可同时比色检测硫离子和高半胱氨酸/半胱氨酸的化学探针的合成及应用

doi:10.7503/cjcu20170806

摘要/Abstract

摘要：

合成了一个能同时检测硫离子(S^2-)和高半胱氨酸/半胱氨酸(Hcy/Cys)的化学探针1, 采用核磁共振波谱(NMR)、高分辨质谱(HRMS)、元素分析和傅里叶换红外光谱(FTIR)等表征了其分子结构, 并通过高效液相色谱、质谱以及核磁滴定氢谱实验研究了探针1检测S^2-和Hcy/Cys的机理. 实验结果表明, 探针1能同时对S^2-和Hcy/Cys进行快速、高敏感性和选择性识别, 并且是通过硫醇亲核反应识别的. 在Tris-HCl缓冲溶液[10 mmol/L, pH=7.40, 50%(体积分数)乙醇]中, 探针1对S^2-于552 nm处表现了显著的吸收强度增强并伴随溶液颜色由无色变为紫红色; 对Hcy/Cys于552 nm处表现了特异的荧光增强, 而对Cys的检测还可见溶液颜色由无色变为酒红色, 据此制备了负载探针1的滤纸传感器用于比色检测S^2-和Cys. 此外, 将探针1用于人血清的加标回收率测定, 获得了较好的结果.

关键词: 探针, 比色检测, 硫离子, 高半胱氨酸/半胱氨酸

Abstract:

A new but versatile probe 1 was developed, which can be used for simultaneous detection and discrimination of sulphion(S^2-) and homocysteine(Hcy)/cysteine(Cys). 4-Bromine-1,8-naphthalenedicarboxy-licanhydride was used as the starting material to prepare probe 1 by the reactions of condensation and Williamson ether synthesis. Meanwhile, probe 1 was characterized by means of ¹H NMR, ¹³C NMR, HRMS, elemental analysis and FTIR spectra. The recognition mechanism of the colorimetric probe 1 responses to S^2- and Hcy/Cys was verified by high performance liquid chromatography, mass spectrum and ¹H NMR titration. A lot of detailed experiment results indicate that the colorimetric probe 1 responses of these three analytes are most likely the result of the thiol nucleophilic reaction. Probe 1 shows rapid, selective and sensitive response for these three analytes with unique absorbance enhancement at 552 nm for S^2- and distinct fluorescent turn on signal changes at 552 nm for Hcy/Cys. At the same time, after reaction of probe 1 with S^2- or Cys in Tris-HCl(10 mmol/L, pH=7.4, 50% ethanol) buffer solution, the color of solution changed from colorless to purple-red or wine, respectively. Therefore, probe 1 could be used to develop a colorimetric test kit for S^2- and cysteine. The detection limits of probe 1 for S^2-, Hcy and Cys were respectively determined as 8.83×10^-6, 1.26×10^-6 and 7.65×10^-7 mol/L, indicating that the probe 1 has a good sensitivity for S^2- and Hcy/Cys. In addition, the competitive experiments results show that the probe 1 is highly selective for S^2- and Hcy/Cys over other competitive. Probe 1 could be used to detect S^2- and Hcy/Cys in the serum samples with satisfactory results.

Key words: Probe, Colorimetric detection, Sulphion, Homocysteine/cysteine

中图分类号:

O657

TrendMD:

夏玉婷, 江波, 吴庆, 胡庆红, 袁泽利. 可同时比色检测硫离子和高半胱氨酸/半胱氨酸的化学探针的合成及应用. 高等学校化学学报, 2018, 39(8): 1647.

XIA Yuting, JIANG Bo, WU Qing, HU Qinghong, YUAN Zeli. Synthesis and Application of Colorimetric Probe for the Simultaneous Detection of Sulphion and Homocysteine/Cysteine in the Serum Samples^†. Chem. J. Chinese Universities, 2018, 39(8): 1647.

图/表 9

Scheme 1 Synthesis route of probe 1

Fig.1 Absorbance spectra change of probe 1(20 μmol/L) in Tris-HCl(10 mmol/L, pH=7.4, 50% ethanol) upon individual addition of 100 μmol/L different anions(A) and fluorescence spectra change of probe 1(20 μmol/L) in Tris-HCl(10 mmol/L, pH=7.4, 50% ethanol) upon individual addition of 100 μmol/L different amino acids(B)Inset: color changes of probe 1 solution before(left) and after(right) the addition of S2-(A) and Cys(B). (A) Other anions include F-, Cl-, Br-, I-, NO3-, CN-, PO43-, HPO42-, H2PO4-, SCN-, SO42-, HSO4-, CO32-, HCO3-, ClO3-, IO4-, CH3COO-; (B) other amino acids include glycine, serine, aspartic acid, glutathione, tyrosine, glutamic acid, arginine, tryptophan, valine, histidine, methionine, phenylalanine, phenylglycine, alanine, leucine, methionine.

