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 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.