Note: The schematics, pictures and Tables in this document are mostly from the publications of skilled scientists and professionals. The reader is implored to read the original manuscripts.
The requirement for this project stems from the use of opioids by addicted patients. These opioids such as fentanyl, carfentanyl and other derivatives of fentanyl are deadly in small concentrations and highly addictive. A convenient means is required to quickly detect the presence and concentration of an opioid at the point of exposure to alert the patient and the caretaker or doctor of a potentially lethal dose and possibly which type of derivative was used. The demand for devices that can quickly indicate the presence and concentration of opioids is high. Not only is such a device important for the patient (addict) but also for anyone that comes into contact with the drug cocktail due to the fact that fentanyl and other drugs may be absorbed through the skin of doctors, patients and law enforcement officers to name but a few.
Private companies are investing a lot of resources in devices that can detect lethal drug doses. Examples include the Apiject[1] pre-filled injection system and the smartdose injection platform from Westpharma.[2] While the developments from these companies are probably more concerned with “legal” screening of drug injection into subjects such as screening of blood samples, there remains a need to assist drug addicted patients to prevent overdosing and injecting potential lethal levels of fentanyl and fentanyl derivatives.
Given the nature of the addiction problem, clinics and safe needle suppliers[3] need technology that can distinguish between fentanyl and its derivatives from other drugs while using the same syringe technology where possible to deliver the drugs to the addicts’ system. The requirements are therefore for a robust delivery system and detection system. In addition, the technology should be cost effective to make it financially viable. Some of the ideas that have been suggested regarding possible technologies include:
The areas of interest as outlined above will be investigated in order to provide an idea of the efficacy of each option as well as the sensitivity in measuring the opioid drug, the ability to discriminate between different opioids and other types of drugs. Separately, while the method may prove viable, some way is required to validate the method and one might have to consider independent validators.
[2] https://www.westpharma.com/products/self-injection-platforms/smartdose
[3] https://www.cdc.gov/ssp/syringe-services-programs-summary.html
One of the latest descriptions of the use of a dye to detect opioids is described in WO2021081063A1 (Loane, 2020). In this case, the dye tetrabromophenolphthalein ethyl ester (TBPP) undergoes a specific color change in the presence of any opioid derivative. It appears that this type of dye can yield a discernable color change from 50 to 5 µmg.ml-1 of an opioid narcotic. In the presence of an opioid substance it undergoes a purple to pink-red color change while remaining yielding a blue to yellow color if there are no opioids present. It will be appreciated that there are many different derivatives of the TBPP dye that could be considered. In the same patent, the use of diazonium dyes is also disclosed such as 4-bromobenzene diazonium tetrafluoroborate. The type of color change is not disclosed.
Another dye suggested for fentanyl detection is Eosin Y (J Kangas, 2017). Further selection of dyes is described in US 2011/0117664 A1 (Amisar, 2011). In this case the use of an azo dye as well as several other types of dyes are disclosed. For example, the azo dye is described as having an electron withdrawing group on the aromatic ring such as nitroso group while the second dye includes examples such as bromophenol red, bromocresol purple and tetrabromophenol blue. Some of the chemical structures of the dyes mentioned to date are presented below. The patent mentions the use of milligrams of dyestuff which in turn is dissolved in organic solvents or water (ml) for the detection of fentanyl-based narcotics.
Figure 1:
Figure 2:
In both patents the use of a cooperating azo dye is used. The number of azo dyes is numerous so only the general structure will be given below.
Figure 3:
In terms of toxicity, the range for azo dyes is 250 to 2000 mg/kg.[1] Most non-azo dyes and pigments lie in similar ranges. Examples of some dyes that illustrate the toxicity levels are Basic Violet 14 and Direct Red 28 that show acute toxicity with a LC50 value at 60.63 and 476.84 µg.ml-1 respectively, whereas the LC50 of Acid Red 26 is between 2500 and 2800 µg.ml-1.[2] These values are based on toxicity for zebra fish but can be accepted as indicative for humans as well. If dyes are therefore to be used in opioid detection, upper levels of around 50 µg.ml-1 should be contemplated. In US 2011/0117664 A1 dyes are used in the amount of 0.0006 g per 0.5 ml (Fast Corinth V). These small amounts could yield less toxic means of detecting opioids. A similarity search was conducted on Pubchem with regard to TBPP and at least 26 structures similar to TBPP were found. Similarly for Eosin Y, 223 similar structures were reported.[3] It appears Eosin can detect various types of drugs in the range from 0.1 – 10 µg.ml-1, depending on what type of drug it is (Rahman, 2017).
[1] https://www2.mst.dk/udgiv/publications/1999/87-7909-548-8/html/kap05_eng.htm
[2] https://pubmed.ncbi.nlm.nih.gov/25727789/
[3] https://pubchem.ncbi.nlm.nih.gov/#query=O%3DC1C(Br)%3DC2Oc3c(Br)c(O)c(Br)cc3C(c3ccccc3CC(O)%3DO)%3DC2C%3DC1Br&tab=similarity
Figure 4:
The structure of Eosin Y is sensitive to pH. This property has been used to discern between fentanyl and other drugs (J Kangas, 2017). With regard to the toxicity of eosin (LD50), the level of toxicity lies between 4000 to 5000 mg/kg.[1]
Figure 5:
The possible candidates for fentanyl detection seem formidable and it is possibly best to do some computer modelling to determine the most viable candidates. For in vivo drug detection it can be assumed that all dyes are toxic (Klemola, 2008). For this reason it is highly recommended that dyes should be immobilized/supported in some way without diminishing their functionality and efficacy as indicators. So of the techniques to deal with the toxicity of dyes rely on absorbing or tethering the dye molecule to a specific carrier such as nano particles.
