Detection of Ketamine and Norketamine Using an Aptamer-Functionalized Graphene Oxide Fluorescent Sensor

doi:10.12116/j.issn.1004-5619.2025.350409

Abstract

Abstract:

Objective To construct an aptamer-functionalized carboxylated graphene oxide （CGO） fluorescent sensor to achieve highly sensitive and specific detection of ketamine （KET） and its metabolite norketamine （NK） using an aptamer capable of simultaneously recognizing KET and NK. Methods A specific aptamer for simultaneous recognition of KET and NK was screened using graphene oxide-systematic evolution of ligand by exponential enrichment （GO-SELEX） and molecular docking techniques. The aptamer, labeled with Cy5 fluorescence, was chemically conjugated to CGO to construct an aptamer-functionalized CGO fluorescent sensor. By optimizing detection conditions, including the mass concentration of CGO, aptamer concentration, reaction temperature, and incubation time, quantitative analysis of the target analytes was achieved using the ratio of fluorescence intensity changes before and after target addition. The stability of the sensor in biological matrices was evaluated by monitoring fluorescence intensity changes over incubation time in blank blood and urine, in comparison with the traditional physical adsorption-based CGO fluorescent sensor. Spiked recovery experiments in blank blood and urine were conducted to compare performance with that of HPLC-MS/MS. Results A specific aptamer A5 was selected and chemically conjugated with CGO to construct the aptamer-functionalized CGO fluorescent sensor. Under optimized conditions, the proposed fluorescent sensor exhibited a linear detection range of 1.0-5.0 ng/mL for KET, with a limit of detection （LOD） of 0.86 ng/mL; while for NK, the linear detection range was 1.0-5.0 ng/mL, with an LOD of 0.70 ng/mL. Compared with the CGO fluorescent sensor constructed via physical adsorption, this sensor demonstrated greater stability in blood and urine. The spiked recovery rates of KET and NK in blank blood and urine ranged from 81.50% to 110.03%, exhibiting detection performance comparable to that of HPLC-MS/MS. Conclusion The aptamer screening method offers a novel approach for selecting aptamers targeting drugs and their metabolites. The constructed aptamer-functionalized CGO fluorescent sensor provides an efficient and reliable strategy for the high-performance detection of KET and NK.

Key words: forensic medicine, toxicological analysis, ketamine, norketamine, graphene oxide-systematic evolution of ligand by exponential enrichment （GO-SELEX）, aptamer, chemical conjugation, fluorescent sensor

CLC Number:

Li-xia WEI, Bo LIU, Xiao-yuan YANG, Xi ZHANG, Yi-feng LAN, Chao ZHANG, Juan JIA, Dan ZHANG, Zhi-wen WEI, Ke-ming YUN, Zhe CHEN. Detection of Ketamine and Norketamine Using an Aptamer-Functionalized Graphene Oxide Fluorescent Sensor[J]. Journal of Forensic Medicine, 2025, 41(4): 326-339.

Figures/Tables 14

Fig. 1 The screening of ketamine-specific aptamers based on GO-SELEX

Fig. 2 Incubation results of GO and ssDNAat different mass ratios

Fig. 3 Amplification results under different annealingtemperatures and cycle numbers

Fig. 4 Recovery rates of ketamine-binding ssDNAin the library after each round of screening

Fig. 5 Gel electrophoretogram of the PCR amplificationproducts after each round of screening

Fig. 6 Fitting curves for the binding of candidateaptamers （A5， A14） to ketamine

Fig. 7 Fitting curve for the binding ofcandidate aptamer A5 to norketamine

Tab. 1 Kd values of candidate aptamers toward ketamine before and after truncation of primer sequences

核酸适配体	引物序列剪切前（78 nt）		引物序列剪切后（40 nt）
核酸适配体	解离常数（K_d）	R²	解离常数（K_d）	R²
A5	12.00±3.20	0.94	13.09±1.79	0.98
A7	22.83±5.93	0.95	10.80±1.67	0.97
A8	24.02±10.94	0.86	4.97±1.05	0.93
A10	2.42±0.59	0.85	5.30±2.18	0.78
A12	9.36±3.55	0.85	3.82±0.70	0.94
A14	20.97±4.36	0.97	8.06±0.94	0.98
A18	3.42±0.78	0.90	1.90±0.97	0.62
A19	19.86±4.18	0.97	3.78±1.18	0.85

Tab. 2 Kd values of candidate aptamers toward norketamine before and after truncation of primer sequences

核酸适配体	引物序列剪切前（78 nt）		引物序列剪切后（40 nt）
核酸适配体	解离常数（K_d）	R²	解离常数（K_d）	R²
A5	40.12±5.47	0.99	5.49±0.88	0.96
A7	11.34±1.18	0.99	5.67±2.06	0.83
A14	20.25±7.45	0.91	5.45±1.13	0.94
A19	42.23±9.63	0.97	3.51±0.93	0.89

Fig. 8 Schematic diagram for the synthesis of aptamer-functionalized CGO fluorescent sensorand the detection principle diagram of ketamine and norketamine

Fig. 9 Optimization of the CGO mass concentration and the concentration of double-stranded DNA containing aptamer A5

Fig. 10 Optimization of the reaction temperature and incubation time

Fig. 11 Fluorescence response changes over incubation time for the aptamer-functionalized CGO fluorescent sensor basedon chemical conjugation and the CGO fluorescent sensor based on physical adsorption in blank blood and urine samples

Tab. 3 Results of the spiked recovery experiments for ketamine and norketaminein blank blood and urine samples （added at a mass concentration of 1 ng/mL）

样本	目标物	本研究检测方法		HPLC-MS/MS		RSD/%
样本	目标物	测量值/（ $x ¯$ ±s，ng·mL^-1）	回收率/%	测量值/（ $x ¯$ ±s，ng·mL^-1）	回收率/%	RSD/%
血液	氯胺酮	0.82±0.07	81.50	0.87±0.03	87.34	9.39
	去甲氯胺酮	1.04±0.05	104.09	1.12±0.02	112.00	5.34
尿液	氯胺酮	0.87±0.04	86.97	0.91±0.04	91.25	4.03
	去甲氯胺酮	1.10±0.02	110.03	1.20±0.05	120.00	6.87

Tab. 3 Results of the spiked recovery experiments for ketamine and norketaminein blank blood and urine samples （added at a mass concentration of 1 ng/mL）

样本	目标物	本研究检测方法		HPLC-MS/MS		RSD/%
样本	目标物	测量值/（ $x ¯$ ±s，ng·mL^-1）	回收率/%	测量值/（ $x ¯$ ±s，ng·mL^-1）	回收率/%	RSD/%
血液	氯胺酮	0.82±0.07	81.50	0.87±0.03	87.34	9.39
	去甲氯胺酮	1.04±0.05	104.09	1.12±0.02	112.00	5.34
尿液	氯胺酮	0.87±0.04	86.97	0.91±0.04	91.25	4.03
	去甲氯胺酮	1.10±0.02	110.03	1.20±0.05	120.00	6.87

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