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Article Cite This: ACS Omega 2019, 4, 801−809
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Disposable Immunosensor Based on Electrochemiluminescence for Ultrasensitive Detection of Ketamine in Human Hair Ya Yang,*,†,‡ Suyan Zhai,‡ Chao Liu,‡ Xiaoshu Wang,‡ and Yifeng Tu*,§ Department of Forensic Medicine, ‡Institute of Forensic Sciences, and §Institute of Analytical Chemistry, Soochow University, Dushu Lake Campus of Soochow University, Suzhou Industrial Park, 215123 Suzhou, P. R. China
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†
ABSTRACT: An ultrasensitive disposable immunosensor based on a gold nanoparticles (AuNPs)/indium tin oxide (ITO) substrate has been constructed to detect ketamine (KET) conveniently. In this study, we obtained a AuNPs/ITO basic electrode by only a two-step ecofriendly drip-coating procedure. The self-assembled monolayers of AuNPs were used as a hetero-bifunctional cross-linker and activator, respectively. Ketamine antibody was used as a biorecognition determinant and immobilized onto the AuNPs/ITO through electrostatic interaction or Au−S or Au−N bonds. The architectures of AuNPs/ITO were characterized by transmission electron microscopy, scanning electron microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy mainly. Electrochemiluminescence (ECL) was used to monitor the specific immune response between the antigen and antibody by measuring changes of intensity in the process of ECL. When the immune complex was incubated on the electrode, it blocked the electron communication and mass transfer, and the ECL signal was quenched. According to the quenched ECL signal, the quantification of KET can be realized. Under optimal conditions, the immunoassay showed a linear range from 10 to 100 pg/g. The low detection limit is 5.73 pg/g. The sensor has been applied to detect ketamine in human hair successfully. In addition, the immunosensor has excellent selectivity, outstanding reproducibility, and acceptable stability. What’s more, the immunosensor exhibits low-cost, simplicity, small sample consumption, and easy miniaturization, which allows it to be integrated into portable platforms to be utilized on the site of crime easily. its stimulant, dissociative, and hallucinogenic effects.10 For these reasons, ketamine is now one of the controlled drugs in most countries. For rapid identification in suspected samples, the demand for tests at the sites of trafficking and amusement and roadside tests is increasing. Currently, gas chromatography−mass spectrometry (GC− MS),11−17 liquid chromatography−mass spectrometry (LC− MS),18−23 and solid-phase extraction−liquid chromatography−mass spectrometry (SPE−LC−MS)24 have been used for ketamine detection in human hair samples because of their advantages such as sensitivity, high stability, and accuracy.25 However, these methods still have some limitations such as the requirement of expensive instruments and professional personage, time-consuming sample pretreatment, and high cost, which limit their widespread application.26,27 Thus, there is a critical demand to establish a simple, efficient, sensitive, and inexpensive test tool for its routine examination in human hair samples. Electrochemiluminescence (ECL) has attracted tremendous attention because of its high sensitivity, lower background signal, better temporal controllability, simple operation and
1. INTRODUCTION Hair is being approved as an impactful biological specimen for forensic toxicology analysis besides urine or blood.1 In recent years, drug testing in hair has gained tremendous attention and occupied an important position because of its wide and factual time window of detection mainly depends on its growth period. Hair samples have their advantages compared with other biological samples including noninvasive sample collection and the convenience of sample handling, transferring, and storage (no special packaging, refrigeration, or preservatives required), especially to be supervised easily in forensic cases.2−4 However, the hair sample’s pretreatment is time consuming, with tedious steps in most of the hair analysis reports. Ketamine (KET) is an analgesic and anesthetic drug used in the medical field widely.5,6 However, the abuse of ketamine is increasing in recent years. According to the inspection of many countries, ketamine is one of the frequently found compounds in drug samples alone or in combination with other illicit drugs.7,8 Besides, ketamine has become a major concern due to its intake for recreational purposes, especially among the young people. Moreover, it can facilitate sexual assaults when it is added to illicit drug formulas or drinks because it is colorless, odorless, and tasteless.9 Meanwhile, ketamine monitoring has also received extensive attention in traffic accidents because of © 2019 American Chemical Society
Received: October 8, 2018 Accepted: December 20, 2018 Published: January 10, 2019 801
DOI: 10.1021/acsomega.8b02693 ACS Omega 2019, 4, 801−809
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Figure 1. Optimization of the AuNPs/ITO electrode, Cluminol = 4 × 10−8 M, pH = 8.0. (A) Concentration of APTMS; (B) hydrolysis time of APTMS; (C) amount of AuNPs; (D) incubation time of AuNPs.
