Rapid Analysis of Cyanide Based on a Ratiometric Fluorescent Probe Using Gold Nanoclusters-Fluorescein

doi:10.12116/j.issn.1004-5619.2025.350403

Abstract

Abstract:

Objective To establish a rapid analysis method for cyanide based on a ratiometric fluorescent probe, providing a quantitative strategy for on-site visual and rapid detection of cyanide. Methods A dual-emission ratiometric fluorescent probe （AuNCs-FL） was constructed by using bovine serum albumin （BSA）-stabilized gold nanoclusters （AuNCs, fluorescence emission at 660 nm） as the responsive signal unit and fluorescein （FL, emission at 515 nm） as the internal reference. Results The etching effect of cyanide on AuNCs resulted in fluorescence quenching at 660 nm, while the fluorescence intensity of FL at 515 nm remained unchanged, enabling a rapid response analysis of cyanide shift from red to green fluorescence. The developed probe enabled rapid analysis of cyanide within 3 min, with a limit of detection （LOD） of 3.4 mg/L and a visual detection range of 10-100 mg/L. Conclusion The AuNCs-FL fluorescent probe is structurally simple, low-cost, and easy to operate, delivering rapid and accurate results. It also avoids the interference from sulfides encountered in commercial cyanide test kits, making it suitable for the on-site rapid detection of suspected powder samples in cyanide poisoning cases.

Key words: forensic medicine, toxicological analysis, gold nanoclusters （AuNCs）, fluorescein, cyanide, ratiometric fluorescent probe, rapid analysis, portable device

CLC Number:

Tai-shen HE, Zhong-jiang LÜ, Yi-ming SUN, Yu-yang LI, Yi YE, Yao LIN, Lin-chuan LIAO. Rapid Analysis of Cyanide Based on a Ratiometric Fluorescent Probe Using Gold Nanoclusters-Fluorescein[J]. Journal of Forensic Medicine, 2025, 41(4): 340-347.

Figures/Tables 10

Fig. 1 Photographs of the self-made portable fluorescence reading device

Fig. 2 Spectral characterization images of AuNCs

Fig. 3 Spectral characterization images of the constructed ratiometric fluorescent probe

Fig. 4 Optimization of reaction conditions for the AuNCs-FL probe and cyanide

Fig. 5 Fluorescence images of single AuNCs， FL，as well as AuNCs-FL after reacting with cyanideat a series of mass concentrations

Fig. 6 Standard curve of cyanide massconcentration versus G/R ratio

Tab. 1 Performance comparison between this method and other fluorescent analysis methods for cyanide

氰化物荧光分析方法	检出限	反应时间	便携式检测装置	数据来源
碳点-AuNCs法	0.15 μmol/L	-	无	[17]
香豆素探针法	0.22 μmol/L	-	无	[23]
氰乙烯基探针法	12.4 nmol/L	30 s	无	[24]
三重荧光探针法	45 nmol/L	>13 min	无	[25]
Benzo-Hemicyanine法	0.43 μmol/L	-	无	[26]
罗丹明B衍生物法	0.33 μmol/L	30 min	无	[27]
Hg-石墨烯量子点法	3.1 μmol/L	5 min	无	[28]
Calixarene法	0.115 μmol/L	-	无	[29]
HBT-Br-thiazolium法	1.79 μmol/L	2 min	试纸	[30]
三苯胺有机探针法	36 nmol/L	4 min	试纸	[31]
聚集诱导发光活性分子法	6.17 nmol/L	-	试纸	[32]
AuNCs-FL法	52.3 μmol/L	2 min	便携式装置（可读数）	本研究

Fig. 7 Results of performance analysis forcyanide using this method

Fig. 8 Fluorescence images of three types of samplesbefore and after the addition of cyanide

Tab. 2 Detection values and recovery rates of three types of samples spiked with cyanide

样品	氰化物加标质量浓度/（mg·L^-1）	检测值/（ $x ¯$ ±s，mg·L^-1）	回收率/%	RSD/%
白糖	30	30.7±0.6	102.3	1.2
淀粉	30	26.0±1.8	86.7	4.5
泥土	30	29.9±2.2	99.7	4.4

Tab. 2 Detection values and recovery rates of three types of samples spiked with cyanide

样品	氰化物加标质量浓度/（mg·L^-1）	检测值/（ $x ¯$ ±s，mg·L^-1）	回收率/%	RSD/%
白糖	30	30.7±0.6	102.3	1.2
淀粉	30	26.0±1.8	86.7	4.5
泥土	30	29.9±2.2	99.7	4.4

