Europium Nanoparticles-Based Fluorescence Immunochromatographic Detection of Three Abused Drugs in Hair

Drug abuse is becoming increasingly dangerous nowadays. Morphine (MOP), methamphetamine (MET) and ketamine (KET) are the most commonly abused drugs. The abuse of these drugs without supervision can cause serious harm to the human body and also endanger public safety. Developing a rapid and accurate method to screen drug suspects and thus control these drugs is essential to public safety. This paper presents a method for the simultaneous quantitative detection of these three drugs in hair by a europium nanoparticles-based fluorescence immunochromatographic assay (EuNPs−FIA). In our study, the test area of the nitrocellulose membrane was composed of three equally spaced detection lines and a quality control line. The test strip realized the quantitative analysis of the samples by detecting the fluorescence brightness of the europium nanoparticles captured on the test line within 15 min. For the triple test strip, the limits of detection of MOP, KET and MET were 0.219, 0.079 and 0.329 ng/mL, respectively. At the same time, it also showed strong specificity. The strip was stable and could be stored at room temperature for up to one year, and the average recovery rate was 85.98–115.92%. In addition, the EuNPs−FIA was validated by high-performance liquid chromatography (HPLC) analysis, and a satisfactory consistency was obtained. Compared to the current immunochromatographic methods used for detecting abused drugs in hair, this method not only increased the number of detection targets, but also ensured sensitivity, improving detection efficiency to a certain extent. The approach can also be used as an alternative to chromatography. It provides a rapid and accurate screening method for the detection of abused drugs in hair and has great application prospects in regard to public safety.


Introduction
The incidence of drug overdose deaths has increased continuously and quickly over the past 40 years, affecting people all over the world [1]. The phenomenon of narcotic drug and psychotropic substance abuse has emerged as one of the most serious threats to human health in the times of calmness. The United Nations Office on Drugs and Crime (UNODC) has published the World Drug Report 2022 [2]. According to the report, in 2020, an estimated 284 million people worldwide aged 15-64 years had used a drug within the last 12 months, representing a 26 percent increase from 2010. The global drug epidemic situation will worsen as the epidemic spreads; at the same time, with the progress of society, more and more new drugs have emerged. Drug testing has also become the focus of research. Gas chromatography/mass spectrometry (GC/MS) [19], liquid chromatography-tandem mass spectrometry (LC−MS/MS) [20], high-performance liquid chromatography (HPLC) [21], UPLC−MS/MS [22], nano−HPLC coupled with tandem mass spectrometry (nano−HPLC−Chip−MS/MS) [23], surface plasmon resonance (SPR) [24], surface-enhanced raman scattering (SERS) [25,26] and electrochemical methods are commonly used to detect drugs. These technologies have a high accuracy, but require a large number of fluid samples, expensive equipment and high skill. Immunoassays have attracted extensive attention in drug detection in recent years due to their simple operation, high efficiency, high sensitivity, strong specificity and applicability to large-scale detection. Examples include enzyme-linked immunosorbent assay (ELISA) [27], capillary electrophoresis (CE) immunoassay [28], chemiluminescence immunoassay [29] and lateral flow immunoassay (LFIA) [30,31]. LFIA is a method that combines immunoreactivity with chromatography, the most common form of which is based on gold nanoparticles [32]. However, the poor repeatability and low sensitivity of colloidal gold nanoparticles also limit its applications. In order to meet the requirements of a more accurate and sensitive analysis, the combination of fluorescent microspheres as labelling probes and portable strip readers has become a research hotspot in recent years. Quantum dots (QDs), carbon nanodots- In modern clinical diagnosis practice, the demand for the rapid quantitative or qualitative detection of various substances in samples is increasing. In this paper, we propose a triple fluorescence immunochromatographic method based on europium nanoparticles for the simultaneous quantitative detection of MOP, MET and KET. By detecting multiple abused drugs in a triple test strip, this screening method provides a shorter analysis time and higher efficiency, and can be used as an alternative to chromatography. In the future, the rapid detection of multiple targets in one sample has great application prospects.
The blank samples were collected from our volunteers, and these negative samples were tested using the GC method. The detection results were consistent with the previous interpretation method. According to the recommendations for drug detection in hair by the Society of Hair Testing (SOHT) [46], the sample should be cut with scissors from the posterior vertex region of the head, as close as possible to the scalp. The proximal portion of hair (approximately 3 cm) was used for analysis, since this region has the least variation in growth rate. All samples were stored at room temperature.

