AttributionsRathbone, D. A., et al. (1996). The Use of a Novel Recombinant Heroin Esterase in the Development of an Illicit Drugs Biosensora. Annals of the New York Academy of Sciences, 799(1), 90-96. Zhou, M. & Panchuk-Voloshina, N. (1997). A one-step fluorometric method for the continuous measurement of monoamine oxidase activity. Analytical Biochemistry, 253, 169-174.

OutreachOur biohack crowdsourced the making of a smart phone app to detect the intensity of the colour for our biosensor.

Design DevelopmentWhen designing this paper-based biosensor we considered our stakeholders’ input.

Enzyme ImmobilisationAfter production the enzymes diffused on the paper, undermining the results. This led to us fusing the\enzymes to a suite of 5 Cellulose Binding Domains (CBDs), on both the N and C terminus to ensure functional folding leading to submitting 72 parts.

LegislationThe UK Misuse of Drugs Act of 1971 does not ban the possession of drug paraphernalia. Thus, possession of drug testing kits is not a crime. Under this Act, Schedule 2 substances (such as heroin) are permitted in universities for research purposes.

AbstractIn 2015 ˜80 million Europeans tried an illicit drug. We have developed a multiplexed, enzyme and paper-based biosensor to semi-quantitatively measure heroin purity, detect PMA (p-methoxyamphetamine) in ecstasy and DNP (2,4-Dinitrophenol) in unregulated diet pills. This will contribute to harm reduction efforts related to illicit drug use. Drug Consumption Rooms and pill testing services have agreed to use our biosensor in their attempts to lessen morbidity and mortality.

This assay can be coupled with Nitrotetrazolium blue, which produces a blue dye with varying intensity depending on NADPH production.

NCH3

CH3COO

CH3COO

O

Heroin(Diamorphine)

Heroin Esterase

O

CH3COO

HO

NCH3

6-Acetylmorphine

Heroin Esterase

HO

O

NCH3

HOMorphine

Morphine Dehydrogenase

NADP+

NADPH

O

HO

O

NCH3 Morphinone

SecondaryMDMA

Primary

PMA

Amplex® RedH2O2 O2

DNP

Laccase

ABTS ABTS˚ˉ

degraded product

Heroin

Time (seconds)0 100 200 300 400 600500 700

00.010.020.030.040.050.060.070.080.090.1

No Enzyme

No Substrate

Abs

(340

nm

)

Heroin Esterase + Morphine Dehydrogenase

NADPH Production in the Presence of Heroin

Heroin Esterase(BBa_K1615045)

0 200 400 6000

10

20

30

40

p-nitrophenyl acetate (µM)

Rate

of R

eact

ion

(V)

In countries where the responsibility of drug safety is placed on the individual, our biosensor will be available in needle exchanges. When taught how to administer naloxone, information and education on how to use our biosensor could be included.

Drug Consumption Rooms (DCRs) provide a safe space to consume drugs in the presence of trained social workers. Our visit to a DCR in Amsterdam confirmed that the staff and clients would use our biosensor to test their samples.

Prototype 1: Simple design. Easy to use

Prototype 2:Multiplexed

Prototype 3:Contained colour

Final: Small sample size

Drug deaths are mostly due to varying purity (13.5-98%). Scotland has the highest rate of 112.5 drug-induced deaths per million citizens.

Stakeholders’ Input

a) b)

A demonstration of the PMA biosensor detecting H2O2 with a 3 minute time to detection. HRP and Amplex Red were applied to each bioactive zone. 20µl of 10µM H2O2 solution was added to the application zone.

a) Heroin esterase was active with p-nitrophenyl acetate, a heroin analogue.

b) This demonstrates that heroin can be detected colourimetrically as NADPH is produced in the presence of NADP+, heroin esterase, morphine dehydrogenase and heroin.

RFP

RFP-CipA

CipA-RFP

CipA

CenA-RFP

RFP-CenA

CenA

Note how the N terminal fusion of cenA folds incorrectly and does not fluoresce.

Our public debate brought together a panel of experts, including a Member of Scottish Parliament, to share their opinions on drug related issues.

The difference in RFU shows that the CBD immobilises RFP on the paper.

Red countries have Drug Consumption Rooms Blue countries are working on Drug Consumption Rooms

Dehghanifard, E., et al. (2013). Biodegradation of 2,4-dinitrophenol with laccase immobilized on nano-porous silica beads. Iranian Journal of Environmental Health Science and Engineering, 10(1), 25.

Individuals should have the choice to consume drugs with absolute impunity.

Resorufin

Monoamine oxidase A oxidises primary monoamines. When the H2O2 is

coupled with horseradish peroxidase (HRP) and Amplex® Red, the red chemical resorufin is produced.

Monoamine Oxidase A

O2 H2O2

aldehyde + ammonia

Resorufin

Detection Methods

Looks Safe on Paper: A Biosensor for Illicit Drugs

IntroductionPMA in ecstasyThe use of ecstasy, commonly composed of MDMA, has risen. MDMA production can result in the formation of the acutely toxic compound PMA.

DNP in Diet PillsDNP is often found in unregulated weight loss pills. There are many adverse side effects of DNP, leading to a Global Health Warning.

Laccase degrades DNP, with the concurrent production of ABTS radicals when ABTS, 100 mM sodium acetate buffer and DNP is present. This can be detected by measuring absorbance at a wavelength of 420 nm.

HRP DNP

Bioactive ZoneApplication Zone

DNP degradation is undetectable visually, so it requires the reporter substrate, ABTS. When DNP is being degraded there will be a less intense ABTS colour. A control strip will be rehydrated with water to produce a baseline colour.

Laccase Activity over Time

Time (seconds)

Abso

rban

ce

0 200 600 800

0.2

0.5

1

1.5

900400

Implementation - Harm Reduction

DanceSafe, a US company that sells drug testing kits, are very supportive and see our biosensor as an improvement on the tests they currently provide.

Where do you think you are?

Total government control over, and condemnation of, drug use

Results

Laccase + ABTS

Laccase + ABTS + DNP

Market biosensor online where a majority of diet pills are purchased.

Patrick Arant, Andreea Cucu, Ivana Lapsanska, Michelle Marufu, Vicky Moynihan, Aseem Narang, Liusaidh Owen, Dominika Pelegrinova, Brooke Rothschild-Mancinelli