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A Novel Approach for Mobile Phone Sensing of Cocaine in Lateral Flow Assay Combined with a Biomimetic Material Emine Guler, Tulay Yilmaz Sengel, Z. Pinar Gumus, Mustafa Arslan, Hakan Coskunol, Suna Timur, and Yusuf Yagci Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03017 • Publication Date (Web): 23 Aug 2017 Downloaded from http://pubs.acs.org on August 23, 2017
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Analytical Chemistry
A Novel Approach for Mobile Phone Sensing of Cocaine in Lateral Flow Assay Combined with a Biomimetic Material Emine Gulera,b,c, Tulay Yilmaz Sengela,b, Z. Pinar Gumusb, Mustafa Arsland, Hakan Coskunole, Suna Timura,f*, Yusuf Yagcid,g* a
Ege University, Faculty of Science, Biochemistry Department, 35100, Bornova, Izmir/Turkey Ege University, Institute of Drug Abuse Toxicology & Pharmaceutical Sciences, 35100, Bornova, Izmir/Turkey c Ege Life Sciences (EGE-LS), Cigli, 35620, Izmir/Turkey d Istanbul Technical University, Department of Chemistry, Maslak, 34469 Istanbul/Turkey e Ege University, Faculty of Medicine, Addiction Treatment Center, 35100, Bornova, Izmir/Turkey f Central Research Testing and Analysis Laboratory Research and Application Center, Ege University, Bornova, Izmir/Turkey g King Abdulaziz University, Faculty of Science, Chemistry Department, Jeddah/Saudi Arabia b
*E-mail:
[email protected];
[email protected]. Phone: +90 2323115487; +90 2122853241
ABSTRACT: Lateral flow assays (LFAs) are ideal choice for drug abuse testing favored by their practicability, portability and rapidity. LFAs based on-site rapid screening devices provide positive/negative judgment in a short response time. Conventionally applied competitive assay format used for small molecule analysis such as abused drugs restricts the quantitation ability of LFA strips. We report herein for the first time, a new strategy using non-competitive assay format via a biomimetic material, namely poly(p-phenylene) β-cyclodextrin poly(ethylene glycol) (PPP-CD-g-PEG) combined with gold nanoparticle (AuNP) conjugates as labeling agent to recognize target cocaine molecule in test zone. The intensities of visualized red color in test line that indicates the cocaine concentration were analyzed via a smartphone application. Significantly, combination of this platform with a smartphone application provides quantitative data on the cocaine amount that makes it very inventive and attractive approach especially for onsite applications at critical points such as traffic and working places.
Lateral flow immunoassay (LFA) based on-site tests are consistently expanding trend within the scope of point-of-care testing (POCT). LFA products manufactured by more than 100 companies occupy the majority of the POCT market.1 The reason for the high interest of POC detection technologies is that it provides rapid access to analytical information with sensitive, user-friendly, portable and low-cost devices.1,2 Highly desirable POCT devices offer analysis near patient from clinical/nonclinical conditions to home of patients or site of accidents.3 To provide this practice, POCT developers make continuous efforts to develop novel strategies including miniaturization affording portability, sensitivity and quantification for reliability, simplicity for ease of handling, and rapidity for shorter result time.4 By the cumulative improvement in modern variety of laboratory devices, smartphone based handheld platforms have emerged as one of the ideal systems for POC monitoring.5 Under the umbrella of on-site diagnostics, connection of smartphone technologies to LFAs is promising as it combines low-cost and naturally abundant papers with ubiquitous mobile phones. Mobile phones as real-time digital platforms with their high-resolution cameras, Wi-Fi or Bluetooth connectivity, and high storage capacity can advance opportunities from healthcare systems to environmental, safety and security, and forensic analysis.6–8 POC diagnostics have an impact on drug abuse for the rapid detection of illegal substances such as cocaine, cannabis, opi-
ates etc.9 On-site screening of illicit drugs is especially critical requirement for road-side field testing10 and workplace drug testing.11 Cocaine, a strong stimulant of central nervous system, is the second most used prohibited drug in Europe and United States. For this reason, detection of cocaine as well as other drugs is critical to prevent serious health and social problems associated with drug abuse. A variety of methods have been employed to detect cocaine, including enzymelinked immunosorbent assay (ELISA), radioimmunoassay and chromatography techniques.10,12–15 LFAs is the most valuable on-site assay system due to non-sophisticated procedure, low cost, visualized results by naked eye, easy operation and small size. One of the main drawbacks of immunoflow assay based on-site drug tests is its limitation to only positive/negative judgment. The conventionally small molecule detection is a competitive format. In this detection format, test line is not visualized for positive sample. It is, therefore, not possible to obtain quantitative results using traditional competitive LFA testing.10,16 On the basis of this background, we designed a LFA assay with sandwich format composed of conjugated polymer containing β-cyclodextrin (β-CD) units, poly(p-phenylene) βcyclodextrin poly(ethylene glycol) (PPP-CD-g-PEG) and gold nanoparticle (AuNP) conjugates. The designed material allows to measure color intensities, directly correlated with cocaine concentration in test line by a smart phone application. The
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strong affinity of β-CD was reported in the literature and biomimetic property of β-CD was successfully adapted to both µwell assay17 and biomolecule free electrochemical sensing system (submitted) in our previous studies. PPP-CD-g-PEG ensues target recognition while AuNPs act as labeling agent. The designed system features integration of PPP-CD-g-PEG polymer to LFA platform which can be adapted to phone sensing and immunoflow test form for on-site applications.
