KR20230094601A - Substrate composition for quantitative analysis of metabolites comprising amino acids and analysis method using the same - Google Patents
Substrate composition for quantitative analysis of metabolites comprising amino acids and analysis method using the same Download PDFInfo
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- KR20230094601A KR20230094601A KR1020210183892A KR20210183892A KR20230094601A KR 20230094601 A KR20230094601 A KR 20230094601A KR 1020210183892 A KR1020210183892 A KR 1020210183892A KR 20210183892 A KR20210183892 A KR 20210183892A KR 20230094601 A KR20230094601 A KR 20230094601A
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- quantitative analysis
- substrate
- metabolites
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- phenylalanine
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Abstract
본 발명은 아미노산을 유효성분으로 함유하는 대사체 정량분석용 기질(Matrix) 조성물 및 이를 이용한 대사체 정량분석 방법에 관한 것으로, 보다 상세하게는 안정한 동위원소로 표지된 페닐알라닌을 기질로 사용하여 시간비행형 이차이온질량분석법(ToF-SIMS)을 수행하여 아미노산 대사체의 정량분석을 수행한 결과, 상기 페닐알라닌 기질은 분석용 기판 위에 균일한 층을 형성하였으며, 시료 건조 후에도 낮은 농도의 아미노산 대사체의 정밀한 정량분석이 가능한 것을 확인함에 따라, 상기 안정한 동위원소로 표지된 페닐알라닌을 유효성분으로 함유하는 조성물은 질량분석법을 이용한 대사체 정량분석용 기질 조성물로 제공될 수 있다.The present invention relates to a matrix composition for quantitative analysis of metabolites containing amino acids as an active ingredient and a method for quantitative analysis of metabolites using the same, and more particularly, to a time flight using phenylalanine labeled with a stable isotope as a substrate As a result of performing quantitative analysis of amino acid metabolites by performing ToF-SIMS, the phenylalanine substrate formed a uniform layer on the substrate for analysis, and even after drying the sample, precise determination of amino acid metabolites at low concentrations was obtained. As confirmed that quantitative analysis is possible, the composition containing the stable isotope-labeled phenylalanine as an active ingredient can be provided as a substrate composition for quantitative analysis of metabolites using mass spectrometry.
Description
본 발명은 아미노산을 유효성분으로 함유하는 대사체 정량분석용 기질(Matrix) 조성물 및 이를 이용한 대사체 정량분석 방법에 관한 것이다.The present invention relates to a matrix composition for quantitative analysis of metabolites containing amino acids as an active ingredient and a method for quantitative analysis of metabolites using the same.
이차이온질량분석법(Secondary Ion Mass Spectrometry, SIMS)은 가속 이온빔을 시료에 조사하여 시료 표면과 충돌하여 탈착되어 나오는 이차 이온의 질량을 분석하는 방식으로서 표면에 흡착된 분자의 질량 분석 기술로 최근 이온빔과 질량분석기의 발달로 조직과 세포와 같은 생체시료의 지질과 대사체를 높은 공간분해능과 질량분해능의 이미징 분석을 가능하게 한다. Secondary Ion Mass Spectrometry (SIMS) is a method of analyzing the mass of secondary ions that collide with and desorb from the sample surface by irradiating a sample with an accelerating ion beam. With the development of mass spectrometers, it is possible to analyze lipids and metabolites of biological samples such as tissues and cells with high spatial resolution and mass resolution.
이러한 SIMS 분석의 주요 장점은 극미량 분석으로서, 검출한계는 수소에서 우라늄까지 거의 모든 원소를 ppm 내지 ppb 수준으로 정밀 분석이 가능하고, 또한 안정한 동위원소로 분석할 수 있으며, 뎁스 프로파일(depth profile)과 이온 이미지(ion image)가 가능하며, 미량 성분의 깊이 분포 측정과 3차원 분석을 할 수 있는 것이다. 그러나 이차 이온의 방출량이 기질(matrix)과 표면의 전자 상태에 극히 민감하여 매질에 따라 스퍼터링된 이차 이온의 양이 현저히 다르게 나타나는 기질 효과(matrix effect)가 있고, 또한 본질적으로 파괴적인 분석 방법이라는 단점이 있다.The main advantage of SIMS analysis is ultra-trace analysis, and the detection limit is that almost all elements from hydrogen to uranium can be precisely analyzed at the ppm to ppb level, and can also be analyzed with stable isotopes. Ion image is possible, and depth distribution measurement and 3D analysis of trace components can be performed. However, the emission of secondary ions is extremely sensitive to the electronic state of the matrix and the surface, so there is a matrix effect in which the amount of sputtered secondary ions is significantly different depending on the medium, and it is also an inherently destructive analysis method. there is
액체생검(liquid biopsy)은 천자나 절개 등의 침습적인 시술 없이 비침습적으로 획득할 수 있는 혈액이나 복수 등의 액체 상태의 체액 시료를 활용하여 질병을 진단하거나 분석하는 방법이다. 액체생검은 환자로부터 비교적 간편하게 체액을 채취하여 암 발생 및 전이 여부를 신속하고 상세하게 파악할 수 있으며, 액체생검 내에 존재하는 핵산 또는 엑소좀과 같은 생체물질은 다양한 질병에 대한 다각적인 분석을 가능하게 하여 질병의 원인 및 치료에 폭넓게 활용될 것으로 전망된다.Liquid biopsy is a method of diagnosing or analyzing a disease by using a body fluid sample in a liquid state, such as blood or ascites, which can be obtained noninvasively without an invasive procedure such as puncture or incision. Liquid biopsy is a relatively simple way to collect bodily fluid from a patient to quickly and accurately determine whether cancer has occurred or metastasized, and biomaterials such as nucleic acids or exosomes present in liquid biopsies enable multilateral analysis of various diseases. It is expected to be widely used in the cause and treatment of diseases.
