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Portable Kit for Identification and Detection of Drugs in Human Urine Using Surface-Enhanced Raman Spectroscopy Zhenzhen Han,† Honglin Liu,*,† Juan Meng,† Liangbao Yang,*,† Jing Liu,‡ and Jinhuai Liu† †

Institute of Intelligent Machines and ‡Cancer Hospital, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, China

Downloaded by UNIV OF NEBRASKA-LINCOLN on September 8, 2015 | http://pubs.acs.org Publication Date (Web): September 1, 2015 | doi: 10.1021/acs.analchem.5b02899

S Supporting Information *

ABSTRACT: A portable kit was demonstrated for rapid and reliable surface-enhanced Raman scattering (SERS) detection of drugs in human urine. This kit contains two sealed reagent tubes, a packet of standardized SERS substrates, and a mini Raman device. A 3 min pretreatment for separating amphetamines from human urine was developed with an extraction rate of >80% examined by ultraperformance liquid chromatography (UPLC). Simultaneously, highly reproducible twodimensional (2D) gold nanorod (GNR) arrays were assembled by the use of methoxymercaptopoly(ethylene glycol) (mPEGSH) capping. Thirty batches of GNR arrays produced the 1001 cm−1 intensity of methamphetamine (MA) molecules with a relative standard deviation (RSD) of 7.9%, and a 21 × 21 μm2 area mapping on a 2D GNR array produced a statistical RSD of 80%. The strong alkaline condition can improve the separating effect of drugs from real human urine because amphetamines are a weak alkaline organic amine.30 A large amount of NaCl powder would reduce the solubility of the components in the water, making them precipitate. The CYH with poor water solubility served as the separation solvent; possessed the higher extraction recovery of amphetamines with less interference from impurities; and was not affected by pH, as compared with hexane, diethyl ether, toluene, and ethyl acetate.31 SERS Performance of GNR Arrays for Detecting Drugs. First, SERS performance of these four different GNR arrays was experimentally confirmed by measuring 50 ppm of MA in aqueous solution. The AR3.7 GNR arrays generated much stronger SERS signals than others (Figure S3). Hence, the AR3.7 GNR arrays are used as a SERS substrate in the following research, and we considered that the interaction between the exciting line of 785 nm with the AR3.7 GNR conduction band electrons with the LSPR of 785 nm could lead to the strongest electromagnetic near fields associated with hot spots among adjacent metal surfaces, which enabled the highly sensitive and reproducible SERS detection. To examine the sensitivity of 2D GNR arrays, SERS experiments were conducted at different concentration of

Figure 1. (a) UV−vis spectra of the prepared GNRs with different aspect ratios (AR) of 2.3, 2.8, 3.3, and 3.7. (b) SEM image and particle size distribution of AR3.7 GNRs. (c) The relationship between the absorbance maximum of GNRs and the amounts of AgNO3 used in the synthesis. (d) TEM image of AR3.7 GNRs.

changes in the LSPR maximum with AgNO3 amounts are shown in Figure 1c. SEM and TEM observations on AR3.7 GNRs indicate good uniformity of the as-prepared GNR sols, with an average diameter of 59.97 nm, as illustrated by particle size statistics (Figure 1b,d). The assembled 2D GNRs were uniform and in good order (inserted histogram in Figure 1b). Notably, the assembled densely arranged 2D arrays are promising to serve as SERS substrates in terms of sensitivity and reproducibility.24−27 We assembled the 2D GNR arrays through a mPEG-SH-mediated procedure (Figure 2).28 The use of mPEG-SH can displace the CTAB molecules from the surface of GNRs. CTAB is a cationic surfactant and makes GNRs positively charged, but the mPEG-induced ordered packing could be explained by dominant van der Waals forces and a possible steric hindrance around the nanoparticles. Considering that the MA/MDMA/MC are all monoamine alkaloids with a secondary amine group, the lone pair electron of the N atom will adsorb the proton in aqueous solution and makes the molecule positively charged. Compared with CTAB, it could be much easier for the drug molecules to enter into the interparticle gaps between mPEG-capped GNRs. The PEGC

DOI: 10.1021/acs.analchem.5b02899 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Figure S4 presents the SERS characteristic spectra of MA with a concentration range from 100 to 0.1 ppm in human urine, and each set of the Raman spectrum is composed of 10 quantitative data values from 10 random spots on the dried sample. The blank sample is the extractant from a urine sample without drug additives and is also measured as the background SERS signal. Ten spectra from different sites of each sample produced a standard deviation (σ) of the 1001 cm−1 intensity, which has important implications for signal reproducibility. Interestingly, the reproducibility shows a concentration dependence: 100 ppm of MA in urine produced a σ value of 3.1%, indicating excellent reproducibility of SERS signals, and the σ value increased from 3.1% to 14.0% as the MA concentration decreased from 100 to 0.1 ppm. The SERS intensity of the 1001 cm−1 peak was plotted as a function of the MA concentration within a fine range of 1−100 ppm, which produced good linearity (Figure S4). The calibrated linear regression equation is I1001 = [MA] × (24.51 ± 0.90) + (2111.62 ± 50.06), and the correlation coefficient (R2) was 0.9947. It is highly acceptable for signal recognition and the fingerprint peaks in each spectrum to be facilely identified. Practicability of the Portable Kit. Thirty volunteers from Hefei Cancer Hospital of the Chinese Academy of Sciences provided urine samples. Routine urinalysis reports were performed (Table S1) and indicated in-detail different components, such as urobilinogen, glucose, bilirubin, ascorbic acid, ketone body, urine specific gravity, pH, protein, nitrite, and hemameba. Small amounts of MA were added to these sample to produce a final concentration of 10 ppm, and we used our developed portable kit for SERS analysis. Figure 4a shows the SERS spectra of 30 volunteers’ blank samples, that is, the extractant from urine samples without drug