[1] New material transforms light, creating new possibilities for sensors (phys.org)
Figure 6:
Another example would be to modify the dye molecule to contain a reactive group that can be polymerized or crosslinked.[1] In this case, the ability to control molecular structure and molecular weight of the chemical tethers become important and chemistries such as click chemistry and controlled free radical polymerization can play a role.
[1] Chemistry of Crosslinking | Thermo Fisher Scientific – ZA, Methacrylate-tethered analogs of the laser dye PM567–synthesis, copolymerization with methyl methacrylate and photostability of the copolymers – PubMed (nih.gov)
Figure 7:
Turning dye molecules into surfactants leads to another possible way of incorporating them into a plastic matrix (Smith McWilliams et al., 2020). Many additives in plastics such as those used in polyethylene and polypropylene act in much the same way as surfactants in that they have both a hydrophobic and hydrophilic nature. Examples include anti-static additives and plasticizers.
Figure 8:
[1] New material transforms light, creating new possibilities for sensors (phys.org)
[1] Chemistry of Crosslinking | Thermo Fisher Scientific – ZA, Methacrylate-tethered analogs of the laser dye PM567–synthesis, copolymerization with methyl methacrylate and photostability of the copolymers – PubMed (nih.gov)
The basic structure of an alkenyl anhydride is given below in Figure 9. The R1 and R2 groups are normally methene units that yield the hydrophobic nature of this molecule. The anhydride moiety makes for a versatile way to modify the molecule to make surfactants and additives.
Figure 9:
The anhydride functionality of PIBSA is an example of tethering the dye molecule for inclusion in a polymer matrix such as polyethylene or polypropylene. A concern is that it will migrate to the surface of the polymer irrespective of the side of the delivery device being used. One side may therefore be covered with an additional plastic film to limit migration.
Figure 10:
The reaction is likely to proceed through any phenolic OH as this provides acidic hydrogens to open and react with the anhydride functionality
In Figure 11 yet another example is shown of a tethered dye (Amat-Guerri et al., 2003). What this means is that the possibility exists to tether the otherwise toxic dyes to polymers and/or nano particles that should to a large extent control or lower the toxicity.
Fugure 11:
As always, the most cost-effective method must be found. A good reference that may be applied to dyes in this regard is the immobilization of enzymes (Hassan et al., 2019). Some of the mechanisms commonly employed in supporting/immobilizing enzymes that may be extrapolated to dyes are shown below in Figure 12. Methods specifically related to foodstuff dyes are covered in (Özkan & Ersus Bilek, 2014). Some examples are taken directly from this reference in Table 1 below.
Table 1:
Spray drying and emulsion polymerization followed by lyophilization (freeze drying) are established techniques to derive encapsulated proteins and the like. The same should be possible for dyes. Although the dyes would be exposed to some heat during spray drying, the dye itself should never experience a temperature higher than 100 °C. Most dyes seem to have a melting or decomposition temperature above 200 °C.[1] For example, TBPP melts/decomposes above 270 °C. This is also good to know if these dyes were to be exposed to extrusion and injection moulding conditions. In terms of cost, using well-established processes such as spray drying also ensures that the costs remain low (Domínguez-Niño et al., 2018). It is not possible to directly point to a cost in any of the options considered to date but certainly emulsification, crosslinking and spray drying could be regarded as the most cost effective of the means to support dyes intended to detect opioids. The reader is also urged to look at the way peptides are supported and delivered in a way that makes economic sense (Li et al., 2023). Wall materials mentioned in the encapsulation of dyes are dextrin and alginates. By lightly crosslinking these materials they could swell when exposed to water or an aqueous phase such as in the case of an aqueous narcotic mixture.
On the subject of a mixture of narcotics (interfering background color), it may not always be easy to identify the color change brought about by the identifying dye and less so when the dye is encapsulated or otherwise suspended. One way to identify the level of opioid would be to look at fluorescence (Beatty et al., 2019). Every dye will have its own unique fluorescence spectrum fingerprint and this will be influenced by the type of drug it complexes with (Tao et al., 2020) (Mohammadzadeh & Karimzadeh, 2022). Interestingly the use of nanoparticles is discussed as a means of fluorescent detection of opioids. It appears that gold nanoparticles are among the type of nanoparticles used in detecting opioids but certainly not the only ones. Copper sulfide nanoparticles and quantum dots should yield appreciable methods to detect very small amounts of illicit drugs. Indeed, mention is made of detecting 0.01 µg.ml-1 fentanyl. If the ability to detect fentanyl-based drugs is therefore a problem by means of the naked eye, a simple fluorescence/uv-detector should be able to do the job.
Figure 12:
Using the distance (and size) dependent ability of gold nanoparticles to undergo visual and fluorescent colour change, opioids can be determined quantitatively. For a very thorough and well described review on the use of chromophores in the determination of drugs, the reader is referred to the work of Garrido (Garrido et al., 2018). While many of the ideas in this publication may seem new and untested nevertheless the principles on which dye determination of illicit drugs and other detection methods work are described in detail.
Figure 13:
While it is possible to detect very low levels of opioids using dyes it remains a question whether the narcotics can be discerned quantitatively or qualitatively. The linear detection range for many dyes interacting with drugs is not known and will require separate experimental verification.
Figure 14:
Dyes could be considered a cost-effective means to detect opioids. Direct pricing information is scarce and it is only one or two Indian companies that disclose pricing and of course the web based retail store Alibaba. One company that disclose some pricing information for a range of dyes is Loba Chemicals.[1] While the pricing is in Rupees, the dollar translated price makes for a compelling case to consider dyes on a per dollar basis. In addition, when the exports of dyes for the USA is taken into consideration it is indicative of a mature industry where pricing has to a large extent stabilized.
