# Limits of Detection

Finding the limit of detection for the heroin purity biosensor.
The heroin purity biosensor depends on a linear relationship between the colorimetric output of the dye produced from a coupled reaction involving the original concentration of heroin1.
The maximum limit of detection for this biosensor is limited by the maximum solubility of heroin in water which is 0.2mgml -1 . It is also limited by the maximum amount of liquid that can be retained on the bioactive zones on the biosensor.

The bioactive zones are based on the area of a paper chad from Whatman 54 and an iso-standard hole punch, which is 113mm22. This area can retain approximately 10μl.

These two limits can be used to mathematically determine the maximum limits of detection; one bioactive zone can only hold 2μg of heroin in its 10μl. The next step is to determine the concentration of dye produced when all this heroin reacts, which requires the stoichiometry of reaction to be known. When two moles of heroin react, this leads to the formation of two moles of NADPH, which react with one mole of PMS to make one mole of a blue-purple formazan dye 2 .

The next stage is to find out what the intensity of this colour output is, which is the maximum that can be achieved. We then perform a series of dilutions from this point till the point where visual detection is no longer possible; this is the minimum limit of our detection. After performing this test we discovered a bioactive zone with an area of 113mm 2 is able to detect as little as 40% heroin by weight (0.8μg), assuming that the original sample tested is 2μg. The weight of the sample tested is important because if all the 2μg is 100% heroin then the solution on the zone is 100% saturated in terms of heroin and this will show the most intense colour change. Only the soluble amount of heroin is able to diffuse into our bioactive zones, hence if the weight of the sample is more than specified then it may give false positives. Since there are 4 bioactive zones, an application zone and 4 lanes then the volume required is ~ 50μl which equates to 10μg of sample.

1 Mayer, K. M., & Arnold, F. H. (2002). A colorimetric assay to quantify dehydrogenase activity in crude cell lysates. Journal of biomolecular screening,7(2), 135-140.

2Sjövall, K. (1967). A tetrazolium technique for the histochemical localization of ATP: creatine phosphotransferase. Histochemie, 10(4), 336-340.

Initially ABTS was chosen as an indicator of DNP degradation by Laccase because we believed the build up of ABTS radicals would cause the colour of the system to go from yellow (the colour of DNP) to green (the colour of ABTS radicals.) We ran assays, adding DNP and ABTS with purified laccase enzyme from Trametes versicolor to a cuvette for spectrophotometry, and found the result not to be what we expected. Laccase and ABTS alone produced a similar colour change to Laccase, ABTS and DNP. As observed in the figure below the major difference is that DNP causes the colour change to happen over a longer time period. This result, though unexpected could still provide the basis for a biosensor…

The biosensor could still work if provided with a control. Two bioactive zone with freeze-dried laccase and ABTS would exist alongside one another, but to one DNP is added and to the other only water. The difference in the colour change after a defined period (90-600 seconds) would be a good indicator of whether DNP was present.

The flaw in this is that we didn’t get the chance to test the effects of other non-toxic components of diet pills with laccase and ABTS. If DNP is the only compound that has this effect, then the DNP diet pill biosensor is functional and specific.