installation, rapidity, and low cost. It has been extensively employed in a variety of fields including food and water safety,28 clinical diagnosis,29−31 environmental pollutant detection,32 etc. The ECL immunoassay (ECLIA), which combines the unique advantage of high specific immunerecognition between antibody and antigen and the remarkable features of ECL, has shown an outstanding capability to meet these demands.33,34 To obtain a label-free immunosensor, it is essential to immobilize one of the immune components on the electrode surface with intrinsic activity.35,36 Thus, a large surface area on the electrode substrate to fix more antibodies, good conductivity to assist transferring the electron, and improved biological nontoxicity and stability are essential. Gold nanoparticle (AuNP), a well-known nanomaterial, because of its large surface area, strong adsorption ability, high chemical stability, good conductivity, and good biocompatibility,37,38 is a good candidate to build biosensors. Moreover, AuNPs can easily be functionalized with biomolecules through electrostatic interaction or Au−N and Au−S bonds, while efficiently retaining the activity of biomolecules in the construction of the biosensor.39−42 Therefore, it is key to design and prepare a AuNP-functionalized substrate for immunosensor development. In this paper, indium tin oxide (ITO) glass was used as the basic conductive material. The AuNPs/APTMS/ITO electrode was prepared by assembling AuNPs on the ITO surface utilizing hydrolyzed (3-aminopropyl) trimethoxysilane (APTMS) as a linker. The electrode shows excellent electrochemical activity and catalytic performance with all parameters optimized. The antibody proteins of ketamine were immobilized onto this electrode to construct a novel label-free electrochemiluminescent immunosensor. After only a two-step drip-coating procedure, the AuNPs/APTMS/ITO electrode was obtained. Then, the antibody proteins were immobilized through direct coating without any auxiliary means. On this sensor, the specific affinity reaction toward ketamine can be
monitored and quantified by the quenched ECL signal of luminol.
2. RESULTS AND DISCUSSION 2.1. Optimization of the Conditions for Sensor Preparation. The functionalization of ITO glass with AuNPs is very important in sensor construction. To prepare an AuNPs/ITO electrode with good performance, some significant parameters are required to be investigated and optimized including the time of hydrolysis of APTMS, the content of APTMS, the quantity of AuNPs, and the period of AuNP decoration. Figure 1A reveals that the ECL emission ability of luminol on the resultant AuNP-functionalized electrode was related to the dosage of APTMS: there was a peak emission at 10 μL of solution containing 0.01% APTMS. It means that moderate ATPMS is favorable for AuNP deposition, but just the opposite if there was a troppo amount because of its insulation. The effect of the hydrolysis time of APTMS at the hygrothermostat was examined, and the result is displayed in Figure 1B. The result showed that the largest ECL intensity occurred at 30 min. Similarly, the effects of the AuNP amount and its operating time were studied, as shown in Figure 1C,D. It is related to the amount of AuNPs closely, reaching the highest at 0.17 μmole, and a duration of 20 min is enough. The concentration of the ketamine antibody (KET-Ab) solution used for its immobilization on the above-mentioned substrate electrode also affects the sensing output of the immunosensor,43 reasonably in virtue of the acquired loading quantity of antibody. With the concentration increased, the response signal increased gradually and reached a maximum at 3.0 μg/mL, indicating a saturated bonding of KET-Ab on the electrode surface (Figure 2A). The pH of the buffer solution has immense influence on the activity of the immobilized antibody on the electrodes44 and is an important parameter for analyzing the performance of immunosensors. 45 The optimal pH for the obtained 802
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Figure 2. Effects of the experimental parameters on the sensor response: (A) the immobilized quantity of KET-Ab; (B) the value of pH; (C) pulse period; (D) lower limiting potential; and (E) upper limiting potential.