그러나 환자 유래 액체생검(혈액, 소변, 침, 눈물, 뇌척수액, 땀, 폐포 세척액(Bronchoalveolar Lavage Fluid, BALF) 등) 내의 대사체 분석을 위한 종래 기술(Nuclear magnetic response(NMR) spectroscopy, gas or liquid chromatography mass spectroscopy(GC-MS or LC-LS) 등)은 시편 당 적어도 수십 ~ 수백 μL(마이크로리터) 용량이 필요하고 각 시편을 개별적으로 한 개씩만 분석하기 때문에 수 마이크로리터 이하 나노리터 수준의 극미량 다수 시편을 신속히 분석하는 데 한계가 있다.However, conventional techniques (Nuclear magnetic response (NMR) spectroscopy, gas or liquid chromatography) for metabolome analysis in patient-derived liquid biopsies (blood, urine, saliva, tears, cerebrospinal fluid, sweat, Bronchoalveolar Lavage Fluid (BALF), etc.) mass spectroscopy (GC-MS or LC-LS), etc.) requires at least tens to hundreds of μL (microliter) capacity per specimen and analyzes only one of each specimen individually, so a very small number of microliters or less nanoliters There are limitations to rapid analysis of specimens.
종래 분석용 기질로 사용된 그래핀 옥사이드(Graphene oxide)는 균일성 및 분석 물질 신호 향상과 같은 기질로서 좋은 장점을 가지고 있지만, 정량분석이 불가능한 문제점이 있으며, Paper-based microarray를 이용한 정량분석은 충분한 방법 검증이 이루어지지 않아 교정 곡선이 정밀하지 않고, 정량한계가 확인되지 않은 문제점이 있는 바, 본 발명은 상기 문제점을 해결하기 위해 아미노산을 유효성분으로 함유하는 대사체 정량분석용 기질(Matrix) 조성물을 제공하고자 한다.Graphene oxide, which has been used as a conventional substrate for analysis, has good advantages as a substrate such as uniformity and analyte signal enhancement, but has a problem in that quantitative analysis is impossible, and quantitative analysis using paper-based microarray is not sufficient. There are problems in that the calibration curve is not precise because the method verification is not performed and the limit of quantification is not confirmed. In order to solve the above problems, the present invention is a matrix composition for quantitative analysis of metabolites containing amino acids as an active ingredient. want to provide
본 발명은 안정한 동위원소로 표지된 아미노산을 유효성분으로 함유하는 대사체 정량분석용 기질(Matrix) 조성물을 제공한다.The present invention provides a matrix composition for quantitative analysis of a metabolite containing an amino acid labeled with a stable isotope as an active ingredient.
또한, 본 발명은 안정한 동위원소로 표지된 아미노산을 유효성분으로 함유하는 기질(Matrix)과 분석물질을 혼합하여 시료 혼합물을 준비하는 단계; 및In addition, the present invention comprises the steps of preparing a sample mixture by mixing a matrix containing an amino acid labeled with a stable isotope as an active ingredient and an analyte; and
상기 시료 혼합물을 분석용 기판에 주입 후 건조시키는 단계를 포함하는, 질량분석법을 이용한 대사체 정량분석 방법을 제공한다.Provided is a method for quantitative analysis of metabolites using mass spectrometry, comprising injecting the sample mixture into a substrate for analysis and then drying it.