Instruments
We used a XYZ3000 distribution platform and CM2000 guillotine cutter to prepare test strips (BioDot, Irvine, CA, USA). A Hitachi F−4500 fluorescence spectrometer system (Hitachi, Tokyo, Japan) was used to record the fluorescence spectra. An FIC−S2011−B14 Dry Fluorescence Immunoassay Analyzer was used to read the test strips (Suzhou Hellman Precision Instruments, Suzhou, China). The immunochromatographic strip results were compared with the data obtained using an Agilent 1100 high-performance liquid chromatography system (Agilent Tech, Santa Clara, CA, USA). Hair was crushed with a ball mill (Beijing Wanfu Intelligent Technology Co., Ltd., Beijing, China).

Preparation of Fluorescent Probes
According to the specific scheme of europium nano-labeled antibodies in our laboratory in the early stages and the related literature reports [47], the specific operation was as follows: 2 mg of fluorescent microspheres was added to 1 mL of MES (0.05 M, pH 5.0) solution and well mixed, centrifuged for 20 min at 12,000 rpm/min; then, the supernatant was removed and we added 1 mL MES, mixed and sonicated. We repeated the appeal operation and washed EuNPs three times. After that, we added 200 µL of MES solution, stirred thoroughly and sonicated. We used the MES solution to produce 10 mg/mL NHS, and 15 µL was added to the sonicated fluorescent microspheres. We prepared 15 mg/mL of EDC with ultrapure water; then, 2 µL of 15 mg/mL of EDC was added to the previously mentioned fluorescent microspheres. In order to enhance antibody attachment, the carboxyl group on the EuNP surface was incubated for 30 min with moderate shaking. Excessed EDC was removed after activation by centrifugation at 12,000 rpm/min for 25 min. We added 1 mL PBS solution (0.01M, pH 7.4) to the precipitate, mixed well and then added MOP−mAb. After that, the fluorescent microspheres were activated and mixed for 2 h. An amount of 50 µL of BB (0.05M, pH 8.0 containing 10% (w/v) BSA) was used to inhibit the remaining active sites of the complex. Finally, fluorescent compounds were dissolved in 50 µL of BB (0.05 M, pH 8.0, including 1% (w/v) BSA and 0.5% (v/v) Tween−20) and kept at 4 • C in the dark for further use. EuNPs−KET−mAb and EuNPs−MET−mAb were prepared using a similar method.

Preparation and Assembly of the Single-Channel Strip
The immunoassay strip was made up of five pieces, as illustrated in Figure 1: an absorbent pad, a backing pad, a conjugate pad, a sample pad and a nitrocellulose membrane (NC).
The conjugate pad and the sample pad were both composed of the same gauge fiberglass. The sample pads were cut into 30 cm long and 2 cm wide strips before being preprocessed with 5 mL of PBS (0.05 M, pH 7.4, containing 2% (w/v) sucrose, 0.5% (w/v) BSA and 0.5% (v/v) Tween−20). The artificial antigens (MOP−BSA, KET−BSA or MET−BSA) and goat anti-mouse IgG (1 µL/cm) were sprayed on the NC membrane through the XYZ3000 distribution platform to become the test line (T line) and the control line (C line). Then, we dried the NC films for 2 h in an oven at 37 • C. In the BBS buffer (0.05 M, pH 8.0, including 8% (w/v) sucrose and 1% (w/v) BSA), europium nanoparticles were conjugated with monoclonal antibody (EuNPs−MOP−mAb, EuNPs−KET−mAb or EuNPs−MET−mAb). They were sprayed on the conjugate pad and placed in an oven at 37 • C to dry for 24 h. The whole immunochromatography system was built after the aforementioned components were processed. We assembled the sample pad, conjugate pad, NC membrane and absorption pad in the sequence specified, leaving a 2 mm space between each component, and then we overlapped and sliced them to a width of 3 mm. Then, the test strips were put into a cartridge for further investigation. Cutting and packing were conducted in a separate room with a temperature of 20-22 • C and a relative humidity of less than 30%.