ITO) appears similar to a layer of gel after immobilizing KETAb, as shown in Figure 3C, and demonstrated successful conglutination of the antibody on the AuNPs/ITO surface. 2.3. Electrochemical Investigation of the Resultant Biosensor. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were considered as optimal tools to characterize the stepwise assembly of the immunosensor,46,47 to show the changes of electrode behaviors through each step of functionalization. As shown in Figure 4A, the welldemarcated redox peaks of Fe(CN)63−/Fe(CN)64− were acquired at bare ITO (curve a). With the coating of APTMS, the nonconductive macromolecules obstructed electron transfer and the peak current decreased (curve b). Instead, after the deposition of AuNPs, related peak currents were remarkably enlarged (curve c), indicating that electron transference for redox of the probe on AuNPs is easier. By further attaching KET-Ab (curve d) and bovine serum albumin (BSA) (curve e), the peak height decreased gradually because of the immobilized Ab and BSA acting as insulators, hindering the electron transfer between the electrode and the probe. As expected, after KET combined on the antibody specifically, the peak current further decreased (curve f), which displayed that the assembly procedure of the biosensor was successful. Electron-transfer resistance (Ret), a sensitive and directive factor that reflects the surface variation of the electrode, can be
immunosensor response should be a balance between the ECL behavior of luminol (a basophilic compound) and the activity of the antibody. In Figure 2B, when KET-Ab attached to AuNPs/ITO, the highest sensing output on the immunosensor was obtained at pH 8.0. The applied potentials and width of the pulsed electrolytic power play important roles in ECL detection. For the highest signal/noise ratio, Figure 2C demonstrates a 3 s optimal pulse period, and Figure 2D,E illustrates the greatest response at the lower limiting potential of −0.1 V and the upper limiting potential of 1.0 V, respectively. 2.2. Morphology of the Nanomaterial and Sensor. Observation of the sensing matrix by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) is helpful and straightforward. The changes of the electrode surface morphology in pace with the obtaining process of the sensor are displayed in Figure 3 clearly. Figure 3A shows the surface of the ITO substrate. The SEM image of the AuNPs/ITO shows that a monodispersed homogeneous AuNP single layer was formed on the surface of the ITO by the linkage of hydrolyzed APTMS (Figure 3B). From the inserted TEM photo, we can see that the size of the AuNPs is 15 ± 2 nm. This shows that the hydrolyzed APTMS can attach AuNPs onto the ITO substrate effectively with excellent dispersion. The surface of the resultant immunosensor (KET-Ab/AuNPs/ 803
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Figure 3. (A) SEM image of bare ITO; (B) SEM image of the AuNPs/ITO electrode (the inset displays a TEM image of the AuNPs); (C) SEM image of the KET-Ab/AuNPs/ITO electrode.
Figure 4. (A) CV (at the scan rate of 50 mV/s) and (B) Nyquist plot of different modified electrodes in 0.1 M KCl containing 5 mM [Fe(CN)6]3−/4−. Electrodes used from curves a to f are bare ITO, APTMS/ITO, AuNPs/ITO, KET-Ab/AuNPs/ITO, BSA/KET-Ab/AuNPs/ITO, and KET/BSA/KET-Ab/AuNPs/ITO, respectively. The equivalent circuit corresponding to the impedance is shown in the inset of (B). (C) CVs of 2.5 × 10−5 M luminol on (a) bare ITO, (b) APTMS/ITO, (c) AuNPs/ITO, (d) KET-Ab/AuNPs/ITO, (e) BSA/KET-Ab/AuNPs/ITO, and (f) KET/BSA/KET-Ab/AuNPs/ITO in phosphate buffered saline (PBS) (pH = 8.0) with a scan rate of 50 mV/s; (D) variation of luminol redox current at the (a) proposed AuNPs electrode and (b) purchased Au electrode by CV with a scan rate of 50 mV/s in 2.5 × 10−5 M luminol of PBS solution (pH = 8.0).