[1] https://www.lobachemie.com/flipbooks/lobachemie-pricelist/Loba-PriceList-2022-23.pdf
[1] https://tradingeconomics.com/united-states/exports-of-chemicals-dyeing
Figure 15:
The history of dyes is a long one with thousands of dyes being developed over a very long period of time (Hagan & Poulin, 2021).
[1] https://www.lobachemie.com/flipbooks/lobachemie-pricelist/Loba-PriceList-2022-23.pdf
Figure 16:
It is also interesting to note that a lot of the dyes being used today was developed many decades ago. Today a large segment of dyes for local consumption and export is controlled by China and eastern countries such as India.
The detection of Debrafenib, an anti-cancer drug is eloquently described using molecular imprinted polymers (MIP) (He et al., 2020). It does not require a large stretch of the imagination to consider MIPs for fentanyl and derivatives derived in the same way.
Figure 17:
The individual molecules and the templates used are shown below in Figure 18.
Figure 18:
The template structures in Figure 18 is similar to some fentanyl derivatives such as those detected using optical fibre grated sensors (Liu et al., 2021). Detection limits using optical fibre grated sensors of a 1000 ng/ml is reported.
Figure 19:
Figure 20:
It appears that MIP technology has been successfully used to detect a fentanyl derivative using long period optical fibre grating (Liu et al., 2021). It is reported that the sensor exhibits a gradual response over increasing concentrations of carboxyl-fentanyl from 0 to 1000 ng/mL with a minimal detected concentration of 50 ng/mL which that corresponds to a wavelength shift of 1.20 ± 0.2 nm. Unfortunately, the LPG technology is designed to detect butyrylfentanyl and it is not reported whether other types of fentanyl derivatives can be detected although the synthesis to other types of fentanyl derivatives should be possible. A patent describes the use of MIP technology to detect, amongst other things drugs (Raphael Levi, Ido Margalit, n.d.). In the patent, the procedure to detect substances can be divided into a number of steps including of which the first step requires fixing the MIP to a solid support. The technology could work in the case of opioid detection but one would have to mix in a number of different imprinted MIPs to differentiate between various fentanyl derivatives. A promising application to detect multiple types of dyes in waste water has been reported (Okutucu et al., 2010). In this publication it is reported that dyes could be detected at microgram level. This same technology could be modified to detect opioids. A patent by Murray (Murray, 2003) uses a molecularly imprinted film containing a lanthanide complex on a substrate such as quartz crystal in order for the molecularly imprinted film to bind to narcotics. This particular patent also describes how to synthesise the MLP and describes a host of narcotics that can be detected including opioids. Detection limits appear to be in the ppm range. The synthesis of MIPs can be achieved in numerous ways. Table 3 details some of the ways to obtain MIPs using mostly cots effective free radical polymerization (Suzaei et al., 2023).
Table 2:
All of the imprinting methods listed in Table 2 have their advantages and disadvantages. For example, emulsion polymerization, while simple and reproducible, leaves copious amounts of surfactants that can interfere with molecular recognition. It is suggested by multiple publications that MIPs are much safer and far less toxic compared to other assay techniques such as dyes. They can be used in vivo and for example, targeted drug delivery is possible using MIPs. It is reported that magnetic nano MIPs do not affect cells (Boitard et al., 2019). Similar studies with mice have shown the effects of nano MIPs to be negligible (Kassem et al., 2022).
The low toxicity bodes well for MIPs as a means of recognizing narcotics such as cocaine in vivo (D’Aurelio et al., 2020). The use of MIPs in solid phase extraction (SPE) for high performance liquid chromatography has been described in detail (Ayerdurai et al., 2022). It is also this technology that requires MIPs to be supported and a number of companies and means of supporting MIPs are discussed. A fact that seems clear at this stage is that MIPs and nano technology cannot readily be separated. There are ways to immobilize and support MIPs in resins which in turn can furnish particles with controlled porosity in the range of 1 µm to 5 µm or larger. In addition, MIPs are already included in thin film design. Overall, the ready availability of MLPs and the ability to detect minute amounts of narcotics make this technology very versatile.
Metal nanoparticles exhibit an effect called the plasmon effect. This effect is due to the interaction of the electrons of nano-metals such as gold with incident light. The electromagnetic field of the incident photons cause a collective oscillation of the free electrons on the nanoparticle’s surface. This collective oscillation generates a resonance condition at a specific wavelength, where the absorption and scattering of light by the nanoparticle are significantly enhanced. Any surface disturbance such an antibody or dye interaction will therefore shift the visible wavelength and result in a color change. Such a change is depicted in Figure 13 where gold particles aggregate with an accompanying color change. Since the antibodies and accompanying nano support require a stable environment, a delivery system is required that is robust and cost effective. Starch can be effective to anchor functionalized gold nanoparticles (Heredia et al., 2020). This may be a way of safely using commercially available nanoparticles.
Heredia et al explore the development of a micro-fluidic device to detect free DNA. The concepts expressed by Heredia can be assimilated into the possible devices discussed so far As mentioned previously regarding MLPs the topic of nanoparticles does not necessarily constitute a separate technology for the detection of drugs but can be combined with both dyes (Jarrahi et al., 2022) (Mohammadzadeh & Karimzadeh, 2022) and MLPs (Ayerdurai et al., 2022). Below is a depiction taken from Jarrahi et al of localizing eosin on a magnetic nano particle. While eosin is immobilized on a magnetic nanoparticle to serve as a catalyst in the case of Jarrahi, the same principle can be applied to tether dyes sensitive to fentanyl derivatives.
Figure 21:
Similarly, the outright utilization of the plasmon effect to improve the detection of azo dyes has been reported (Elhani et al., 2020). The detection of opioids using gold nanoparticles has recently been discussed (Razlansari et al., 2022). The detection limit for fentanyl and other drugs is mentioned Table 3).