본 발명에 따르면, 안정한 동위원소로 표지된 페닐알라닌을 기질로 사용하여 시간비행형 이차이온질량분석법(ToF-SIMS)을 수행하여 아미노산 대사체의 정량분석을 수행한 결과, 상기 페닐알라닌 기질은 분석용 기판 위에 균일한 층을 형성하였으며, 시료 건조 후에도 낮은 농도의 아미노산 대사체의 정밀한 정량분석이 가능한 것을 확인함에 따라, 상기 안정한 동위원소로 표지된 페닐알라닌을 유효성분으로 함유하는 조성물은 질량분석법을 이용한 대사체 정량분석용 기질 조성물로 제공될 수 있다.According to the present invention, as a result of quantitative analysis of amino acid metabolites by performing time-flight secondary ion mass spectrometry (ToF-SIMS) using phenylalanine labeled with a stable isotope as a substrate, the phenylalanine substrate is a substrate for analysis A uniform layer was formed on the top, and as it was confirmed that precise quantitative analysis of amino acid metabolites at low concentrations was possible even after sample drying, the composition containing the stable isotope-labeled phenylalanine as an active ingredient metabolites using mass spectrometry It may be provided as a matrix composition for quantitative analysis.
도 1은 패럴린 우물 배열 기판 제작 과정을 나타낸 것이다.
도 2는 패럴린 우물 배열 기판 위에 아미노산 시료를 준비하는 과정 및 시간비행형 이차이온질량분석법(TOF-SIMS) 분석 결과이다.
도 3은 그래핀 옥사이드(GO) 기질 내의 아미노산 알라닌(alanine)의 농도별 ToF-SIMS 분석 결과이며, 패럴린 기판의 우물 패턴 위에 건조된 GO 기질을 확인한 이미지이다.
도 4는 패럴린 기판의 우물 패턴 위에 건조된 20 종의 아미노산을 확인한 이미지이다.
도 5는 알라닌(alanine), 트레오닌(threonine), 시스테인(cysteine), 이소루이신(isoleucine), 루이신(leucine), 페닐알라닌(phenylalanine), 트레오신(tyrosine) 및 트립토판(tryptophan)의 ToF-SIMS 분석 결과이다.
도 6은 프롤린(proline), 발린(valine), 아스파라긴(asparagine), 글루탐산(glutamic acid), 글루타민(glutamine) 및 히스티딘(histidine)의 ToF-SIMS 분석 결과이다.1 shows a process of manufacturing a Parylene well array substrate.
2 is a process of preparing an amino acid sample on a Parylene well array substrate and a result of time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis.
3 is a ToF-SIMS analysis result for each concentration of the amino acid alanine in a graphene oxide (GO) substrate, and is an image confirming the dried GO substrate on the well pattern of the parylene substrate.
4 is an image confirming 20 types of amino acids dried on a well pattern of a parylene substrate.
5 is ToF-SIMS of alanine, threonine, cysteine, isoleucine, leucine, phenylalanine, tyrosine and tryptophan is the result of the analysis.
6 is a ToF-SIMS analysis result of proline, valine, asparagine, glutamic acid, glutamine, and histidine.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 안정한 동위원소로 표지된 아미노산을 유효성분으로 함유하는 대사체 정량분석용 기질(Matrix) 조성물을 제공할 수 있다.The present invention can provide a matrix composition for quantitative analysis of a metabolite containing an amino acid labeled with a stable isotope as an active ingredient.
상기 아미노산은 페닐알라닌(phenylalanine), 발린(valine), 메티오닌(methionine), 이소루이신(isoleucine), 루이신(leucine) 및 트립토판(tryptophan)으로 이루어진 군에서 선택되는 것일 수 있으며, 보다 바람직하게는 페닐알라닌일 수 있으나, 이에 제한되지 않는다.The amino acid may be selected from the group consisting of phenylalanine, valine, methionine, isoleucine, leucine and tryptophan, more preferably phenylalanine It may be, but is not limited thereto.
또한, 본 발명은 안정한 동위원소로 표지된 아미노산을 유효성분으로 함유하는 기질(Matrix)과 분석물질을 혼합하여 시료 혼합물을 준비하는 단계; 및In addition, the present invention comprises the steps of preparing a sample mixture by mixing a matrix containing an amino acid labeled with a stable isotope as an active ingredient and an analyte; and
상기 시료 혼합물을 분석용 기판에 주입 후 건조시키는 단계를 포함하는, 질량분석법을 이용한 대사체 정량분석 방법을 제공할 수 있다.A metabolite quantitative analysis method using mass spectrometry may be provided, comprising injecting the sample mixture into a substrate for analysis and then drying it.