References 33

[1]	廖林川. 法医毒物分析[M].5版.北京：人民卫生出版社，2016：119-121.
	LIAO L C. Forensic toxicological analysis[M]. 5th ed. Beijing： People’s Medical Publishing House，2016：119-121.
[2]	陈东. 医源性氰化物积蓄中毒案例鉴定探讨[J].中国法医学杂志，2024，39（S1）：75-76. doi：10.13618/j.issn.1001-5728.2024.S.039 .
	CHEN D. Discussion on identification cases of iatrogenic cyanide accumulation and poisoning[J]. Zhongguo Fayixue Zazhi，2024，39（S1）：75-76.
[3]	HENDRY-HOFER T B， NG P C， WITEOF A E， et al. A review on ingested cyanide： Risks， clinical presentation， diagnostics， and treatment challenges[J]. J Med Toxicol，2019，15（2）：128-133. doi：10.1007/s13181-018-0688-y .
[4]	OSAK M， BUSZEWICZ G， BAJ J， et al. Determination of cyanide in blood for forensic toxicology purposes — A novel NCI GC-MS/MS technique[J]. Molecules，2021，26（18）：5638. doi：10.3390/molecules26185638 .
[5]	AKHGARI M， BAGHDADI F， KADKHODAEI A. Cyanide poisoning related deaths， a four-year experience and review of the literature[J]. Aust J Forensic Sci，2016，48（2）：186-194. doi：10.1080/00450618.2015.1045552 .
[6]	魏鑫，王遥雪，凌约涛，等. 顶空气相色谱法测定固体废物中氰化物[J].化学分析计量，2020，29（6）：15-18. doi：10.3969/j.issn.1008-6145.2020.06.004 .
	WEI X， WANG Y X， LING Y T， et al. Determination of cyanide in solid wastes by headspace gas chromatography[J]. Huaxue Fenxi Jiliang，2020，29（6）：15-18.
[7]	左家信，范翔，李欣，等. 顶空-气相色谱法测定饮用水中的氰化物和氯化氰[J].分析仪器，2023（5）：36-40. doi：10.3969/j.issn.1001-232x.2023.05.008 .
	ZUO J X， FAN X， LI X， et al. Determination of cyanide and cyanogen chloride in drinking water by headspace-gas chromatography[J]. Fenxi Yiqi，2023（5）：36-40.
[8]	温尚龙，陈欣义，庞兆东，等. 一种快速定性测试废水处理中氰化物含量的检测方法：CN112798577A[P].2021-05-14.
	WEN S L， CHEN X Y， PANG Z D， et al. A rapid qualitative detection method for cyanide content in wastewater treatment： CN112798577A[P]. 2021-05-14.
[9]	王晓芳，陈美，杨春亮，等. 木薯中氰化物含量的异烟酸-吡唑林酮分光光度法测定[J].分析仪器，2009（1）：32-34.
	doi：10.3969/j.issn.1001-232X.2009.01.010.WANG X F， CHEN M， YANG C L， et al. Determination of cyanide in cassava by isonicotinic acid-pyrazolone spectrophotometry[J]. Fenxi Yiqi，2009（1）：32-34.
[10]	WEI Y， TANG J， ZHANG J， et al. A label-free fluorescent-hydrogel sensor for heparin detection in diluted whole blood[J]. Chem Commun （Camb），2025，61（6）：1215-1218. doi：10.1039/d4cc03780d .
[11]	YANG W， YE L， WU Y， et al. Arsenic field test kits based on solid-phase fluorescence filter effect induced by silver nanoparticle formation[J]. J Hazard Mater，2024，470：134038. doi：10.1016/j.jhazmat.2024.134038 .
[12]	JACKSON R， ODA R P， BHANDARI R K， et al. Development of a fluorescence-based sensor for rapid diagnosis of cyanide exposure[J]. Anal Chem，2014，86（3）：1845-1852. doi：10.1021/ac403846s .
[13]	LIN Y， YE S， TIAN J， et al. Paper-assisted ratiometric fluorescent sensors for on-site sensing of sulfide based on the target-induced inner filter effect[J]. J Hazard Mater，2023，459：132201. doi：10.1016/j.jhazmat.2023.132201 .
[14]	LIN Y， LI Y， CHANG H， et al. Rapid testing of Δ9-tetrahydrocannabinol and its metabolite on-site using a label-free ratiometric fluorescence assay on a smartphone[J]. Anal Chem，2023，95（18）： 7363-7371. doi：10.1021/acs.analchem.3c00666 .
[15]	ZHOU J， CHEN X， WEI Y， et al. Portable and rapid fluorescence turn-on detection of total pepsin in saliva based on strong electrostatic interactions[J]. Anal Chem，2023，95（49）：18303-18308. doi：10. 1021/acs.analchem.3c04723 .
[16]	LONG L， YUAN X， CAO S， et al. Determination of cyanide in water and food samples using an efficient naphthalene-based ratiometric fluorescent probe[J]. ACS Omega，2019，4（6）：10784-10790. doi：10.1021/acsomega.9b01308 .
[17]	HU Y， LU X， JIANG X， et al. Carbon dots and AuNCs co-doped electrospun membranes for ratiometric fluorescent determination of cyanide[J]. J Hazard Mater，2020，384：121368. doi：10.1016/j.jhaz mat.2019.121368 .