APTMS/ITO (curve c), showing the superior electrical conductivity of AuNPs. Excitingly, the Ret values greatly increase successively in the cases of KET-Ab (curve d) and BSA (curve e) affixed, indicating that the immobilized Ab and BSA severely obstruct the electron exchange between the probe and the electrodes. The Ret value got larger when KET
illustrated by a Nyquist plot involving a semicircle and a Warberg line. EIS curves were obtained to monitor the fabricating procedures for the immunosensor as shown in Figure 4B. In comparison with the ITO substrate (curve a), the Ret increased obviously after the APTMS film was covered on it (curve b). However, a much smaller Ret emerged on AuNPs/ 804
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Figure 5. (A) ECL responses for different concentrations of KET in PBS at pH 8.0; (B) the calibration curve of KET from the immunosensor.
Table 1. Comparison of the Analytical Performance of Some Different Methods for the Detection of Ketamine detection methods
sample
linear range
detection limit
GC−MS LC/MS/MS PCI/GC/MS CE GC−MS/MS
urine/plasma hair urine hair urine plasma hair
0.04−20 mg/mL 0.1−100 ng/mg 50−100 ng/mL 0.5−8.0 ng/mg 10−250 ng/mL 10−500 ng/mL 10−100 pg/g
0.01 mg/mL 20 ng/g 25 ng/mL 80 ng/g 5 ng/mL
49 50 51 52 53
5.73 pg/g
this study
ECLIA
references
Figure 6. (A) Specificity of the proposed immunosensor; (B) stability of the immunosensor over a 15 day period; (C) reproducibility of the immunosensor (evaluated from the response to 0.05 ng/mL KET on five different electrodes); (D) interferences of hair and urine components along with the dilution multiple. Error bars represent the relative standard deviations (RSDs) from three independent determinations.
was combined on the sensor (curve f), attributing to the formation of the immune complex, which blocked mass transfer and electron conduction.48 The results were consistent with CV measurements. Figure 4C shows the curves of luminol (0.1 mM) on various electrodes in PBS buffer. As can be seen, compared with bare ITO (curve a) and APTMS/ITO (curve b), the peak current on AuNPs/ITO (curve c) is higher, and evidenced the very clear catalytic ability of AuNPs for the oxidation of luminol. It is rational that the AuNPs not only amplified the surface area of the electrode, but also greatly accelerated electron transfer
from luminol to electrode. With continuous catching of KETAb (curve d), BSA (curve e), and KET (curve f) on the electrode, the peak current decreases gradually, which proves the acquisition of these biomolecules on the electrode surface. In addition, the performance of the AuNPs/APTMS/ITO electrode significantly influences the preparation of the immunosensor, and thus, it was further characterized. In Figure 4D, on AuNPs-decorated ITO, there is a very clear oxidation peak of luminol at a potential of 0.45 V (curve a), other than nothing on the bulk Au electrode (curve b). This result demonstrates that AuNPs/ITO is a better electrode 805
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material than the bulk Au electrode for the oxidation of luminol. 2.4. Analytical Performance of the Proposed Immune-Biosensor. To examine the sensitivity of the soobtained immune-biosensor, it was tested by the correlation of the ECL response for various concentrations of KET under optimal experimental conditions. While KET specifically combined with the antibody on the sensor, the ECL signal was collected. With increase of the KET concentration from 10 to 100 pg/g, the ECL intensity decreased (Figure 5A). As shown in Figure 5B, a linear correlation between the responding signals of the sensor and the KET concentration was obtained. The associated calibration curve is described as ΔECL = 0.223 + 0.0157CKET (pg/g) (R = 0.994), with a detection limit of 5.73 pg/g (S/N = 3). According to the SF/Z JD107004-2016 technical specifications of Judicial authentication, the detection limits of LC−MS/MS or GC/MS in hair analysis are 2 and 0.5 μg/g, respectively, which clearly showed that the sensor has high enough sensitivity for forensic toxicology detection of ketamine in hair. Table 1 shows the comparison of the proposed immunebiosensor for the detection of KET with those of previously reported papers. It clearly indicates that the immunosensor displays comparable or even better analytical performance (limit of detection, linear range, etc.). Some very important characteristics of this immunosensor, including stability, selectivity, and reproducibility, need to be verified. The selectivity of the obtained immunosensor was tested by the ECL response with some other potential interferents such as glucose, uric acid, ascorbic acid, and cysteine. As shown in Figure 6A, the ECL intensity almost remained unchanged after incubation with the above-given interfering substances. This was due to the specific recognition between antibody and antigen.54 These results prove the high selectivity of the obtained immunosensor. The stability of the immunosensor was also examined. The sensor was stored in PBS buffer solution at 4 °C during the whole testing period. The recorded signal of the KET immunosensor also retained about 93% of its initial value after 2 weeks (see Figure 6B). It indicated the excellent stability of this immunosensor. Finally, five parallelly prepared sensors were utilized to investigate the reproducibility of the immunosensor by detecting the same concentration of KET (50 pg/g). As the results in Figure 6C show, all sensors gave close ECL responses with a relative standard deviation (RSD) of 5.4%, demonstrating the good reproducibility and precision of the obtained immunosensor. 