Table 3:
Figure 22:
Figure 22 illustrates the use of modified gold nanoparticles to detect opioids. It is suggested that lower detection limits (LODs) of 10-9 M can be reached. In addition, this technique allows for quantitative determination of the amount of opioid. Apart from gold nanoparticles, copper sulfide nanoparticles have shown an LOD of 0.008 µg.ml-1 and a linear detection range of 0.01 – 2.0 µg.ml–
[1] https://www.researchgate.net/publication/317013180_Price_tag_in_nanomaterials/figures?lo=1
Figure 23:
Compared to dyes and MLPs the cost of nano technology is relatively high. In Figure 26, some data is shown for selected nano materials. Gold nanoparticle prices are determined by the size of the nanoparticles and the amounts.[1] Similarly, there seems to be a relationship between the type of nanoparticles and their applications as seen in Figure 27.
[1] https://nn-labs.com/products/gold-nanoparticles-in-water-au
Figure 24:
Although nano technology seems to become well established in developed countries the gap in utilizing this technology elsewhere in the world is still a challenge. More development is required to make it accessible worldwide.
[1] https://www.researchgate.net/publication/317013180_Price_tag_in_nanomaterials/figures?lo=1
[1] https://nn-labs.com/products/gold-nanoparticles-in-water-au
Fentanyl polyclonal antibodies are already available through large chemical companies such as Merck/Sigma-Aldrich. For example, the fentanyl ELISA fentanyl detection assay is sensitive to 0.2 ng.ml-1 fentanyl. Antibodies can be adsorbed onto gold nano-particles (Lew et al., 2021). Gold nanoparticles[1] show an affinity for sulphur in the antibody structure. Depending on the nanoparticle size and distance between adjacent gold nanoparticles, a colour change can be achieved when the antibody binds to a fentanyl molecule.
Enzyme linked immunosorbent assay (ELISA) is probably one of the best known uses of antibodies to detect molecules.[2] While this technique is used to detect pathogens and viruses, recently the detection of opioids has been mentioned (Rashighi & Harris, 2017). Limiting concentrations of about 1 µg.ml-1 could be detected. The DAI Fentanyl ELISA Kit is based upon the competitive binding to antibody of enzyme labeled antigen and unlabeled antigen in proportion to their concentration in the reaction mixture. Typically a 20 μl. aliquot of a diluted unknown specimen is incubated with a 100 μl. dilution of enzyme (Horseradish peroxidase) labeled Fentanyl derivative in micro-plate wells and coated with fixed amounts of high affinity purified polyclonal anti-Fentanyl. The wells are washed thoroughly and a chromogenic substrate added. The color produced is stopped using a dilute acid stop solution and the wells read at 450 nm. The intensity of the color developed is inversely proportional to the concentration of drug in the sample. The technique is sensitive to 0.1 ng/ml Fentanyl. The DAI Fentanyl ELISA Kit avoids extraction of urine or blood sample for measurement. Thirty industrially available immunoassays were evaluated using carfentanyl as base opioid for cross referencing (Wharton et al., 2021).
[1] https://www.americanelements.com/gold-nanoparticle-dispersion-7440-57-5
[2]https://www.thermofisher.com/za/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-elisa.html#10
Figure 25:
Figure 25 shows that although some specific ELISA assays may be very sensitive to specific fentanyls, available immunoassays are not necessarily able to detect all variants of fentanyl. The ELISA-fentanyl assay seems to be the best all round. Further development of opioid detection using Fentanyl Analogue Screening (FAS) kits is ongoing.[1]
The way that dyes and MLPs are packaged for final use may allow for quantitative determination of opioids. If it is correct to assume that a specific amount of dye or MLP react with a specific amount of narcotic then packaging a known amount of dye or MLP should yield some means of discerning the amount of narcotic quantitatively by linking the color obtained before modification directly to a zero setpoint. One way of dealing with the MIP’s or dyes is to consider Solid Phase Extraction (SPE) technology as a blueprint for the future. In Figure 19, MIPs are packaged in resin particles and then put in a cartridge. Instead of several steps, employing such cartridge technology in combination with a syringe could allow direct visual detection of an opioid or by applying uv-light, fluorescence could be used.
[1] https://www.cdc.gov/nceh/dls/erb_fas_kits.html
Figure 26:
Figure 27 suggests one way of attaching a cartridge containing a dye, MLP or any other type of sensing method to a syringe.
Figure 27:
Previously it has been suggested to co-extrude polyethylene or polypropylene or any suitable polymer with starch as a carrier for dyes or nanoparticles (MLPs). The technology to incorporate starch into polymer composites with conventional polymers such as polyethylene and polypropylene is well known (Obasi & Igwe, 2012), (Li Nie et al., 2006). Although starch and modified starch are good all-round methods to contain water-sensitive materials, other types of polymers can also be used for this purpose. These include various natural resins such as xanthan gum, alginates and the like as well as synthetic water-soluble polymers such as polyacrylic acid and polyvinyl pyrrolidone completely biodegradable polymers such as polylactic acid (PLA). By balancing the ratio of reactive species carrier to polyethylene, polypropylene or PLA for example, enough surface area may be exposed of the carrier to wet the reactive species and lead to some means of detection such as a color change. In this case the reactive specie may be a dye, antibody or any other suitable detection system already discussed that is specific for opioids while the carrier may be the aforementioned water-soluble or water swellable polymer.
Figure 28:
Suggested delivery device and method of incorporating a reactive specie such as a drug either as a salt or supported/immobilized in a carrier such as starch. The water-soluble salts of dyes or a carrier such as starch may then allow the syringe wall to become porous and facilitate the coloration of the aqueous phase either by coloring the liquid itself or the walls of the syringe. It also becomes clear that MLPs supported in a resin may be incorporated in this way.
Figure 29:
Design of delivery system may incorporate the release of an agent that detects an opioid and turns the aqueous phase a certain color or the walls of the delivery device may come into contact with the aqueous phase and upon contact may activate a suitable detection agent that may change color.