Test Strip Detection Process
A total of 80 µL of standard solution or sample solution was dropped on the sample pad for EuNPs−FIA. Then, the findings were viewed under a portable 365 nm UV light within the immune response time. Simultaneously, we inserted the test strip into the fluorescent dry immunoassay analyzer to read the data and record the fluorescence intensity on the T and C lines.

Preparation of the Triple-Immunochromatographic Test Strip
These were prepared using the single-component immunoassay strip preparation method. We increased the number of T lines on the NC membrane based on the singlechannel immunological test strip. We sprayed MET−BSA, KET−BSA, MOP−BSA and goat anti-mouse IgG on the NC membrane (Figure 1b,c). We diluted EuNPs−MET−mAb, EuNPs−KET−mAb and EuNPs−MOP−mAb with the BBS solution. After that, we sprayed them on the conjugate pad according to a certain concentration.

Optimization of Parameters of the Triple-Immunochromatographic System
Placing multiple analytes on a strip may affect the color rendering results. In order to select the best location of the binding area, the color rendering intensity of MOP, MET and KET were tested in three different binding areas on the basis of the single optimization results (referred to in Supplementary Materials, Figure S1). According to the obtained data, we selected the best combination position of the three analytes.

Sensitivity and Specificity
To create varying concentrations of the mixed standard solutions, the three standards (MOP, KET and MET) were diluted with the BBS buffer (0.01 M, pH 8.0). The amounts of MOP/KET/MET were 0.2/0.2/0.2 ng/mL, 0.4/0.4/0.4 ng/mL, 0.6/0.6/0.6 ng/mL, 0.8/0.8/0.8 ng/mL, 1/1/1 ng/mL, 2/2/2 ng/mL, 4/4/8 ng/mL, 10/10/10 ng/mL, 12/12/20 ng/mL and 14/14/25 ng/mL. Under optimal conditions, MOP, KET and MET were measured simultaneously using triple-immunochromatographic strips, and the fluorescence values of the T or C lines were read by a fluorometric reader. A standard curve was created, and a linear equation was fitted. The half-maximal inhibitory concentration (IC 50 ) and the limit of detection (LOD) were utilized as quantitative measures to evaluate the performance of the test strip. The linear range of this analysis was set at the concentration that results in 10-80% inhibition.
The specificity of the triple-test strip was determined by testing a negative sample or a standard (4 ng/mL) containing only one or more abused drugs, and observing whether the three test lines produced a crossover result. In order to further evaluate the specificity of the strip, other drugs were tested, including cannabidiol, phenobarbital, diazepam, mephedrone, caffeine, taurine, papaverine, (1R, 2S)-(-)-ephedrine hydrochloride, pseudoephedrine hydrochloride, tetrahydrocannabinol, norketamine, sodium pentachlorophenol, R(+)-cathinone hydrochloride, methcathinone, tetrahydro-cannabinolic acid, methadone, methoxyphenamine, phenylpropanolamine hydrochloride, methylenedioxymethamphetamine, 6-monoacetylmorphine and F-ketamine. For cross-reactivity investigation, we diluted each medication to 500 ng/mL and recorded the fluorescence intensity using the reader.