상기 기질은 안정한 동위원소로 표지된 페닐알라닌일 수 있으며, 보다 바람직하게는 안정한 동위원소로 표지된 L-페닐알라닌일 수 있으나, 이에 제한되지 않는다.The substrate may be phenylalanine labeled with a stable isotope, more preferably L-phenylalanine labeled with a stable isotope, but is not limited thereto.
상기 기질은 5 내지 20mM 농도로 포함되는 것일 수 있다.The substrate may be included in a concentration of 5 to 20 mM.
상기 시료 혼합물은 신호표준화 물질로 발린을 추가로 더 포함하는 것일 수 있으며, 보다 상세하게는 상기 발린은 안정한 동위원소로 표지된 L-발린일 수 있으며, 상기 신호표준화 물질은 50 내지 150μM 농도로 포함될 수 있다. The sample mixture may further include valine as a signal standardization material, and more specifically, the valine may be L-valine labeled with a stable isotope, and the signal standardization material may be included at a concentration of 50 to 150 μM. can
상기 분석물질은 생체물질일 수 있으며, 보다 상세하게는 혈액, 혈장, 혈청, 소변, 침, 눈물, 뇌척수액 폐포 세척액(Bronchoalveolar Lavage Fluid, BALF) 및 땀으로 이루어진 군에서 선택되는 것일 수 있으나, 이에 제한되는 것은 아니다.The analyte may be a biological material, and more specifically, may be one selected from the group consisting of blood, plasma, serum, urine, saliva, tears, cerebrospinal fluid alveolar lavage fluid (BALF), and sweat, but is limited thereto. it is not going to be
상기 시료 혼합물을 분석용 기판에 주입 후 건조시키는 단계는 시료 혼합물이 주입된 분석용 기판을 -20℃에서 동결시킨 후 4 내지 25℃에서 건조시키는 것일 수 있다.The step of injecting the sample mixture onto the substrate for analysis and drying may include freezing the substrate for analysis into which the sample mixture is injected at -20°C and then drying at 4 to 25°C.
보다 상세하게는 안정한 동위원소로 표지된 L-페닐알라닌(F*) 10mM, 안정한 동위원소로 표지된 L-발린(V*) 100μM과 한 종류의 아미노산을 3차 수에 녹인 표준 용액을 제조하였다. 상기 표준 용액 내의 아미노산의 농도는 0, 0.1, 0.5, 1, 5, 10, 20, 50, 100, 200 및 500μM로 준비하였다. 상기 표준 용액을 패럴린 기판의 우물 패턴(직경: 1mm) 위에 200nL씩 주입하였으며, 농도별로 3번 반복하였다. 기판 위의 용액 방울을 냉동실 -20℃에서 24시간 동안 동결시킨 후, 4℃ 냉장고에서 1시간 동안 천천히 건조하였으며, 이후 남은 수분을 공기(20℃) 중에 완전히 건조시켰다.More specifically, a standard solution was prepared by dissolving 10 mM of L-phenylalanine (F * ) labeled with a stable isotope, 100 μM of L-valine (V * ) labeled with a stable isotope, and one amino acid in tertiary water. Amino acid concentrations in the standard solution were prepared at 0, 0.1, 0.5, 1, 5, 10, 20, 50, 100, 200 and 500 μM. 200 nL of the standard solution was injected onto the well pattern (diameter: 1 mm) of the parylene substrate, and repeated three times for each concentration. The solution droplet on the substrate was frozen in a freezer at -20 ° C for 24 hours, then slowly dried in a 4 ° C refrigerator for 1 hour, and then the remaining moisture was completely dried in air (20 ° C).
상기 대사체는 아미노산일 수 있으며, 보다 상세하게는 상기 아미노산은 알라닌(alanine), 트레오닌(threonine), 시스테인(cysteine), 이소루이신(isoleucine), 루이신(leucine), 페닐알라닌(phenylalanine), 티로신(tyrosine) 및 트립토판(tryptophan)으로 이루어진 군에서 선택되는 것일 수 있다.The metabolite may be an amino acid, and more specifically, the amino acid is alanine, threonine, cysteine, isoleucine, leucine, phenylalanine, tyrosine (tyrosine) and tryptophan (tryptophan) may be selected from the group consisting of.
본 발명의 실시예에 따르면, 페닐알라닌(F*)을 기질(matrix) 물질로, 발린(V*)을 ToF-SIMS 신호표준화 물질로 사용하여 20 종의 아미노산을 농도 0 ~ 500μM 범위에서 분석하여 교정 곡선을 그리고, 검출한계, 정량 하한, 분석 농도 범위 및 직선성을 확인하였다. According to an embodiment of the present invention, using phenylalanine (F * ) as a matrix material and valine (V * ) as a ToF-SIMS signal standardization material, 20 amino acids are analyzed and calibrated in a concentration range of 0 to 500 μM A curve was drawn, and the detection limit, lower limit of quantification, assay concentration range and linearity were checked.