[18]	LIU Y， AI K， CHENG X， et al. Gold-nanocluster-based fluorescent sensors for highly sensitive and selective detection of cyanide in water[J]. Adv Funct Mater，2010，20（6）：951-956. doi：10.1002/adfm.200902062 .
[19]	SUN Z， WU Z， ZONG Y， et al. Construction of metal-organic framework as a novel platform for ratiometric determination of cyanide[J]. Biosensors （Basel），2024，14（6）：276. doi：10.3390/bios14060276 .
[20]	YANG H， YANG Y， LIU S， et al. Ratiometric and sensitive cyanide sensing using dual-emissive gold nanoclusters[J]. Anal Bioanal Chem，2020，412（23）：5819-5826. doi：10.1007/s00216-020-02806-2 .
[21]	WEI Y， YANG L， YE Y， et al. A simple aptamer-dye fluorescence sensor for detecting Δ9-tetrahydrocannabinol and its metabolite in urban sewage[J]. Chem Commun （Camb），2024，60（39）：5205-5208. doi：10.1039/d4cc00824c .
[22]	YE S， YU B， REN T， et al. Point-of-care platform based on solid-phase fluorescence filter effect for urinary iodine testing in children and pregnant women[J]. Anal Chem，2023，95（37）：13949-13956. doi：10.1021/acs.analchem.3c02531 .
[23]	PAN W， HAN L， CAO X， et al. Dual-response near-infrared fluorescent probe for detecting cyanide and mitochondrial viscosity and its application in bioimaging[J]. Food Chem，2023，407：135163. doi：10.1016/j.foodchem.2022.135163 .
[24]	PENG T， LI S， ZHOU Y， et al. Two cyanoethylene-based fluorescence probes for highly efficient cyanide detection and practical applications in drinking water and living cells[J]. Talanta，2021，234：122615. doi：10.1016/j.talanta.2021.122615 .
[25]	LI Q， NIE J， SHAN Y， et al. Water-soluble fluorescent probe for simultaneous detection of cyanide， hypochlorite and bisulfite at different emission wavelengths[J]. Anal Biochem，2020，591：113539. doi：10. 1016/j.ab.2019.113539 .
[26]	MAGESH K， VIJAY N， WU S P， et al. Dual-responsive benzo-hemicyanine-based fluorescent probe for detection of cyanide and hydrogen sulfide： Real-time application in identification of food spoilage[J]. J Agric Food Chem，2023，71（2）：1190-1200. doi：10.1021/acs.jafc.2c05567 .
[27]	MU S， GAO H， LI C， et al. A dual-response fluorescent probe for detection and bioimaging of hydrazine and cyanide with different fluorescence signals[J]. Talanta，2021，221：121606. doi：10.1016/j.talanta.2020.121606 .
[28]	KONGSANAN N， PIMSIN N， KEAWPROM C， et al. A fluorescence switching sensor for sensitive and selective detections of cyanide and ferricyanide using mercuric cation-graphene quantum dots[J]. ACS Omega，2021，6（22）：14379-14393. doi：10.1021/acso mega.1c01242 .
[29]	OGUZ A， OGUZ M， KURSUNLU A N， et al. A fully water-soluble Calix[4]arene probe for fluorometric and colorimetric detection of toxic hydrosulfide and cyanide ions： Practicability in living cells and food samples[J]. Food Chem，2023，401：134132. doi：10.1016/j.foodchem.2022.134132 .
[30]	ERDEMIR S， MALKONDU S. Visual and quantitative detection of CN^- ion in aqueous media by an HBT-Br and thiazolium conjugated fluorometric and colorimetric probe： Real samples and useful applications[J]. Talanta，2021，221：121639. doi：10. 1016/j.talanta.2020.121639 .
[31]	SERT A， ERDEMIR S， MALKONDU S. Ratiometric detection and monitoring of cyanide in biological， environmental and food samples by a novel triphenylamine-xhantane based fluorescent probe[J]. Anal Chim Acta，2024，1320：343000. doi：10.1016/j.aca.2024.343000 .
[32]	MAJEED S， WASEEM M T， KHAN G S， et al. Development of AIEE active fluorescent and colorimetric probe for the solid， solution， and vapor phase detection of cyanide： Smartphone and food applications[J]. Analyst，2022，147（17）：3885-3893. doi：10.1039/d2an00937d .
[33]	NG B， QUINETE N， GARDINALI P R. Assessing accuracy， precision and selectivity using quality controls for non-targeted analysis[J]. Sci Total Environ，2020，713：136568. doi：10.1016/j.scitotenv.2020. 136568 .