Stability
According to the stability inspection method of in vitro diagnostic test strips, we put the test strips in an oven at 50 • C for 21 days for accelerated destruction stability test to detect the stability of the reagent card.

Precision and Accuracy
To evaluate the precision and accuracy of the triple-immunochromatographic test strip, eight groups from the same batch were selected for negative detection by using a manner similar to that of the single strips. The inter-batch experiment was conducted with eight strips from different batches. We recorded the fluorescence intensity and then determined the inter-assay and intra-assay coefficient of variation. The process was repeated three times.

Spiked Recovery Tests and Actual Sample Analysis
The spiked samples were analyzed by HPLC and EuNPs−FIA to confirm the reliability. To create spiked samples, negative hair samples were spiked with varied amounts of MOP (0.5, 1, 2 and 4 ng/mL), MET (0.5, 1, 2 and 4 ng/mL) and KET (0.5, 1, 2 and 4 ng/mL) standards.
To investigate the practical usefulness of this EuNPs−FIA further, 30 positive samples were analyzed utilizing a multitarget fluorescent immunochromatographic assay, with references to various procedures for test material processing as follows [48]: the hair was firstly washed twice with 10 mL of deionized water, then twice with 5 mL of acetone for 2 min. After drying at room temperature, we cut the hair into small pieces of around 2-3 mm in size, accurately weighed 20 mg of hair and placed it in a dedicated grinding tube. The samples were shaken in 1 mL of methanol in the grinding tube. We used a ball mill to grind the sample for 3 min at a frequency of 3000 r/min, then ultrasound assisted the shaking for 2 h. Then, we transferred 500 µL of the grinding liquid to a centrifuge tube, centrifuged it at 4000 r/min for 5 min, transferred the supernatant to a 0.22 µm organic membrane filtration and finally analyzed the filtrate. We used the above hair treatment method to treat 530 negative hair samples from volunteers, and then used the EuNPs−FIA for detection. All actual samples were analyzed and compared using an Agilent 1100 HPLC system (Agilent Tech, Santa Clara, CA, USA). Phenomenex Kinetex Bighenyl (100 mm 2.1 mm, 1.7 µm) chromatographic column, 0.1% formic acid (1 mmol/L ammonium formate) (phase A) and acetonitrile (phase B) were employed as mobile phases, and the flow rate was 0.1 mL/min. The injection volume was 1 µL, and the column temperature was 35 • C.

Data Analysis
Standard curves, sample means and standard deviations (SD) were determined using OriginPro 9.1 (OriginLab, Northampton, MA, USA) and Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). Schematic diagrams of the test strips were drawn using the Photoshop software (Adobe Systems, San Jose, CA, USA).

Detection Principle of the Immunochromatographic Detection System
This test strip employs the competitive immunochromatography experimental principle. Three distinct antigens are encapsulated on the NC membrane and serve as capture reagents. To serve as detection probes, antibody pairs labeled with EuNPs are sprayed over the conjugate pad. A drop of 80 µL of the test material is placed on the sample pad, and it flows to the other side by capillary action. The fluorescence signal values at the characteristic excitation (365 nm) and emission (610 nm) wavelengths are measured with an immunofluorescence detector. When a negative sample is identified, the conjugated pad's nano-labeled antibody attaches to the appropriate solid-phase antigen on the nitrocellulose membrane, generating a fluorescent detection line visible under UV light, as illustrated in Figure 1d,e. When the target analyte is present in the sample, it binds to the binding pad's nano-fluorescent-labeled monoclonal antibody. Excess antibody conjugate continues to travel along the liquid chromatography column due to capillary action and binds to the antigen on the T line, resulting in less fluorescently labeled antibodies on the detection line and a drop in fluorescence intensity. If there are enough target analytes, they will completely prohibit the detection reagent from binding to their respective captures, resulting in no visible detection line at the relevant point on the T line for positive samples. The target analyte content in the test sample is inversely linked with the T line to C line ratio. The lower the target analyte content, the higher the T/C.