그 결과, 도 5 및 표 2와 같이 20 종의 아미노산 중 8 종의 아미노산 알라닌(alanine), 트레오닌(threonine), 시스테인(cysteine), 이소루이신(isoleucine), 루이신(leucine), 페닐알라닌(phenylalanine), 티로신(tyrosine) 및 트립토판(tryptophan)은 농도 500μM 범위 내에서 높은 직선성(R2: 0.991~0.999)으로 정량 분석이 가능한 것을 확인할 수 있었다. 또한, 농도별 신호 세기가 일정하여 편차가 적고, 정량 하한은 농도 수 μM 정도까지 가능한 것이 확인되었다.As a result, as shown in Figure 5 and Table 2, 8 of the 20 amino acids alanine, threonine, cysteine, isoleucine, leucine, phenylalanine ), tyrosine (tyrosine) and tryptophan (tryptophan), it was confirmed that quantitative analysis is possible with high linearity (R 2 : 0.991 ~ 0.999) within the concentration range of 500 μM. In addition, it was confirmed that the signal intensity for each concentration was constant and the deviation was small, and the lower limit of quantification was possible up to several μM of concentration.
상기 질량분석법은 시간비행형 이차이온질량분광법(TOF-SIMS), 매트릭스 보조 레이저 탈착 이온화 비행시간형 질량분석법(MALDI-TOF MS) 및 탈착 전기 분무 이온화 질량분석법(DESI-MS)으로 이루어진 군에서 선택되는 것일 수 있으며, 보다 바람직하게는 시간비행형 이차이온질량분광법(TOF-SIMS)일 수 있으나, 이에 제한되는 것은 아니다.The mass spectrometry is selected from the group consisting of time-of-flight secondary ion mass spectrometry (TOF-SIMS), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and desorption electrospray ionization mass spectrometry (DESI-MS). It may be, more preferably time-flight secondary ion mass spectroscopy (TOF-SIMS), but is not limited thereto.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
<참고예> <Reference example>
하기의 참고예는 본 발명에 따른 각각의 실시예에 공통적으로 적용되는 참고예를 제공하기 위한 것이다.The following reference examples are intended to provide reference examples commonly applied to each embodiment according to the present invention.
1. 물질1. Matter
아미노산 글리신(glycine, G7126), L-알라닌(L-alanine, A7627), L-세린(L-serine, S4500), L-프롤린(L-proline, P0380), L-발린(L-valine, V0500), L-트레오닌(L-threonine, T8625), L-시스테인염산(L-cysteine hydrocholoride, C1276), L-아이소루신(L-isoluecine, I2752), L-루신(L-leucine, L8000), L-아스파라진(L-asparagine, A0884), L-아스파트산(L-aspartic acid, A9256), L-글루타민(L-glutamine, G3126), L-라이신 염산염(L-lysine monohydrochloride, L5626), L-글루탐산(L-glutamic acid, G1251), L-메티오닌(L-methionine, M9625), L-히스티딘 일염화물(L-histidine monohydrochloride monohydrate, H8125), L-페닐알라닌(L-phenylalanine, P2126), L-아르기닌(L-arginine, A5006), L-티로신(L-tyrosine, T3754) 및 그래핀 옥사이드(graphene oxide, GO, 777676)를 Sigma Aldrich에서 구매하였고, 동위원소가 표지된 L-페닐알라닌(L-phenylalanine, 13C9, 99%; 15N, 99%)과 L-발린(L-valine, 13C5, 99%; 15N, 99%)은 Cambridge Isotope Laboratories Inc. 제품을 사용하였다. Amino Acids Glycine (G7126), L-alanine (A7627), L-serine (S4500), L-proline (P0380), L-valine (V0500) ), L-threonine (T8625), L-cysteine hydrocholoride (C1276), L-isoluecine (I2752), L-leucine (L8000), L -Asparagine (A0884), L-aspartic acid (A9256), L-glutamine (G3126), L-lysine monohydrochloride (L5626), L -Glutamic acid (L-glutamic acid, G1251), L-methionine (L-methionine, M9625), L-histidine monohydrochloride monohydrate (H8125), L-phenylalanine (P2126), L- Arginine (A5006), L-tyrosine (T3754) and graphene oxide (GO, 777676) were purchased from Sigma Aldrich and isotopically labeled L-phenylalanine , 13C9, 99%; 15N, 99%) and L-valine (13C5, 99%; 15N, 99%) were purchased from Cambridge Isotope Laboratories Inc. product was used.
