Disposable screen printed sensor for the electrochemical detection of methamphetamine in undiluted saliva
© Bartlett et al. 2016
Received: 7 August 2015
Accepted: 7 January 2016
Published: 1 February 2016
Methamphetamine has an adverse effect on the ability to drive safely. Police need to quickly screen potentially impaired drivers therefore a rapid disposable test for methamphetamine is highly desirable. This is the first proof-of-concept report of a disposable electrochemical test for methamphetamine in undiluted saliva.
A screen printed carbon electrode is used for the N,N′-(1,4-phenylene)-dibenzenesulfonamide mediated detection of methamphetamine in saliva buffer and saliva. The oxidized mediator reacts with methamphetamine to give an electrochemically active adduct which can undergo electrochemical reduction. Galvanostatic oxidation in combination with a double square wave reduction technique resulted in detection of methamphetamine in undiluted saliva with a response time of 55 s and lower detection limit of 400 ng/mL.
Using a double square wave voltammetry technique, rapid detection of methamphetamine in undiluted saliva can be achieved, however there is significant donor variation in response and the detection limit is significantly higher than desired. Further optimization of the assay and sensor format is required to improve the detection limit and reduce donor effects.
KeywordsSquare wave voltammetry SWV Galvanostatic oxidation Screen printed electrode Mediator Methamphetamine Saliva Detection
Two thirds of US trauma centre admissions are due to motor vehicle accidents with almost 60 % of such patients testing positive for drugs or alcohol . Cannabis, cocaine and methamphetamine are the drugs most frequently detected in drivers randomly stopped for roadside drug screening [2–5]. In Norway prior to the year 2000 there was almost no methamphetamine on the Norwegian market. There was a steady increase in methamphetamine usage till 2010 where it appeared to have stabilized. The data for this study was confirmed by testing venous blood of convicted motorists, customs seizures and wastewater analysis . A US survey, using a questionnaire which annually monitored adolescent drug use, showed a gradual decline in methamphetamine use from 3.7 % in 1981 (peak year) to 1.2 % in 2008 . A recent study showed conflicting trends when comparing the questionnaire survey approach and wastewater analysis. Over the period 2010–2013 the population survey showed a slight decline in methamphetamine use while wastewater analysis showed a doubling of methamphetamine usage .
Methamphetamine remains a significant public health concern with known neurotoxic and neurocognitive effects to the user . It is frequently abused as a recreational drug due to its stimulant and euphoric effects. The physiological and psychological side effects of methamphetamine include confusion, paranoia, depression, nausea and blurred vision. Driving a vehicle while under the influence of methamphetamine is thus clearly undesirable.
Roadside screening for methamphetamine in oral fluid has a number of requirements: it needs to be fast, ideally 15–30 s, i.e. ideally the same speed as a breath alcohol test; it must be very sensitive, ideally <10 ng/mL (25 ng/mL was used as the cut-off concentration in the European DRUID [Driving under the influence of Drugs, Alcohol, and Medicines] project ); and it should be non-invasive, difficult to tamper with and be portable. The currently available drug screening products require a minimum of 5–10 min for a test . Test time and cost are restricting the roadside drug screening market to <10 % the volume of the alcohol screening market.
Oral fluid which contains saliva and other liquid substances present in the oral cavity are of great interest for roadside drug screening. Although this fluid is easy to collect there is considerable inter-sample variability in the fluid matrix that generates issues when developing a testing methodology . Dilution of the sample can reduce the donor variability, however this dilutes the drug of interest and therefore requires the device to have greater sensitivity. The current devices on the market are primarily lateral flow immunodiagnostic tests, where the presence or absence of a coloured bar can be read either visually or in a meter in response to the drug of interest; these were used in the DRUID project. The response times are typically several minutes. The clinical sensitivity of these devices in saliva can be relatively poor at 16–75 % although clinical specificity can be close to 100 % .
There are only a few reports of the electrochemical sensing of amphetamines, and there are no reports of the determination of amphetamines in undiluted saliva using disposable electrochemical sensors. Electrochemical sensing of methamphetamine by direct oxidation has been reported at a pretreated pencil graphite electrode [LOD 50 nM (7.5 ng/mL) in aqueous solution, response time >10 min] , at a self-assembled boron-doped diamond electrode [LOD 50 nM (7.5 ng/mL) in aqueous solution, response time not given] , and in alkaline solution using a gold nanoparticle-multiwalled carbon nanotube modified screen printed electrode [LOD 0.3 nM (0.05 ng/mL), response time not given] . The indirect electrochemical detection of amphetamine in saliva has been reported using 1,2-naphthoquinone-4-sulfonate at an edge plane pyrolytic graphite electrode [LOD 41 μM (6.2 μg/mL) in aqueous solution, response time not given] .