Optimization of the Triple-Immunochromatographic System
The single-component test strip was successfully prepared, laying a good experimental foundation for further increasing the number of detection lines at the later stage. The Supplementary Materials provide the optimization details of the single-channel test strip.
To optimize the antigen concentration in the early stage, we conducted the experimental analysis and optimization of the antigen location of the triple fluorescent immunochromatography system; the results are shown in Table 1. When MOP−BSA was in the T 3 binding region, KET was in the T 2 binding region and MET was in the T 1 position, the best color effect was achieved.

Sensitivity and Specificity
In Figure 2a, it can be observed that as the concentration of the standard increased, the fluorescence intensities of T 1 , T 2 and T 3 lines gradually weakened, until they disappeared. Among other drugs, 6-monoacetylmorphine showed cross reaction in the MOP binding region. Norketamine and F-ketamine combined in the KET detection area. Methyenedioxymethamphetamine and (1R, 2S)-(-)-ephedrine hydrochloride showed weak fluorescence signals in the MET detection area. These were mainly due to the structural similarity of these substances (Table S7), but EuNPs−FIA did not react with other drugs, showing great specificity (Figure 3). The triple test strip was used to detect a standard sample containing only one or more MOP, KET or MET medicines. It showed that there was no cross reactivity between the three substances of abuse (Figure 2c).
Among other drugs, 6-monoacetylmorphine showed cross reaction in the MOP binding region. Norketamine and F-ketamine combined in the KET detection area. Methyenedioxymethamphetamine and (1R, 2S)-(-)-ephedrine hydrochloride showed weak fluorescence signals in the MET detection area. These were mainly due to the structural similarity of these substances (Table S7), but EuNPs−FIA did not react with other drugs, showing great specificity (Figure 3).
Among other drugs, 6-monoacetylmorphine showed cross reaction in the MOP binding region. Norketamine and F-ketamine combined in the KET detection area. Methyenedioxymethamphetamine and (1R, 2S)-(-)-ephedrine hydrochloride showed weak fluorescence signals in the MET detection area. These were mainly due to the structural similarity of these substances (Table S7), but EuNPs−FIA did not react with other drugs, showing great specificity (Figure 3).  Figure 4a depicts the stability test findings of the EuNPs−FIA. The triple-test strip was held at 50 °C for 21 days, and the T/C value almost did not change. The coefficient of variation (CV) was less than 12.03%. It demonstrated that the newly created strip was stable and may be stored at room temperature for up to one year [49].

Precision and Accuracy
The precision of the triple immunochromatographic strip was evaluated by detecting the intra-assay and inter-assay. As shown in Figure 4b, the CV% of the triple-detection strip was less than 10.96%. The small CV value indicated a high accuracy, which meant that the prepared triple strip can meet the requirements of quantitative detection.  Figure 4a depicts the stability test findings of the EuNPs−FIA. The triple-test strip was held at 50 • C for 21 days, and the T/C value almost did not change. The coefficient of variation (CV) was less than 12.03%. It demonstrated that the newly created strip was stable and may be stored at room temperature for up to one year [49].

Spiked Recovery Tests and Actual Sample Analysis
The accuracy and repeatability of the developed triple immunoassay was checked by testing spiked blank samples. The experiment was repeated six times for each concentration. The results showed that the average recoveries of MOP ranged from 98.61% to 115.92%, with a relative standard deviation (RSD) of less than 6.85% at the spiked concentrations of 0.5-4 ng/mL ( Table 2). The average recovery rate of KET was 85.98-111.03%, RSD < 8.19%. The average recovery rate of MET was 94.16-112.34%, RSD < 6.04%. The determination results were verified by HPLC analysis, with a correlation coefficient of R 2 > 0.97 (Figure 5a).