Fig.2 Absorbance spectra of probe 1(20 μmol/L) in the presence of S2-(0—42 μmol/L)(A) and luorescece spectra of probe 1(20 μmol/L) in the presence of Hcy(0—100 μmol/L)(B)

Fig.3 Absorbance(552 nm) changes of probe 1(20 μmol/L) in Tris-HCl(10 mmol/L, pH=7.40, 50% ethanol) solution upon individual addition of 44 μmol/L miscellaneous anions and followed by further addition of 44 μmol/L S2-(A) and fluorescence intensity(552 nm) changes of probe 1(20 μmol/L) in Tris-HCl(10 mmol/L, pH=7.40, 50% ethanol) solution upon individual addition of 100 μmol/L amino acids and followed by further addition of 100 μmol/L of Hcy(A) a. S2-; b. S2-+F-; c. S2++SCN-; d. S2-+Cl-; e. S2-+SO42-; f. S2-+Br-; g. S2-+I-; h. S2-+HSO3-; i. S2-+NO3-; j. S2-+CN-; k. S2-+CO32-; l. S2-+PO43-; m. S2-+HCO3-; n. S2-+HPO42-; o. S2-+ClO3-; p. S2-+IO4-; q. S2-+H2PO4-; r. S2-+Ac-. (B) a. Hcy; b. Hcy+GSH; c. Hcy+Try; d. Hcy+Glu; e. Hcy+Arg; f. Hcy+Gly; g. Hcy+Trp; h. Hcy+Asn; i. Hcy+Val; j. Hcy+Hcy; k. Hcy+His; l. Hcy+Met; m. Hcy+Ser; n. Hcy+Phe; o. Hcy+Phg; p. Hcy+Cys; q. Hcy+Ala; r. Hcy+Leu; s. Hcy+Mts.

Fig.4 Partial 1H NMR spectra of probe 1(a), product of probe 1 with 0.835 mol/L(b) and 1.67 mol/L(c) S2- in DMSO-d6/D2O(volume ratio 9:1) and the intermidate(d)

Fig.5 Sensing mechanism of probe 1 to S2-, Hcy and Cys

Fig.6 Color changes of the filter paper coated with probe 1(20 μmol/L) used for the detection of S2-(A) and Cys(B)(A) a. Only probe 1; b. 1+F-; c. 1+Cl-.; d. 1+Br-; e. 1+ I-; f. 1+NO3-; g. 1+S2-; h. 1+SO42-; i. 1+HSO4-; j. 1+CO32-; k.1+HCO3-; l. 1+ClO32-; m. 1+PO43-; n. 1+H2PO4-; o. 1+HPO42-; p. 1+SCN-; q. 1+IO4-; r. 1+CN-; s. 1+AcO-. (B) a. Only probe 1; b. 1+Hcy; c. 1+Met; d. 1+Arg; e. 1+Gly; f. 1+Trp; g. 1+GSH; h. 1+Ser; i. 1+His; j. 1+Lys; k. 1+Val; l. 1+Phg; m. 1+Phe; n. 1+Ala; o. 1+cys; p. 1+Tyr; q. 1+Asn; r. 1+Leu; s. 1+Glu.

Fig.7 Color changes of the filter paper coated with probe 1(20 μmol/L) used for the detection of S2-(A) and Cys(B)(A)c(S2-)/(μmol·L-1): a. 0; b. 6.0; c. 12.0; d. 18.0; e. 24.0; f. 30.0; g. 40.0. (B) c(Cys)/(μmol·L-1): a. 0; b. 10.0; c. 30.0; d. 40.0; e. 50.0; f. 60.0; g. 80.0.

Table 1 Determination of S2-, Hcy and Cys in the serum samples

Analyte	c(Spiked)/(μmol·L^-1)	Quantities measured/(μmol·L^-1)	Average recovery(%)	RSD(%)(n=5)
$S 2 - a$	4	3.9	96.4	2.0
	6	6.3	105.8	7.6
	8	7.6	95.3	2.2
Cys^b	14	13.8	98.2	2.6
	16	15.9	99.5	3.6
	18	17.7	98.3	2.3
Hcy^b	16	15.9	99.3	5.3
	18	17.9	99.4	4.9
	20	19.4	97.2	6.7

Table 1 Determination of S2-, Hcy and Cys in the serum samples

Analyte	c(Spiked)/(μmol·L^-1)	Quantities measured/(μmol·L^-1)	Average recovery(%)	RSD(%)(n=5)
$S 2 - a$	4	3.9	96.4	2.0
	6	6.3	105.8	7.6
	8	7.6	95.3	2.2
Cys^b	14	13.8	98.2	2.6
	16	15.9	99.5	3.6
	18	17.7	98.3	2.3
Hcy^b	16	15.9	99.3	5.3
	18	17.9	99.4	4.9
	20	19.4	97.2	6.7

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