<실시예 1> 패럴린 우물 배열 기판 제작<Example 1> Fabrication of Parylene well array substrate
도 1과 같이 우물 배열의 기판을 제작하였다.As shown in FIG. 1, a substrate having an array of wells was fabricated.
보다 상세하게 화학기상증착법(Chemical Vapor Deposition, CVD)을 이용하여 실리콘(Si) 기판 표면 위에 패럴린 필름(~300nm)을 증착하였다. 포토레지스트(PR, AZ GXR 601)를 1μm 두께로 코팅한 후 노광(UV Dose 100mJ/cm2)과 현상 과정을 거쳐서 직경 1mm 크기의 우물 배열 패턴(4 x 9 array)을 새겼다. 반응성 이온식각 장비(Reactive ion etching, RIE, O2 20 sccm, 50W)으로 패럴린을 식각하여 PR과 동일한 패턴을 만들고, 아세톤(Acetone)으로 기판을 20분 동안 세척하여 패럴린 표면 위에 PR과 불순물을 제거한 후 메탄올(methanol) 및 이소프로필알코올(isopropyl alcohol, IPA)로 각 5분씩 기판을 세척하고 질소(N2) 건으로 건조하였다.In more detail, a parylene film (~300 nm) was deposited on the surface of a silicon (Si) substrate using chemical vapor deposition (CVD). After coating with photoresist (PR, AZ GXR 601) to a thickness of 1 μm, a well array pattern (4 x 9 array) with a diameter of 1 mm was engraved through exposure (
<실시예 2> 시료 분석<Example 2> Sample analysis
1. 시료 준비1. Sample preparation
안정한 동위원소로 표지된 L-페닐알라닌(L-phenylalanine, F*) 10mM, 안정한 동위원소로 표지된 L-발린(L-valine, V*) 100μM과 한 종류의 아미노산을 3차 수에 녹인 표준 용액을 만들었다. 표준 용액 내의 아미노산의 농도는 0, 0.1, 0.5, 1, 5, 10, 20, 50, 100, 200 및 500μM 로 상기 표준 용액을 패럴린 기판의 우물 패턴 (직경: 1mm) 위에 200nL 씩 주입하였으며, 농도별로 3번 반복하였다. 기판 위의 용액 방울을 냉동실 -20℃에서 24시간 동안 동결시킨 후, 4℃ 냉장고에서 1시간 동안 천천히 건조하였다. 이후 남은 수분을 공기(20℃) 중에 완전히 건조하였다. 또한, 아미노산 10mM 용액 및 그래핀 옥사이드(graphene oxide, 1 mg/ml) 용액도 동일한 방법으로 준비하였다. A standard solution in which 10 mM of L-phenylalanine (F * ) labeled with a stable isotope, 100 μM of L-valine (V * ) labeled with a stable isotope, and one amino acid were dissolved in tertiary water. made The concentration of amino acids in the standard solution was 0, 0.1, 0.5, 1, 5, 10, 20, 50, 100, 200 and 500 μM, and the standard solution was injected on a parylene substrate well pattern (diameter: 1 mm) at 200 nL each, Each concentration was repeated 3 times. After the solution droplets on the substrate were frozen in a freezer at -20°C for 24 hours, they were slowly dried in a refrigerator at 4°C for 1 hour. After that, the remaining moisture was completely dried in air (20 ° C.). In addition, an
2. ToF-SIMS 분석2. ToF-SIMS analysis
ToF-SIMS 분석을 위해 독일 ION-TOF 사의 ToF-SIMS 5-100 모델을 사용하였고, Positive mode에서 Pulsed 30 keV Bi3 + 일차 이온빔(0.4pA)을 이용하여 시료 표면의 250 x 250 μm2 영역(128 x 128 pixels)을 각 시료 당 약 1분씩 분석하였다. 측정하여 얻은 모든 mass spectra는 CH3+, C2H3+, C3H5+ 및 13C9H11 15NO2+ peaks를 이용하여 internal calibration 하였으며, 아미노산 ToF-SIMS 분석 정보는 표 1과 같다. 각 아미노산의 ToF-SIMS 분석 측정값을 V* 측정값으로 나누어서 표준화하였다.For the ToF-SIMS analysis, a ToF-SIMS 5-100 model from ION-TOF of Germany was used, and a 250 x 250 μm 2 area ( 128 x 128 pixels) were analyzed for about 1 minute for each sample. All mass spectra obtained from the measurement were internally calibrated using CH 3 +, C 2 H 3 +, C 3 H 5 + and 13 C 9 H 11 15 NO 2 + peaks, and amino acid ToF-SIMS analysis information is shown in Table 1. same. ToF-SIMS analysis measurements of each amino acid were normalized by dividing by V * measurements.