If the dyes or MIPs can be supported and contained in suitable resins, thin film technology could be considered to deposit films containing dyes, MLPs or nano particles or any combination of these on the inside of the syringe or delivery system.[1] Specifically, if a polymeric thin film with controlled permeability is deposited on the inside of a syringe, access to the dye or other type of detection system should be possible.
[1] https://vaccoat.com/blog/thin-film-and-thin-films-types/ , https://korvustech.com/thin-film-applications/
[1] https://www.intechopen.com/chapters/69080
Figure 30:
In Figure 30 some of the concepts associated with MLPs also seem to be associated with thin films such as the loading of active species and targeted delivery. This has been considered in the patent by Murray (Murray, 2003) previously described. It stands to reason that thin film technology can be used to immobilize dyes, dye containing vehicles, MLPs and nano particles.
The fatal level of opioids in humans differ between gender and there is also an age difference (Shigeev, 2007). For the age group above 25, the mean acute opioid toxicity level is 1.5 µg.ml-1 while the lethal range is from 0.1 to 4.1 µg.ml-1. Females above the age of 25 are reported to be more resistant to opioid poisoning and the lethal dose is around 0.98 µg.ml-1, while it is 0.78 µg.ml-1 for males. In the age group below 25 the lethal dose becomes lower for both males and females at 0.39 µg.ml-1. While most dyes will probably indicate the presence of opioids < 4 µg.ml-1, only those that can give a linear response from 0.1 to 5 µg.ml-1 will probably be able to indicate a fatal dosage level.
WO2020/014174 proposes a number of dyes for the detection of opioids (JOY et al., n.d.). These dyes are considered along with previously mentioned dyes (Tsunghsueh WU, n.d.) in terms of thermal stability (melting point), LD50 and detection limits (LDL).
[1] https://www.intechopen.com/chapters/69080
Table 4:
| Dye | Mp (°C) | LDL (µg.ml-1) | LD50 (mg.kg-1) |
| Tetrabromophenolphthalein | 210 | 5 – 50 | 300, dog, intravenous |
| Eosin Y | 300 | 0.1 – 10 | 2344, mouse, oral |
| Bromocresol green | NA | 5 – 40 | 900000 (0.04%), oral, rat |
| Nile Red | 203 – 205 | NA | NA |
| Malachite green | 158 – 160 | NA | NA |
| Methyl orange | >300 | 0.04 – 0.12 | 60, oral, rat |
| Cresol Red | 290 (decomp) | NA | NA |
| Thymol blue | 221 – 224 | 5 – 30 | 10740, oral, rat |
| Azobenzole | 69 | NA | NA |
| m-Cresol purple | >250 | NA | NA |
| Bromochlorophenol blue | 230 | NA | NA |
| Bromophenol blue | 273 | 7 – 660 | >90, (0.04%), ml/kg, oral, rat |
| Congo red | >360 | NA | 143, human |
| 1-Naphtholphthalein | 238 – 240 | NA | NA |
| Bromothymol blue | 202 | 2 – 10 | NA |
The human eye is said to be able to detect color changes down to 100 µM. It is not clear at this stage if this is enough to detect very small changes in color so it is proposed that an alternative strategy be considered. This strategy is dye replacement. A very good visual representation of the technology is given below.[1]
[1] Colorimetric Molecularly Imprinted Polymer Sensor Array using Dye Displacement | Journal of the American Chemical Society (acs.org)
Figure 31:
So while dye-based colorimetry on its own may be used to detect opioids based on the multiple references on the subject, it appears that combining the technology with MIPs greatly improves the application of the technology and takes it from the ability to indicate whether an opioid is present (yes/no) to being able to get an idea of the concentration of the opioid by calibrating the color change (Lowdon, Eersels, et al., 2020).
Figure 31:
Dye displacement technology has for example been developed especially for the detection of nitrogen containing ring structures including for opioids (Redeker et al., 2019). By combining known selective dyes as disclosed so far with MIP technology it should be possible to design very sensitive, cost effective biomedical assays for the detection of opioids. Of course, MIP technology is ideally suited for dye replacement technology but it is also associated with nano technology such as quantum dots (Shamirian et al., 2015), (Deep Sekhar Biswas, Paraskevi Gaki, Elisabete Cruz Da Silva, Antoine Combes, Andreas Reisch, Pascal Didier, 2023).
[1] Förster resonance energy transfer (FRET)
Figure 32:
The use of dye displacement technology also extends to the use of other nano particles including the now familiar gold nanoparticle technology (Wu et al., 2019), (Magdalena Swierczewskaa, Seulki Lee 2011), (Lowdon, Diliën, et al., 2020)
[1] Förster resonance energy transfer (FRET)
With regard to MIPs, stability and re-use has been extensively studied (Meléndez-Marmolejo et al., 2022), (Kupai et al., 2017). Specifically it has been found that MIPs in most cases are more resistant to degradation when they are crosslinked and it has been shown that MIPs can be re-used many times. These observations are a function of the type of analyte and dye and although the preceding statements are general, there may be exceptions. Figure 33 illustrates these findings by means of thermo gravimetric analysis (TGA). The crosslinked decomposition temperature is significantly higher at 345 °C. The decomposition of acrylic polymers occur at the so-called ceiling temperature where polymer chains start to unzip into their original constituent monomers.
Figure 33:
Nano technology has been described as interwoven with dye technology and MIPs. Consequently the stability of nanoparticles is of importance (Andrievski, 2014),(Phan & Haes, 2019).
Figure 34:
With regard to nanoparticles, many variables will influence their stability. Increases in temperature can drive agglomeration both in the liquid state (dispersions) as well as in the solid state. If particles are not stabilized, for example by anchoring metal nanoparticles in a more stable substrate, particle size may increase and particle morphology may be altered. If there is a large particle size distribution, thermodynamic forces may drive agglomeration. pH may influence the stability of suspensions of nanoparticles depending on the surface charge of the particles. In addition, oxidation of the particle surface can change the particle morphology and/or lead to loss of functionality.