Precision and Accuracy
The precision of the triple immunochromatographic strip was evaluated by detecting the intra-assay and inter-assay. As shown in Figure 4b, the CV% of the triple-detection strip was less than 10.96%. The small CV value indicated a high accuracy, which meant that the prepared triple strip can meet the requirements of quantitative detection.

Spiked Recovery Tests and Actual Sample Analysis
The accuracy and repeatability of the developed triple immunoassay was checked by testing spiked blank samples. The experiment was repeated six times for each concentration. The results showed that the average recoveries of MOP ranged from 98.61% to 115.92%, with a relative standard deviation (RSD) of less than 6.85% at the spiked concentrations of 0.5-4 ng/mL ( Table 2). The average recovery rate of KET was 85.98-111.03%, RSD < 8.19%.
The average recovery rate of MET was 94. .34%, RSD < 6.04%. The determination results were verified by HPLC analysis, with a correlation coefficient of R 2 > 0.97 (Figure 5a).

Conclusions
Nowadays, drug users use more than one drug, with the intention of enhanc potentiating the drug effects. In this study, a triple fluorescent immunochromatog strip was successfully developed and optimized for the simultaneous detection o abused drugs (MOP, KET and MET) in hair samples. The test results can be ob

Actual Sample Detection
In order to further verify the reliability of the EuNPs−FIA quantitative method, 30 actual samples were simultaneously analyzed with EuNPs−FIA and HPLC. To ensure accuracy, each sample was tested three times ( Table 3). The results of MOP, KET and MET in the actual samples are highly correlated (Figure 5b). Therefore, the method can be reliably used to detect these three abused drugs in hair.
According to the recommendations for hair testing in forensic cases developed by the International Society for Hair Testing, when the level of morphine, methamphetamine or ketamine is greater than 0.2 ng/mg, a positive result must be produced [46]. The results showed that the negative coincidence rate of 530 samples from volunteers was 100% (Table S6). This result showed that this multiplex test strip analysis was useful for detecting the abused drugs in hair. Compared with the results of 560 actual samples from HPLC analysis, the consistency of the two methods was 100% ( Figure 6).   (S1 represents sample 1, S2 represents sample 2, and so on).

Conclusions
Nowadays, drug users use more than one drug, with the intention of enhancing or potentiating the drug effects. In this study, a triple fluorescent immunochromatographic strip was successfully developed and optimized for the simultaneous detection of three abused drugs (MOP, KET and MET) in hair samples. The test results can be obtained within 15 min of testing. Through the sensitivity standard curve established by the fluorescence intensity, the results showed that the LOD of MOP, KET and MET was 0.219, 0.079 and 0.329 ng/mL, respectively. In terms of reliability, the EuNPs−FIA showed good accuracy and good consistency with HPLC through the recovery and actual sample detection experiments. Compared with the current immunochromatographic methods for detecting abused drugs, this method effectively improved the issues of a single-target sub- (S1 represents sample 1, S2 represents sample 2, and so on).

Conclusions
Nowadays, drug users use more than one drug, with the intention of enhancing or potentiating the drug effects. In this study, a triple fluorescent immunochromatographic strip was successfully developed and optimized for the simultaneous detection of three abused drugs (MOP, KET and MET) in hair samples. The test results can be obtained within 15 min of testing. Through the sensitivity standard curve established by the fluorescence intensity, the results showed that the LOD of MOP, KET and MET was 0.219, 0.079 and 0.329 ng/mL, respectively. In terms of reliability, the EuNPs−FIA showed good accuracy and good consistency with HPLC through the recovery and actual sample detection experiments. Compared with the current immunochromatographic methods for detecting abused drugs, this method effectively improved the issues of a single-target substance and low sensitivity of traditional labelling materials. As an alternative method of chromatography, this screening method can provide help for the detection of clinically abused drugs. In summary, the development of multiple quantitative test strip assays will become a powerful tool for monitoring multiple drugs and use in other health, food safety and environmental tests.