This paper reports a mediated screen printed carbon electrode for the detection of methamphetamine in undiluted saliva using substituted N,N′-(1,4-phenylene)-dibenzenesulfonamide mediator. Screen printed electrodes are well established as cheap and disposable single use sensors which can be manufactured with high reproducibility .
The sensor is optimized for speed of response and for response in undiluted saliva.
Results and discussion
Initial mediator screen
With primary amines such as amphetamine (AMP), 1,2-addition can take place, resulting in elimination of the two benenesulfonamide groups from the mediator and formation of an AMP-mediator adduct. This adduct can also undergo oxidation via II and subsequently undergo electrochemical reduction.
Optimization of electrochemical procedure with dried reagent
It was desired that the mediator and buffer solution be dried down in some way on the sensor. Deposition of mediator solution directly onto the sensor requires tight control of the volume and position of the dispensed reagent. Therefore an alternative technique was used comprising a porous overlayer onto which mediator was dried and which is then secured over the sensor. On application of sample, the mediator dissolves and diffuses to the working electrode where it can be oxidized, react with MAMP and produce a reduction response to MAMP. Sensors with overlayer applied are shown in Fig. 2b.
Galvanostatic oxidation of OX1006 was investigated in combination with the overlayer. The advantage of galvanostatic oxidation compared to potentiostatic oxidation is that the amount of oxidized mediator should be relatively independent of the concentration of mediator which has dissolved off the overlayer and reached the electrode surface, provided there is sufficient mediator. A potential disadvantage of galvanostatic oxidation is that if there is insufficient mediator, other species present will be oxidized, resulting in a large increase in working electrode potential.
There are very few reported examples of galvanostatic oxidation to generate reactant, and these examples are for electrochemical titrations using separate generator-collector electrodes [19, 20]. For example, Tomcik et al.  have reported the galvanostatic generation of hypobromite at an interdigitated microelectrode array, for end-point titration of the drugs Antabus and Celaskon. In our application, the working electrode is used to both generate the reactant (oxidized mediator) and detect the mediator-MAMP adduct.
In order to increase the speed of the SWV technique, the first part of the scan between +0.6 and +0.1 V was conducted at a higher scan rate compared to the second part of the scan between +0.1 V and −0.4 V. Both parts of the scan were optimized for amplitude, step size and frequency. The third peak height is independent of frequency (Additional file 2), therefore a faster scan rate can be used for the first part of the scan without any adverse effect on the 3rd peak height.
The LOD compares favourably with that obtained using indirect electrochemistry with 1,2-naphthoquinone-4-sulfonate , and it is considerably higher than the LODs obtained using direct electrochemical methods [13–15], although all these methods use aqueous solution and not undiluted saliva. Use of microelectrodes should provide greater sensitivity of response, since increased mass transport of MAMP to the electrode should result in increased peak heights i.e. higher nA per ng/mL MAMP. However this would require reproducible screen printed microelectrodes and development of a suitable manufacturing methodology was beyond the time and budgetary restraints of this work.
The response to MAMP and amphetamine in saliva using the split SWV technique showed a new peak formed in response to MAMP at −0.04 V, and no new peak observed in response to amphetamine (Additional file 3). This demonstrates the selectivity of the mediator to secondary amines over primary amines.
Variation in response with different donor saliva samples
The response to MAMP increased with decreasing molecular weight cut-off of the filter e.g. for donor 1, the 3rd peak heights in response to 1 μg/mL MAMP were 15, 142 and 353 nA for unfiltered saliva, 100 and 3 kDa filters. However there is still considerable donor variation in response with the filtrate from the 3 kDa filter (the 3rd peak heights for donors 1 and 2 were 353 and 512 nA respectively). While this filter will have removed larger proteins and mucins, some small proteins and protein fragments will remain, which may compete for adsorption sites on the electrode surface with the mediator MAMP adduct. In addition, the filtrate will contain endogenous amines which may react with the mediator.
Response to saliva buffer containing added protein
Average peak height/nA (±1 SD)
% Decrease in peak height
1st peak (at +0.40 V)
2nd peak (at +0.25 V)
3rd peak (at −0.06 V)
2865 ± 1369
3257 ± 1939
436 ± 30
SSB + 0.021 mg/mL mucin
2665 ± 728
2581 ± 893
481 ± 59
SSB + 0.021 mg/mL mucin + 0.3 mg/mL lysozyme
1952 ± 1009
1781 ± 1018
382 ± 70
SSB + 0.021 mg/mL mucin + 3.0 mg/mL lysozyme
985 ± 275
717 ± 233
54 ± 29
(+)-Methamphetamine hydrochloride (M8750), d-amphetamine sulphate (A5880), human recombinant lysozyme (L1667) and mucin from bovine submaxillary glands (M3895) were obtained from Sigma-Aldrich Co. Ltd (Poole, UK). The mediators were synthesized by Peakdale Molecular (High Peak, UK). All other chemicals were purchased from Sigma-Aldrich Co. Ltd. All chemicals were used as received without further purification. All solutions were prepared using deionized water with resistivity no less than 18.2 MΩ cm.
