en) | Sample plate using MALDI-TOF mass spectrometer and manufacturing method of the sample plate | |
| KR101204342B1 (en) | Sample plate for MALDI-Tof mass spectrometer and a method for analysing a mass using the MALDI-Tof mass spectrometer | |
| Morris et al. | Laser desorption ionization (LDI) silicon nanopost array chips fabricated using deep UV projection lithography and deep reactive ion etching | |
| KR101592517B1 (en) | Substrate for Laser Desorption Ionization Mass Spectrometry and method for manufacturing the same | |
| US9671409B2 (en) | Ultrathin calcinated films on a gold surface for highly effective laser desorption/ionization of biomolecules | |
| JP4918662B2 (en) | Sample holder and mass spectrometry method for MALDI mass spectrometry | |
| KR20170041528A (en) | The nano island solid matrix for matrix assisted laser desorption/ionization time-of-flight mass spectrometry and the method thereof | |
| WO2016116014A1 (en) | Image acquisition semiconductor film for high-resolution mass-spectral imaging system, and preparation method and application | |
| US8080784B2 (en) | Ionization device | |
| US9000361B2 (en) | Nanophotonic production, modulation and switching of ions by silicon microcolumn arrays | |
| Baur et al. | Soft cluster-induced desorption and ionization of biomolecules—Influence of surface load and morphology on desorption efficiency | |
| Allwood et al. | Preparation of ‘near’homogeneous samples for the analysis of matrix-assisted laser desorption/ionisation processes | |
| Yamada et al. | TiN nanopillar-enhanced laser desorption and ionization of various analytes | |
| Zeng et al. | On-plate glycoproteins/glycopeptides selective enrichment and purification based on surface pattern for direct MALDI MS analysis | |
| Kosyakov et al. | Carbon nanocoatings: A new approach to recording mass spectra of low-molecular compounds using surface-assisted laser desorption/ionization mass spectrometry | |
| WO2008032569A1 (en) | Mass analysis using ionizing by surface plasmon | |
| Huo et al. | Surface‐assisted laser desorption and ionization mass spectrometry using low‐cost matrix‐free substrates | |
| KR102062447B1 (en) | Target surfaces for MALDI mass spectrometry using Graphene films and Mass analysis method using the same | |
| KR102097319B1 (en) | Sample plate with adjustable ionization efficiency, method of fabricating the same and mass spectrometer analysis by using the same | |
| KR101761484B1 (en) | Method of organic matrix solution for matrix-assisted laser desorption/ionization-mass spectroscopy and organic matrix manufactured by thereof | |
| RU2531762C1 (en) | Method of determining metals and metal complexes | |
| JP6685587B2 (en) | Ionization aid for mass spectrometry and mass spectrometry method using the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| AMND | Amendment | ||
| E601 | Decision to refuse application | ||
| AMND | Amendment |