CYH-extracted MA from human urine. The intensity of SERS spectra increased with a corresponding MA concentration increase from 0.1 to 100 ppm (Figure 3a). The clear fingerprint

Figure 3. (a) SERS detection of MA with concentrations of 100, 50, 20, 10, 1, and 0.1 ppm, respectively. (b) Thirty SERS spectra collected at a random site on 30 batches of GNR arrays. (c) Histogram of SERS intensity at 1001 cm−1 of the 30 spectra in b. (d) SERS area mapping on a specific GNR array with area of 21 μm × 21 μm and a step size of 1 μm.

peaks of MA molecules at 620, 750, 834, 1001, 1206, and 1582 cm−1 are presented in all of the spectra.32,33 The Raman bands at 1001 and 620 cm−1 are attributed to the phenyl ring breathing mode, and the 1205, 1030, and 834 cm−1 peaks are assigned to the phenyl-C stretch, C−H deformation of phenyl ring, and C−C stretch of isopropyl, respectively. Well-resolved peaks can be identified clearly, even when the concentration of MA is at 0.1 ppm. The limit of detection is at least 0.1 ppm considering three times standard deviations. On the basis of the intensity of the peak at 1001 cm−1, the enhancement factor of AR3.7 GNR arrays was calculated to be ∼8.42 × 10 6 (Supplementary Section 3), indicating excellent sensitivity. Quantitative SERS analysis remains a challenge; one of the main reasons is the reproducibility and uniformity of the fabricated SERS substrates. The problem of reproducibility has hampered the progress of the SERS technique toward a practical analyzer. Therefore, we also examined the uniformity/ reproducibility of this SERS platform by measuring 50 ppm of MA. Figure 3b shows a 3D presentation of 30 SERS spectra, each of which was collected at a random site on different batches of the GNR arrays. All of the spectra produced clear fingerprint peaks of MA molecules. The intensity of the 1001 cm−1 peak from those 30 SERS spectra was plotted as histogram, and the statistical data of 2818 ± 222 cps were indicated by the red line and green area in part c,34 that is, a relative standard deviation (RSD) of 7.9%, indicating good reproducibility of 2D GNR arrays. One would next like to know if these random sites represented discrete hot spots arising from sample preparation heterogeneity. To assess possible fluctuations in every location of each array, a 21 × 21 μm2 area on a 2D GNR array was scanned to produce an area mapping image with a pixel resolution of 1 μm by the use of the SERS intensity at 1001 cm−1 (Figure 3d). A statistical RSD of these 441 pixels is less than 10%, implying excellent uniformity.

Figure 4. SERS detection of the extractant from 30 volunteers’ urine samples (a) without any addition of amphetamines and (b) with the addition of 10 ppm MA. (c and d) Histogram of SERS intensity at 1001 and 1029 cm−1 of the 30 spectra in b.

additives. It clearly proves the feasibility of our pretreatment procedures is not influenced by personal factors. Figure 4b shows the 3D presentation of SERS spectra of the extractant from a urine sample with the addition of 10 ppm MA. All of the spectra presented clear fingerprint peaks of MA molecules compared with the blank sample, that is, MA in 30 volunteers’ urine can be detected successfully. The RSD values of signal intensities at 1001 and 1029 cm−1 are 8.4% and 12.6%, D


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Figure 5. Real detection and recovery test of 20 ppm MA in different urine samples from 30 volunteers.

from the MA fingerprints. We consider that the strong peaks at 812 cm−1 (MDMA) and 1001 cm−1 (MA and MC) can be used for high-throughput screening of drug suspects related to amphetamines. Further confirmation should be by other laboratory methods, but that is the purpose of the portable kit. Dual-analyte detection of MA and MDMA in urine using the portable kit, as compared with single-analyte detection, is shown in Figure 6. The peaks 716, 812, 1248, 1360, 1496, and