Visual changes associated with colorimetric essays to detect analyte molecules can be simple yes/no type of color changes as the color changes in Figure 35 portrays (Alberti et al., 2020).
Figure 35:
More useful information that involve a transient, concentration dependent color change can be accomplished using dye displacement technology combined with MIP technology. An example of this is the indication of trace amounts of bisphenol A using a polyacetylene MIP (Shin & Shin, 2020).
Note the changes in absorption spectra that accompanies the change in bisphenol A concentration.
Figure 36:
Figure 37:
Figure 37 finally shows the change in color of MIPs when the acrylamide group interacts with TNT molecules. Probably the best indication of color changes is provided by (Ayerdurai et al., 2022). The reader is encouraged to consult this comprehensive description on the use of MLPs and other technologies. Principally it illustrates very unambiguously that color changes due to the detection of analytes can be tied directly to the concentration of the detected analyte.
Note the accompanying color changes that allow visual concentration dependent analysis.
Figure 38:
In this document (Ayerdurai et al) several patents have been revealed that tie certain dyes directly to opioids. As such they are known to at least provide a yes/no indication of a specific type of opioid. Specific instances of using these dyes in dye displacement technology combined either with MIPs or nanoparticles will probably be forthcoming but it still means that some work is required to test for the best type of dye or dye combinations (Raghu & Basavaiah, 2012).
Several technologies have been discussed to detect opioids. These include the use of dyes, MIPs, nanoparticles and antibodies. Currently there is great deal of overlap between these technologies and it appears that combining certain technologies for example where dye displacement technology is concerned, detection can be improved in this case using a dye and MIP . Each technology has its advantages and disadvantages. In the case of dyes, there are many dyes that can discern between different types of drugs including opioids but the toxicity of dyes makes it difficult to use in direct patient contact applications. Furthermore, dyes, while able to discern between different kinds of drugs and opioid derivatives, usually can only indicate the presence or not of a drug and although the perception may be somewhat distorted it appears that obtaining an indication of the concentration of a drug is more challenging.
MIPs can be used to interact with different types of drugs by using the drugs as a template during the manufacturing of the MIPs. Manufacturing can be achieved in various ways but it is important that the additives or processes used to manufacture very small MIP particles do not interfere with the ability of the MIP to complex with the correct drug or analyte molecule. MIPs on their own can complex with drugs such as opioids and extract them from a mobile phase. This technology is used in solid phase extraction techniques to obtain samples for analysis using GC, HPLC or many other types of analytic methods. While this is great for SPE applications it does nothing to help with detecting drugs visually. However, by loading MIPs with dyes upfront and then exposing the MIPs to drugs allow for a competition of the drug molecules to displace the dye molecules thereby causing either a fainter color or a different color to form. This combination of technologies is probably the most cost effective way to apply them.
Nano technology has been shown to be effective to discern between drugs and other molecules. To some extent, a continuum in color change (for example blue to red) can take place as an indicator of the concentration of the analyte as in the case of gold nanoparticles where the characteristic colors associated with the plasmon effect can be exploited. As with the dyes, both dyes and MIP technology can be combined with nano technology as well as bio-assays involving enzymes and antibodies. An example of dye displacement technology in the case of nanoparticles is the displacement of dyes from the surfaces of nano dots. This results in the nano dot “switching on” and becoming fluorescent when the dye is displaced or removed through preferential complexation with another molecule such as an opioid.
Antibody assays such as the ELISA is widely used and can, through a color change indicate the presence of drugs. However, as shown in Figure 25 one size does not necessarily fit all. The antibodies used in a specific ELISA kit have to be tailored to detect/interact with a specific opioid which may require developing new or improving existing kits as new opioid drugs are discovered. At least it seems they can give an indication of the presence of some opioids.
If all the technologies are assessed, it appears that the dye displacement technology using a combination of MIPs and dyes could be the most cost effective options for now. However, new technology such as the use of thin films should also be considered. If the same ability to displace dyes for example can be designed into a thin film it might become more cost effective and more efficient at detecting opioids.
Alberti, G., Zanoni, C., Magnaghi, L. R., & Biesuz, R. (2020). Disposable and low-cost colorimetric sensors for environmental analysis. International Journal of Environmental Research and Public Health, 17(22), 1–23. https://doi.org/10.3390/ijerph17228331
Amat-Guerri, F., Liras, M., Luisa Carrascoso, M., & Sastre, R. (2003). Methacrylate-tethered Analogs of the Laser Dye PM567—Synthesis, Copolymerization with Methyl Methacrylate and Photostability of the Copolymers¶. Photochemistry and Photobiology, 77(6), 577. https://doi.org/10.1562/0031-8655(2003)077<0577:maotld>2.0.co;2
Amisar, S. (2011). (12) Patent Application Publication (10) Pub. No.: US 2011/0117664 A1. 1(19).