I, IsoleucineL, Leucine
I, Isoleucine
3. 검증3. Verification
아미노산 농도를 x 값, 표준화한 ToF-SIMS 측정값을 y 값으로 하고, 아미노산 농도별 표준화된 측정값의 평균과 표준 편차를 그래프로 그렸다. 농도별 평균값에 대해 최소 제곱법(method of least square)을 적용하여 직선 교정 곡선(y=mx+b)을 그렸다. 직선의 식에서 m은 기울기(slope)이고, b는 y 절편(y intercept)이다. 직선성(linearity)의 척도를 나타내는 상관계수(correlation coefficient)의 제곱, R2는 다음 식을 이용해서 구하였다.The amino acid concentration was used as the x value and the standardized ToF-SIMS measured value as the y value, and the average and standard deviation of the standardized measured values for each amino acid concentration were graphed. A linear calibration curve (y=mx+b) was drawn by applying the method of least squares to the average value for each concentration. In the equation of a straight line, m is the slope and b is the y intercept. The square of the correlation coefficient representing a measure of linearity, R 2 , was obtained using the following equation.
여기서 는 모든 x 값의 평균, 는 모든 y 값의 평균이다.here is the average of all x values, is the average of all y values.
3개의 바탕(아미노산 0μM)의 신호를 측정하여 표준 편차(s)를 계산하고, 직선 교정 곡선에서 구한 기울기 m을 이용하여 최소 검출 가능 농도(Limit of detection, LOD)와 정량 하한(Lower limit of quantitation, LLOQ)을 다음 식을 통해 얻었다.The standard deviation (s) was calculated by measuring the signals of three blanks (0 μM amino acid), and the minimum detectable concentration (LOD) and lower limit of quantitation (LOD) were calculated using the slope m obtained from the straight line calibration curve. , LLOQ) was obtained through the following equation.
<< 실시예Example 3> 결과 확인 3> Check the result
1. 매트릭스(matrix)로서의 아미노산 평가1. Evaluation of amino acids as a matrix
ToF-SIMS를 이용하여 유기물을 분석하는 데 있어서 분석 신호 세기를 향상시키기 위해 다양한 물질이 기질(matrix)로 제시되었다. 그 중 그래핀 옥사이드 (graphene oxide, GO)는 기판 표면 위에 균일한 층을 형성하고 지질과 같은 생체 분자의 신호를 향상시키는 성질 때문에 ToF-SIMS 분석에 좋은 기질 물질로 간주된다. 그러나 도 3과 같이 패럴린 기판의 우물 패턴 위에 건조된 GO 기질은 아미노산(alanine) 농도별 ToF-SIMS 신호가 일정하지 않고 직선성이 부족하여 정량 분석에는 한계를 나타내었다. Various materials have been suggested as matrices to improve analysis signal strength in analyzing organic matter using ToF-SIMS. Among them, graphene oxide (GO) is considered a good substrate material for ToF-SIMS analysis because of its properties of forming a uniform layer on the substrate surface and enhancing the signal of biomolecules such as lipids. However, as shown in FIG. 3, the ToF-SIMS signal for each amino acid (alanine) concentration of the GO substrate dried on the well pattern of the parylene substrate was not constant and lacked linearity, which showed limitations in quantitative analysis.
상기 결과는 그래핀 옥사이드와 아미노산이 용액상에서 화학적 성질이 달라, 건조된 후 그래핀 옥사이드 내의 아미노산의 비율이 초기 용액의 농도에 부합하지 않기 때문이다.This result is because graphene oxide and amino acids have different chemical properties in the solution phase, so that the ratio of amino acids in graphene oxide after drying does not match the concentration of the initial solution.
ToF-SIMS를 이용한 아미노산 정량 분석을 위해, 용액상에서 화학적 성질이 유사한 형태로 녹아 있는 20 종의 아미노산을 ToF-SIMS 기질로서 확인하였다. For quantitative analysis of amino acids using ToF-SIMS, 20 amino acids dissolved in a form with similar chemical properties in solution were identified as ToF-SIMS substrates.