Screen printed electrodes were fabricated in house with appropriate stencil designs using a DEK Horizon printing machine (DEK, Weymouth, UK). Successive layers of carbon-graphite ink (C2120403D1, modified in house by the addition of 0.1 % TX-100), dielectric ink (D2070423P5) and Ag/AgCl ink (60:40, C2030812P3) obtained from Gwent Electronic Materials Ltd. (Pontypool, UK) were printed onto a polyester substrate. The layers were cured using a tunnel drier at 70 °C (Natgraph, Nottingham, UK). The reproducibility of response of a sample of sensors from each print batch was determined using square wave voltammetry (SWV) with 1 mM OX1006 in 0.4 M sodium carbonate buffer (pH 10.8), 0.23 M NaCl, 0.0018 % TX-100. The SWV settings were as follows: start potential +0.6 V, stop potential −0.5 V, frequency 10 Hz, amplitude 0.05 V and step size 0.00285 V. Each sensor batch comprised 15 sheets with 4 rows of 48 sensors per sheet. A sample of 12 sensors from the second sheet of each batch were tested for SWV response to OX1006, and the responses were characterized for peak position and peak height. The %CVs were typically in the range 0.5–1.7 and 3–5 % for peak position and peak height respectively.
Voltammetric measurements were performed using either a MultiAutolab M101 or a μ-Autolab III potentiostat (Eco Chemie). The screen printed sensors were used as a two electrode system, with a combined counter/reference electrode (Ag/AgCl ink).
The overlayer material was composed of abaca and cellulosic fibres (75 %) in a polypropylene thermoplastic matrix (25 %), dry weight 16.5 g/m2 (CD020010, Ahlstrom) in reel format (1 cm wide) was obtained from Ahlstrom (Duns, UK). The overlayer was coated with OX1006 as follows: 1 mg/mL OX1006 was prepared in 0.4 M sodium carbonate buffer solution (pH 10.8) containing 1 M NaCl and 0.1 % Triton X-100. The solution was dispensed onto the membrane at a loading of 0.1–1 mg/mL and dried at 40 °C. The dried overlayer was heat soldered to each sensor along the edges.
Saliva buffer, which mimics real saliva except for the absence of proteins, consisted of 27.5 mM sodium chloride, 6.3 mM ammonium chloride, 4.9 mM sodium phosphate (monobasic), 2.9 mM potassium chloride, 1.1 mM sodium citrate (anhydrous), 0.02 mM magnesium chloride (anhydrous), 0.27 mM sodium carbonate and 0.2 mM calcium chloride.
Each saliva sample was collected immediately before use by spitting into a pot. Saliva samples containing MAMP were prepared by dissolving MAMP directly into the saliva sample at 1 mg/mL. Saliva samples containing lower MAMP concentrations were obtained by dilution of the 1 mg/mL sample with neat saliva.
Centrifugal filtration of saliva was performed using Amicon Ultra 0.5 mL centrifugal filters with molecular cut-off weights of 100, 30, 10, and 3 kDa. The samples were centrifuged at 14,000g for 10 min. The filters were weighed before and after centrifugation and deionised water was added to each filtrate to adjust for volume lost.
The detection of 400 ng/mL MAMP in undiluted saliva has been reported using mediated disposable screen printed sensors with a response time of 55 s. While the response time is significantly faster than existing lateral flow immunodiagnostic tests, the limit of detection of the sensors is considerably higher (400 ng/mL compared to 10 ng/mL) and is too high to be acceptable as a screening test. The precision of the sensor response is adversely affected by saliva proteins and further development of the sensor is required to overcome these effects and obtain a commercially viable sensor. Saliva samples are notoriously variable in terms of composition and viscosity, even within the same donor sample collected over a short period of time, and it is probable that an on-strip dilution of the sample would decrease adverse effects arising from sample variability and viscosity, however this would require controlled sample dilution. It would also require greater sensitivity of response which may be achieved by the use of microelectrodes and this is a route that should be investigated further. In conclusion, development of a disposable roadside test for the rapid determination of methamphetamine in undiluted saliva is challenging, and requires significant further effort.
square wave voltammetry
differential pulse voltammetry
LM and MB co-directed the study. CF demonstrated the initial concept. EE and AR characterised the electrode performance. DB and CW performed the mediator screen. CAB optimized the electrochemical procedure and ST investigated donor variation. LM and MB wrote the manuscript. All authors read and approved the final manuscript.
The authors gratefully acknowledge Professor Richard Compton and Professor Craig Banks for helpful discussions. Professors Compton and Banks are the company founders and are shareholders in Oxtox.
The authors declare that they have no competing interests.
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