respectively; the difference originated from the remarkable differences in signal intensity of these two bands. The results evidence good reproducibility of this portable kit. Next, the blind tests were performed to analyze the concentration of 20 ppm MA added in real urine samples collected from 30 volunteers from the hospital (Figure 5). UPLC assays revealed that the extraction rates of the 3 min pretreatment ranged from 77.3% to 90.3%, with a statistical value of 84.9% ± 3.1%, although the proposed SERS method produced a MA recovery ranging from 82.7% to 114.1% with a statistical value of 98.8% ± 7.5%. The data indicate that the obtained SERS results were highly consistent with those from the UPLC assays, although the SERS data have a slightly larger value of fluctuation. Nevertheless, these values well confirm that the SERS platform has a high accuracy and reliability to meet the requirements of practical applications. Similar experiments were carried out using MDMA added to urine samples from the 30 volunteers. Figure S5 shows the 2D and 3D presentations of the SERS spectra of the extractant from urine samples with the addition of 10 ppm MDMA. The obvious feature of these spectra is also high similarity. All of the spectra presented clear fingerprint peaks (716, 812, 1280, and 1640 cm−1) of MDMA.35,36 The bands at 716 and 812 cm−1 are assigned to the CC symmetric stretching mode and in-plane C8H deformation, respectively. The in-plane aromatic C−H bending mode appears as a band in the SERS spectrum at 1280 cm−1. A distinction can be achieved by looking in detail at 1640 cm−1, where C−H and C−C vibrations of the skeleton are superimposed on those of the methyl or ethyl group.37 Figure S5c,d shows the variation of the intensity at 1289 and 1640 cm−1 bands with RSD values of 15.0% and 17.0%, respectively. The increased RSD of MDMA compared with MA molecules might originate from the deceased affinity of the MDAM molecule with the methylenedioxy group to metal surface. However, the maximal RSD value of the signal intensities of major SERS peaks is significantly lower than 20%, indicating an acceptable reproducibility. The portable kit for detecting MC molecules in human urine was also verified in a simplified way. Figure S6 shows four different runs of SERS detection of 10 ppm MC in human urine, indicating a good signal intensity and reproducibility. Interestingly, the SERS fingerprint peaks of MC molecules were very similar to that of MA, especially the strongest peaks at 1001 and 1028 cm−1, which could be attributed to the similar molecular structures. Nevertheless, the peaks at 681, 762 and 1595, 1618, and 1686 cm−1 could distinguished themselves

Figure 6. Portable kit for SERS detection of 10 ppm MA (red line), 10 ppm MDMA (green line), the mixture of 5 ppm MA and 5 ppm MDMA (blue line), and the blank (black line) in human urine.

1628 cm−1 can be explicitly indexed to MDMA, whereas the peaks 1001, 1030, 1208, 1312, and 1600 cm−1 can be assigned to MA. Importantly, the SERS intensities of the adjacent bands at 812 and 1000 cm−1 in dual-analyte detection could be used to quantify the molar ratios of MDMA and MA. The blue dualanalyte spectrum has a lower level of intensity than that for sole MDMA and sole MA, which is understandable because of lower concentrations of each analyte in dual-analyte detection. Interestingly, the intensity at 1001 cm−1 assigned to MA decreased to 86.9% the level of the red line, but the 812 cm−1 assigned to MDMA decreased to 57.5% the level of the green line. The results might reveal two features: the first is that 10 ppm of molecules is enough for adsorption on every hot spot, and the second is that MA molecules might have a higher affinity to the metal surface than MDMA, which is consistent with the previous analysis on the larger RSD values of MDMA spectra. E


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CONCLUSIONS We have noticed that mPEG-SH-mediated assembly of 2D GNR arrays promises highly sensitive, robust, and reliable SERS substrates that can be applied for quantitative analysis of pure analytes, but difficult to implement for trace detection of drugs in untreated urine samples. Although it is often difficult to detect the intrinsic SERS spectra of analytes at relatively low concentrations, the poor selectivity in complex mixtures makes sample identification more difficult because of overlapping Raman bands of many interfering components. Our SERS results clearly indicated that our pretreatment procedure here is sufficient to lower the high background signals caused by complex components in urine. This portable kit serves as a proof-of-concept for reliable SERS analysis of drugs in human urine, which can be finished in several minutes and produce an acceptable detection limit. SERS analysis on urine samples with various clinical natures demonstrated the practicability and the resistance to false positives, which is a vital problem for law enforcement applications. Moreover, this portable kit is easy to operate for a nonprofessional, as compared with other analytical technology. In brief, these above experiments in terms of sensitivity, reproducibility, uniformity, practicability, and dual-analyte detection demonstrate the excellent performance of our portable kit for identification and detection of amphetamines in real human urine samples. We believe that this portable kit opens vast possibilities for the rapid and on-spot ultratrace or quantitative sensing in various fields, especially for public safety and healthcare.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02899. Supplementary experimental section, characterization of 2D assembled Au NR arrays, UPLC spectra, SERS enhancement of GNRs with different aspect ratios, enhancement factor calculation, detection of MDMA and MC in urine, routine urinalysis report of 30 volunteers (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interests.

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ACKNOWLEDGMENTS This work was supported by the National Instrumentation Program of China (2011YQ0301241001, 2011YQ0301241101), National Natural Science Foundation of China (21305142, 21271136), Natural Science Foundation of Anhui Province, China (1308085QB27), and the Open Project of State Key Laboratory of Physical Chemistry (201405).

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