20 종의 아미노산 10mM을 각각 패럴린 기판의 우물 패턴 위에 실험 방법의 시료 준비 절차를 따라 건조한 결과, 도 4와 같이 아미노산 페닐알라닌(phenylalanine), 발린(valine), 메티오닌(methionine), 이소루이신(isoleucine), 루이신(leucine) 및 트립토판(tryptophan)이 기판 표면 위에 연속적이고 균일한 층을 형성하였다. 그 중 페닐알라닌이 1mm 직경의 우물 패턴 위 전체에 가장 균일하게 형성되어 있어 기질(matrix)로 적합한 것이 확인되었다. 다만 분석 물질로서 페닐알라닌과의 신호 간섭을 피하기 위해 안정한 동위원소로 표지된 L-페닐알라닌(L-phenylalanine, F*)을 대신 사용하였다.As a result of drying 10 mM of 20 amino acids each according to the sample preparation procedure of the experimental method on the well pattern of the parylene substrate, as shown in FIG. 4, the amino acids phenylalanine, valine, methionine, and isoleucine ), leucine and tryptophan formed a continuous and uniform layer on the substrate surface. Among them, it was confirmed that phenylalanine was most uniformly formed throughout the well pattern with a diameter of 1 mm, and thus suitable as a matrix. However, in order to avoid signal interference with phenylalanine as an analyte, L-phenylalanine (F*) labeled with a stable isotope was used instead.
2. 아미노산 ToF-SIMS 분석2. Amino acid ToF-SIMS analysis
페닐알라닌(F*)을 기질(matrix) 물질로, 발린(V*)을 ToF-SIMS 신호표준화 물질로 사용하여 20 종의 아미노산을 농도 0 ~ 500μM 범위에서 분석하여 교정 곡선을 그리고, 검출한계, 정량 하한, 분석 농도 범위 및 직선성을 평가하였다. Using phenylalanine (F * ) as a matrix material and valine (V * ) as a ToF-SIMS signal standardization material, 20 amino acids were analyzed in the concentration range of 0 ~ 500 μM to draw a calibration curve, detection limit, and quantification The lower limit, assay concentration range and linearity were evaluated.
그 결과, 도 5 및 표 2와 같이 20 종의 아미노산 중 8 종의 아미노산 알라(alanine), 트레오닌(threonine), 시스테인(cysteine), 이소루이신(isoleucine), 루이신(leucine), 페닐알라닌(phenylalanine), 티로신(tyrosine) 및 트립토판(tryptophan)은 농도 500μM 범위 내에서 높은 직선성(R2: 0.991~0.999)으로 정량 분석이 가능한 것을 확인할 수 있었다. 또한, 농도별 신호 세기가 일정하여 편차가 적고, 정량 하한은 농도 수 μM 정도까지 가능하였다. As a result, as shown in FIG. 5 and Table 2, 8 of the 20 amino acids are alanine, threonine, cysteine, isoleucine, leucine, and phenylalanine. ), tyrosine (tyrosine) and tryptophan (tryptophan), it was confirmed that quantitative analysis is possible with high linearity (R 2 : 0.991 ~ 0.999) within the concentration range of 500 μM. In addition, the signal intensity for each concentration was constant, so there was little variation, and the lower limit of quantification was possible up to several μM of concentration.
반면, 도 6과 같이 F* 기질 내 아미노산의 ToF-SIMS 신호 감응의 차이로 나머지 12 종 아미노산 중 6 종의 아미노산 프롤린(proline), 발린(valine), 아스파라긴(asparagine), 글루탐산(glutamic acid), 글루타민(glutamine) 및 히스티딘(histidine)은 농도가 높아질수록 신호 감응이 농도에 비례하지 않아 직선성은 확인되지 않았다. 하지만 농도에 따라 일정한 신호 감응을 보이기 때문에 sigmoid curve와 같은 비선형 모델을 이용하면 정량 분석은 가능할 것으로 예상되었다.On the other hand, as shown in FIG. 6, six of the remaining 12 amino acids are proline, valine, asparagine, glutamic acid, As the concentration of glutamine and histidine increased, the signal response was not proportional to the concentration, so linearity was not confirmed. However, it was expected that quantitative analysis would be possible using a non-linear model such as a sigmoid curve because it shows a constant signal response depending on the concentration.
마지막으로 나머지 6 종의 아미노산 글리신(glycine), 세린(serine), 아스파틱산(aspartic acid), 라이신(lysine), 메티오닌(methionine) 및 아르기닌(arginine)은 기질 F* 의 의한 신호 간섭 및 불안정한 ToF-SIMS 신호로 정량 분석을 위한 일정한 패턴의 신호 세기가 나타나지 않았다.Finally, the remaining six amino acids, glycine, serine, aspartic acid, lysine, methionine, and arginine, are responsible for signal interference by substrate F * and unstable ToF- The SIMS signal did not show a constant pattern of signal intensity for quantitative analysis.
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22
))
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (10)
상기 시료 혼합물을 분석용 기판에 주입 후 건조시키는 단계를 포함하는, 질량분석법을 이용한 대사체 정량분석 방법.Preparing a sample mixture by mixing a matrix containing an amino acid labeled with a stable isotope as an active ingredient and an analyte; and
A method for quantitative analysis of metabolites using mass spectrometry, comprising the step of injecting the sample mixture into a substrate for analysis and then drying it.
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