EXPERIMENTAL SECTION Chemical and Materials. Benzoylecgonine mAb conjugated AuNPs were purchased from Arista Biologicals (St Allentown PA, 18101). Nitrocellulose (NC) membrane was obtained from mdi Membrane Technologies (Ambala Cantt 133 006, India). Conjugate release pad, sample pad and absorbent pad were purchased from Ahlstrom Corporation (Helsinki, Finland). Tetrahydrocannabinol (THC) was obtained from THC Pharm (Biochem.thc-pharm, Germany). The other standards including cocaine, methamphetamine and benzoylecgonine (BE; cocaine metabolite) were purchased from Cerilliant (Cerilliant Corp., Round Rock, TX, USA). Bovine serum albumin (BSA), nicotine and its metabolite cotinine were obtained from Sigma-Aldrich (St Louis, USA). All other chemicals were reagent grade. PPP-CD-g-PEG was used as a biomimetic material for the construction of test lines and synthesized according to previously described procedure via coupling reactions15. Design of β-CD/LFA Strip. One test strip was composed of five parts including PVC backing card, sample pad, conjugate release pad, nitrocellulose (NC) membrane and absorbent pad. Before test components were immobilized onto the membranes, sample pad and NC membrane were pretreated with blocking solutions. Conjugate release pad and absorbent pad were used with their untreated forms. The sample pad was initially, pre-treated with 5.0% sucrose, 5.0% dextran, 0.5% Tween 20 and 0.05% sodium azide for 30 min and then washed with sodium phosphate buffer (10 mM, pH 7.4). On the other hand, the NC membrane was soaked in 1X TBS (50 mM Tris HCl, 150 mM NaCl) containing 0.05% Bovine serum albumin (BSA), 0.005% casein, 0.02% (Polyvinylpyrrolidone (PVP), 0.002% Tween 20 and washed with distilled water. Both sample pad and NC membrane were dried in a desiccator overnight. To impregnate conjugates and analytical membrane lines, a dispenser system (The IsoFlow Dispenser, Imagene Technology, USA) was used. Anti-Bovine IgG antibody (2.0 mg/mL) and PPP-CD-g-PEG (1.0 mg/mL) were dispensed in control line (C-line) and test line (T-line), respectively as detection zones. Anti-Benzoylecgonine antibody/AuNPs conjugate (Anti-BE/AuNPs) was also adsorbed to conjugate pad via a dispenser. Afterwards, the resulted NC membrane and conjugate pad were dried at 37°C and 20% humidified conditions in a test cabinet (Nüve TK120, Turkey) for 4 h. All of the membrane components were then inserted into the PVC backing card. Finally, the assembled LFA components were cut into test strips via a membrane cutter (A-Point Guillotine cutter). Analysis of Cocaine Using LFA. Cocaine samples with different concentrations were prepared in the synthetic saliva solution. Synthetic saliva was prepared according to the previous works.18 Cocaine standard solutions (100 µL) were applied to the sample pad and test results in the analytical window were photographed. Color intensities arising from AuNPs
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in T-line were measured by a smartphone application (Color Analysis). Chromatographic Confirmation. Calculation and verification of the analyte concentration were carried out using LCMS / MS according to the previous work.19 Briefly, the artificial saliva sample containing 500 ng /mL of analyte was diluted 1000- fold and injected to LC-MS/MS system. The used LC-MS/MS system comprises Waters Acquity UPLC instrument using an Acquity UPLC BEH C18 column (1.7 µm, 50 mm x 2.1 mm i.d.). Instrumental conditions for cocine analysis include mobile phase: A) Water contains 0.1 % Acetic Acid B) Acetonitrile contains 0.1 % Acetic Acid; column temperature: 50°C; gas flow rate: 500L/h; flow rate: 0.400 mL/min and injection volume: 5.0 µL.
RESULTS AND DISCUSSION LFA Procedure and Mobile Phone-based Detection. As schematized in Scheme 1A, PPP-CD-g-PEG was adsorbed onto the NC membrane as test zone to capture cocaine molecules due to the affinity of β-CD towards cocaine. In the principle of β-CD/LFA, a liquid sample is applied to the sample pad and migrated in the direction of conjugate pad, NC membrane and absorbent pad, respectively. As the sample reach to the conjugate pad, Anti-BE/AuNPs migrated forward together with liquid sample. When the applied sample is cocaine positive, the cocaine molecules in the sample interact with β-CD residues in T-line and red line appears in the test zone as a result of the attachment of Anti-BE/AuNPs to cocaine. In the case of negative sample, however, Anti-BE/AuNPs conjugates cannot interact with T-line polymer due to the absence of cocaine. In summary, two visible red lines in NC membrane sign to the positive test result for cocaine while one visible red line in the control implies the negative. Test results were not only evaluated with naked eye but also quantitatively analyzed using mobile phone application (Scheme 1B).