Andrievski, R. A. (2014). Review of thermal stability of nanomaterials. Journal of Materials Science, 49(4), 1449–1460. https://doi.org/10.1007/s10853-013-7836-1
Ayerdurai, V., Lach, P., Lis-Cieplak, A., Cieplak, M., Kutner, W., & Sharma, P. S. (2022). An advantageous application of molecularly imprinted polymers in food processing and quality control. Critical Reviews in Food Science and Nutrition. https://doi.org/10.1080/10408398.2022.2132208
Beatty, M. A., Selinger, A. J., Li, Y., & Hof, F. (2019). Parallel Synthesis and Screening of Supramolecular Chemosensors That Achieve Fluorescent Turn-on Detection of Drugs in Saliva. Journal of the American Chemical Society, 141(42), 16763–16771. https://doi.org/10.1021/jacs.9b07073
Boitard, C., Curcio, A., Rollet, A. L., Wilhelm, C., Ménager, C., & Griffete, N. (2019). Biological Fate of Magnetic Protein-Specific Molecularly Imprinted Polymers: Toxicity and Degradation. ACS Applied Materials and Interfaces, 11(39), 35556–35565. https://doi.org/10.1021/acsami.9b11717
Bräuer, B., Unger, C., Werner, M., & Lieberzeit, P. A. (2021). Biomimetic sensors to detect bioanalytes in real-life samples using molecularly imprinted polymers: A review. Sensors, 21(16). https://doi.org/10.3390/s21165550
D’Aurelio, R., Chianella, I., Goode, J. A., & Tothill, I. E. (2020). Molecularly imprinted nanoparticles based sensor for cocaine detection. Biosensors, 10(3), 1–13. https://doi.org/10.3390/bios10030022
Deep Sekhar Biswas, Paraskevi Gaki, Elisabete Cruz Da Silva, Antoine Combes, Andreas Reisch, Pascal Didier, and A. S. K. (2023). Long‐Range Energy Transfer between Dye‐Loaded Nanoparticles Observation and Amplified.pdf. Advanced Materials Research, 2301402, 1–13.
Domínguez-Niño, A., Cantú-Lozano, D., Ragazzo-Sanchez, J. A., Andrade-González, I., & Luna-Solano, G. (2018). Energy requirements and production cost of the spray drying process of cheese whey. Drying Technology, 36(5), 597–608. https://doi.org/10.1080/07373937.2017.1350863
Elhani, S., Ishitobi, H., Inouye, Y., Ono, A., Hayashi, S., & Sekkat, Z. (2020). Surface Enhanced Visible Absorption of Dye Molecules in the Near-Field of Gold Nanoparticles. Scientific Reports, 10(1), 3–5. https://doi.org/10.1038/s41598-020-60839-0
Garrido, E., Pla, L., Lozano-Torres, B., El Sayed, S., Martínez-Máñez, R., & Sancenón, F. (2018). Chromogenic and Fluorogenic Probes for the Detection of Illicit Drugs. ChemistryOpen, 7(5), 401–428. https://doi.org/10.1002/open.201800034
Hagan, E., & Poulin, J. (2021). Statistics of the early synthetic dye industry. Heritage Science, 9(1), 1–14. https://doi.org/10.1186/s40494-021-00493-5
Hassan, M. E., Yang, Q., Xiao, Z., Liu, L., Wang, N., Cui, X., & Yang, L. (2019). Impact of immobilization technology in industrial and pharmaceutical applications. 3 Biotech, 9(12), 1–16. https://doi.org/10.1007/s13205-019-1969-0
He, C., Ledezma, U. H., Gurnani, P., Albelha, T., Thurecht, K. J., Correia, R., Morgan, S. P., Patel, P., Alexander, C., & Korposh, S. (2020). Surface polymer imprinted optical fibre sensor for dose detection of dabrafenib. Analyst, 145(13), 4504–4511. https://doi.org/10.1039/d0an00434k
Heredia, F. L., Resto, P. J., & Parés-Matos, E. I. (2020). Fast Adhesion of Gold Nanoparticles (AuNPs) to a Surface Using Starch Hydrogels for Characterization of Biomolecules in Biosensor Applications. Biosensors, 10(8). https://doi.org/10.3390/bios10080099
J Kangas, M. (2017). A New Possible Alternative Colorimetric Drug Detection Test for Fentanyl. Organic & Medicinal Chemistry International Journal, 4(4), 4–6. https://doi.org/10.19080/omcij.2017.05.555645
Jarrahi, M., Maleki, B., & Tayebee, R. (2022). Magnetic nanoparticle-supported eosin Y salt [SB-DABCO@eosin] as an efficient heterogeneous photocatalyst for the multi-component synthesis of chromeno[4,3-b]chromene in the presence of visible light. RSC Advances, 12(45), 28886–28901. https://doi.org/10.1039/d2ra05122b
JOY, A., JAIN, T., NUN, N., NARAYANAN, A., & CATANIA, R. (n.d.). WO2020/014174 METHODS AND DEVICES THAT CHANGE COLOR TO INDICATE THE PRESENCE OF OPIOIDS AND OTHER NARCOTICS.
Kassem, S., Piletsky, S. S., Yesilkaya, H., Gazioglu, O., Habtom, M., Canfarotta, F., Piletska, E., Spivey, A. C., Aboagye, E. O., & Piletsky, S. A. (2022). Assessing the In Vivo Biocompatibility of Molecularly Imprinted Polymer Nanoparticles. Polymers, 14(21). https://doi.org/10.3390/polym14214582
Klemola, K. (2008). Textile toxicity: cytotoxicity and spermatozoa motility inhibition resulting from reactive dyes and dyed fabrics. In Kuopio University Publications C. Natural and Environmental Sciences (Vol. 241, Issue October).
Kupai, J., Razali, M., Buyuktiryaki, S., Kecili, R., & Szekely, G. (2017). Long-term stability and reusability of molecularly imprinted polymers. Polymer Chemistry, 8(4), 666–673. https://doi.org/10.1039/c6py01853j
Lew, T. T. S., Aung, K. M. M., Ow, S. Y., Amrun, S. N., Sutarlie, L., Ng, L. F. P., & Su, X. (2021). Epitope-Functionalized Gold Nanoparticles for Rapid and Selective Detection of SARS-CoV-2 IgG Antibodies. ACS Nano, 15(7), 12286–12297. https://doi.org/10.1021/acsnano.1c04091
Li, M., Guo, Q., Lin, Y., Bao, H., & Miao, S. (2023). Recent Progress in Microencapsulation of Active Peptides—Wall Material, Preparation, and Application: A Review. Foods, 12(4). https://doi.org/10.3390/foods12040896
Li Nie, P., Bassi, Atchison, C. C. M., & Michael Douglas Parker, Lawrence, K. (2006). US 2006 / 0210960 A1 STARCH-PLASTIC COMPOSITE RESINS AND PROFILES MADE BY EXTRUSION (Patent No. 2006/0194902 A1).