2.5. Real Sample Analysis. To examine the practicability of the immunosensor, five spiked human hair samples were tested. After washing, the hair samples were cut into 1−2 mm segments. In 200 μL of NaOH (10% w/w), 10 mg of hair segments together with different concentrations of the standard solution of KET were digested at 80 °C for 10 min and neutralized to pH 8.0 using hydrochloric acid. Figure 6D exhibits that some components of the blank sample of hair might be potential interferents for detection, but their interference can be avoided by diluting the sample attributed to the high sensitivity of this sensor. When diluted 1000 times, there was almost no impact on the response signal. The results of KET detection in hair are listed in Table 2, indicating that the recovery of detection of the human hair is in the range from 88 to 105%, with the RSD between 2.4 and 8.3%. It
Table 2. Detected Results of KET in Human Hair Samples and the Recoveries sample hair
1 2 3 4 5
taken
found
recovery
RSD %
100 30 50 70 10
97 31.5 47 72.1 8.8
97 105 94 103 88
2.4 4.3 3.7 4.2 8.3
means that the proposed immunosensor is appropriate for the toxicological analysis of KET in human hair.
3. CONCLUSIONS In this work, a novel immunosensor for KET has been fabricated based on the immobilization of antigen on the surface of AuNP-functionalized ITO. The immunosensor showed good selectivity, high sensitivity, good stability, and reproducibility; it lays the foundation for the detection of KET with precision and accuracy in hair samples for toxicological analysis. Furthermore, because of its ultrahigh sensitivity, the amount of sample required is small. This provides a promising solution for detecting biological samples that are more easily collected and contain low levels of drug, such as sweat, saliva, or fingertip blood. Altogether, the construction of an immunosensor that combines the high sensitivity of ECL detection with specific immune responses of antibody to antigen can be regarded as a promising device for ketamine detection for onsite monitoring. It is shown that the assembling procedure is simple, low cost, and reproducible. Thus, the proposed immunosensor-preparing methodology can meet the demand of the rapid screening of ketamine by a disposable immunosensor, especially for onsite monitoring. 4. EXPERIMENTAL SECTION 4.1. Reagents and Materials. Ketamine (KET, 1.0 mg/ mL) was purchased from Sigma-Aldrich Company. Ketamine antibody (KET-Ab, 1.0 mg/mL) was obtained from Beijing Biosynthesis Biotechnology Co., Ltd. (P. R. China). 5-Amino2,3-dihydro-1,4-phthalazinedione (luminol) was purchased from Fluka Chemical Corp. 3-Aminopropyltrimethoxysilane (APTMS) was purchased from Aladdin Industrial Co., Ltd. (P. R. China). Chloroauric acid (HAuCl4·4H2O), trisodium citrate, bovine serum albumin (BSA, 96−99%), and phosphates (Na2HPO4·12H2O and NaH2PO4·2H2O) were purchased from Sinopharm Group Co. Ltd. (P. R. China). Phosphate buffer solutions (PBS 0.2 mol/L) were obtained by mixing NaOH, NaH2PO4, and Na2HPO4 in proportion. ITO glass was obtained from Suzhou Nippon Sheet Glass Electronics Co. Ltd. (P. R. China). All of the other chemicals were of analytical grade. They did not need further purification before using. Throughout the experiment, ultrapure water prepared by a water purification system was used. 4.2. Instruments. The ECL experiments were carried out on a lab-built system as reported in a previous paper.55 An Electrochemical Workstation (RST-5200, Risetest Instruments Co. Ltd., Suzhou, P. R. China) was used to measure cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). All experiments were carried out with a conventional three-electrode system. A bare ITO electrode (cut into 1.0 cm × 5.0 cm pieces) or functionalized ITO glass was used as the working electrode, a platinum wire as the auxiliary electrode, and a silver wire as the reference electrode. 806
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Scheme 1. Schematic Illustration of the Fabrication Process of the Ketamine Immunosensor
test. The conditions for sensor preparation and detection were optimized, including the concentration of APTMS, the time of hydrolysis of APTMS, the amount of AuNPs, incubation of antibodies, the pH value of the buffer solution, the pulse width, the lower/upper limiting potentials, etc. Before and after the immuno-interaction, the ECL was detected based on the difference of ECL intensity under these optimized conditions with a pulsed potential exerting on the sensor to record the ECL signal of luminol. When the concentration of KET increased, which meant that more KET molecules were captured on the surface of the immunosensor, the ECL intensity decreased.