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Analytical Chemistry
Scheme 1. Schematic illustration of A) the design strategy and B) test principle of β-CD based non-competitive cocaine assay. Figure 1A shows the standard curve set up for cocaine using a smart phone. Different concentrations of target analyte in the range of 0.01- 5.0 µg/mL were applied to test strips and test results were photographed (Figure 1B). Red color in T-line was cropped and processed using an android-based mobile application, Color analysis evaluates color group and color percentage in image. Evidently, a good linear trend in a broad detection range (0.01 – 1.0 µg/mL) was attained in the cocaine calibration curve.
Figure 1. A) Calibration curve from a smart phone based detection of color intensities in β-CD/LFA strips. B) Photographs of standard cocaine solutions applied LFA strips in the concentration range of 0.01- 5.0 µg/mL. Cross-reactivity is the most encountered problem for immunochromatographic assay.20 For this reason, the effect of some drugs and metabolites on LFA result was investigated. Potential interferences including benzoylecgonine (BE), nicotine, cotinine, codeine, tetrahydrocannabinol (THC) and methamphetamine (METH) were chosen according to the β-CD interaction properties17 and possibility of availability in biological matrices. As demonstrated in Figure 2, red line appeared in control line, which means that the test result is negative. These results indicated that the tested drugs did not cause falsepositive test response.
Figure 2. Effect of some interferents on β-CD/LFA strips. BE: Benzoylecgonine; THC: Tetrahydrocannabinol; METH: Methamphetamine. As already mentioned, fabrication of these test strips is unique in terms of the usage of biomimetic polymeric material and its integration for mobile phone sensing. Previously reported studies involving β-CD usage for cocaine analysis concern modified electrodes21 or µ-well assay17 through cocaine binding affinity. The latter approach was also practiced in our previous work. When compared to the other β-CDs based approaches, analytical performance of the fabricated βCD/LFA test is quite good with the additional benefit of simple applicability. Finally, to achieve the biological matrix application, synthetic saliva samples spiked with cocaine were applied to β-CD/LFA strips and the color intensities in test zone were measured by smartphone application. In oral fluid differently from urine, parent drugs are dominate species. For this reason, both screen and confirmatory assays target the parent drugs for drug detection in saliva. Hence, immunoassays designed for the detection of hydrophilic drug metabolites in urine cannot be applied for drug screening in saliva.22 In the case of cocaine detection in oral fluid, immunoassays are designed to target cocaine because cocaine predominates in saliva in compared to its metabolism products.23 On the other hand, oral fluid as the noninvasive alternative to blood, is preferable specimen for onsite drug detection such as workplace applications, drug testing in driving.24 In our study, we deliberately selected synthetic saliva for sample application due to the oral fluids properties and advantages. Thus, the cocaine amount in the synthetic saliva was calculated as 0.53 µg/mL with 106% recovery for the 0.5 µg/mL standard addition. This result clearly indicates that β-CD/LFA paper kit simply using a mobile phone holds a good potential for on-site detection of drug abuse. Spiked samples (500 ng/mL) was confirmed via chromatography and cocaine amount was found as 503.24±3.88 ng/mL. Data obtained from both LFA system and reference method were compatible. This finding verified the accuracy of the proposed method.
CONCLUSIONS In summary, a novel approach to construct a non-competitive cocaine assay based on LFA is reported has been accomplished. The adaptation of sandwich format instead of competitive format, which is conventional for small molecules, ena-
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bled quantitative detection through a smartphone application. This LFA production was achieved using a specifically designed, PPP-CD-g-PEG, as a capture in T-line. Because β-CD units in the structure of the conjugated polymer have a strong affinity to cocaine, T-line was visualized in the presence of cocaine in the sample. Concentration dependent color intensities were analyzed by mobile phone application to obtain a calibration curve. After linear graph was successfully made for cocaine in the range of 0.01 – 1.0 µg/mL The effect of some interferences was also investigated. Finally, chromatographic confirmation of the designed cocaine test was carried out by applying cocaine added synthetic saliva samples. All the data is valuable for phone sensing with non-competitive small molecule assay. The reported approach offers a promising methodology for the construction of rapid test kits for practical applications such as road-side drug testing, workplace substance detection.
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Corresponding Author * e-mail:
[email protected] *e-mail:
[email protected] Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / ‡These authors contributed equally. (match statement to author names with a symbol)
Notes Any additional relevant notes should be placed here.
ACKNOWLEDGMENT This study was funded by Industrial Thesis Support Program (SAN-TEZ) of Republic of Turkey, Ministry of Science, Industry and Technology (Project Grant No: 0620.STZ.2014) and Republic of Turkey, Ministry of Development (Project Grant No: 2016K121190). It was also partially supported by Ege University Research Fund through BAP projects (Project No: 16-FEN-004 and Project No: 14-FEN-019) and Istanbul Technical University Research Fund.
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