Size and micromorphology of the nanomaterials and the surface of the sensor were observed by transmission electron microscopy (TEM) (FEI, at an accelerating voltage of 200 kV) and scanning electron microscopy (SEM) (Hitachi, S-4700, Japan). 4.3. Preparation of the AuNPs. AuNPs were obtained by the sodium citrate reduction method according to the previous report.56 First, 100 mL of HAuCl4 (0.01%) was stirred by magnetic stirrers and was heated until 100 °C. Then, 4.5 mL of citrate solution (1%) was added and refluxed until the color of the fluid became wine red. Then, heating was stopped and it was cooled to room temperature. A AuNP colloid was obtained after centrifuging for 10 min at 3000 rpm, and the agglomerated or larger AuNPs were eliminated. To remove the excess sodium citrate, the fluid continued to be centrifuged for 30 min at 10 000 rpm. Redispersing the obtained fluid in water yielded different levels of Au solution. 4.4. Fabrication of the KET Immunosensor. The immunosensor was prepared as shown in Scheme 1. First, the ITO glasses were cut into pieces (1.0 cm × 5.0 cm). Each ITO slide was cleaned by sonication in 1 M NaOH/ethanol mixture (1:1, v/v), acetone, and ultrapure water (15 min each) sequentially, and immersed in NH3·H2O (30%) for 12 h to introduce the hydroxyl groups forming the hydrophilic surface. Next, the plates were thoroughly washed by pure water and dried with N2 gas. Then, APTMS solution (0.01% of anhydrous ethanol, v/v, 10 μL) was dropped on the surface. Within the saturated ethanol vapor, ethanol volatilized slowly in an equilibrium environment. After that, the ITO glass was placed in a hygrothermostat to insure full hydrolysis of APTMS. Subsequently, 50 μL of AuNPs sol was dropped on this APTMS/ITO surface for 30 min to deposit AuNPs, and then washed with deionized water and dried under nitrogen gas. Further, 10 μL of KET-Ab (3 μg/mL) was dripped onto the AuNPs-functionalized electrode surface, immobilized by specific interactions between the available mercapto or amine groups of the antibody and Au,39 in a refrigerator at 4 °C. Following this, 10 μL of bovine serum albumin (BSA) solution (2 wt %) was used to cover nonspecific bound sites on the electrode for 1 h at 37 °C. 4.5. Measurement of ECL and Important Factors’ Optimization. For the detection of KET, the resultant sensor was overlaid with various concentrations of ketamine solution for incubation at 37 °C for 1.5 h, and then put into the ECL
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. Tel: +86-512-65125019 (Y.Y.). *E-mail:
[email protected]. Tel: +86-512-6510-8012 (Y.T.). ORCID
Ya Yang: 0000-0002-1299-655X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (No. 81601642) and Project of Science and Technology of Suzhou (No. SYS201666).
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REFERENCES
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DOI: 10.1021/acsomega.8b02693 ACS Omega 2019, 4, 801−809