Liu, L. L., Grillo, F., Canfarotta, F., Whitcombe, M., Morgan, S. P., Piletsky, S., Correia, R., He, C. Y., Norris, A., & Korposh, S. (2021). Carboxyl-fentanyl detection using optical fibre grating-based sensors functionalised with molecularly imprinted nanoparticles. Biosensors and Bioelectronics, 177(July 2020), 113002. https://doi.org/10.1016/j.bios.2021.113002
Loane, C. (2020). WO2021081063A1 FENTANYL ANALOGUE DETECTION METHODS AND KITS THEREOF (Patent No. PCT/US2020/056613).
Lowdon, J. W., Diliën, H., Singla, P., Peeters, M., Cleij, T. J., van Grinsven, B., & Eersels, K. (2020). MIPs for commercial application in low-cost sensors and assays – An overview of the current status quo. Sensors and Actuators, B: Chemical, 325(September). https://doi.org/10.1016/j.snb.2020.128973
Lowdon, J. W., Eersels, K., Arreguin-Campos, R., Caldara, M., Heidt, B., Rogosic, R., Jimenez-Monroy, K. L., Cleij, T. J., Diliën, H., & van Grinsven, B. (2020). A Molecularly Imprinted Polymer-Based Dye Displacement Assay for the Rapid Visual Detection of Amphetamine in Urine. Molecules, 25(22), 1–16. https://doi.org/10.3390/MOLECULES25225222
Magdalena Swierczewskaa, b, Seulki Lee*, a, and X. C. (2011). The design and application of fluorophore–gold nanoparticle activatable probes. Phys Chem Chem Phys, 13(21), 9929–9941. https://doi.org/10.1039/c0cp02967j.The
Meléndez-Marmolejo, J., Díaz de León-Martínez, L., Galván-Romero, V., Villarreal-Lucio, S., Ocampo-Pérez, R., Medellín-Castillo, N. A., Padilla-Ortega, E., Rodríguez-Torres, I., & Flores-Ramírez, R. (2022). Design and application of molecularly imprinted polymers for adsorption and environmental assessment of anti-inflammatory drugs in wastewater samples. Environmental Science and Pollution Research, 29(30), 45885–45902. https://doi.org/10.1007/s11356-022-19130-0
Mohammadzadeh, S., & Karimzadeh, Z. (2022). Development of a New Method Based on Copper Sulfide Nanoparticles for the Determination of Fentanyl in Biological Samples. ImmunoAnalysis, 2(1), 5. https://doi.org/10.34172/ia.2022.05
Murray, G. M. (2003). US 2003/0003587 A1 MOLECULARLY IMPRINTED POLYMER BASED SENSORS FOR THE DETECTION OF NARCOTICS.
Obasi, H. C., & Igwe, I. O. (2012). Cassava starch-mixed polypropylene bidegradable polymer: Preparation, characterization, and effect biodegradable products on growth of plants. International Journal of Science and Research, 3(7), 802–807.
Okutucu, B., Akkaya, A., & Pazarlioglu, N. K. (2010). Molecularly imprinted polymers for some reactive dyes. Preparative Biochemistry and Biotechnology, 40(4), 366–376. https://doi.org/10.1080/10826068.2010.525428
Özkan, G., & Ersus Bilek, S. (2014). Microencapsulation of Natural Food Colourants. International Journal of Nutrition and Food Sciences, 3(3), 145. https://doi.org/10.11648/j.ijnfs.20140303.13
Phan, H. T., & Haes, A. J. (2019). What Does Nanoparticle Stability Mean? HHS Public Access. J Phys Chem C Nanomater Interfaces, 123(27), 16495–16507. https://doi.org/10.1021/acs.jpcc.9b00913.What
Raghu, M. S., & Basavaiah, K. (2012). Rapid and sensitive extraction-free spectrophotometric methods for the determination of pheniramine maléate using three sulphonthalein dyes. Proceedings of the National Academy of Sciences India Section A – Physical Sciences, 82(3), 187–196. https://doi.org/10.1007/s40010-012-0019-7
Rahman, H. (2017). Utilization of Eosin Dye As an Ion Pairing Agent for Determination of Pharmaceuticals: a Brief Review. International Journal of Pharmacy and Pharmaceutical Sciences, 9(12), 1. https://doi.org/10.22159/ijpps.2017v9i12.21220
Raphael Levi, Ido Margalit, Y. D. (n.d.). EP2041567A2 Rapid diagnostic devices based on molecular imprinted polymers.
Rashighi, M., & Harris, J. E. (2017). Detection of opioids in umbilical cord lysates: an antibody- based rapid screening approach. Physiology & Behavior, 176(3), 139–148. https://doi.org/10.1053/j.gastro.2016.08.014.CagY
Razlansari, M., Ulucan-Karnak, F., Kahrizi, M., Mirinejad, S., Sargazi, S., Mishra, S., Rahdar, A., & Díez-Pascual, A. M. (2022). Nanobiosensors for detection of opioids: A review of latest advancements. European Journal of Pharmaceutics and Biopharmaceutics, 179(September), 79–94. https://doi.org/10.1016/j.ejpb.2022.08.017
Redeker, S., Grinsven, V., Chemical, A. B., Lowdon, J. W., Eersels, K., Rogosic, R., Heidt, B., & Diliën, H. (2019). Thomas ( 2019 ) Substrate displacement colorimetry for the detection of di- Downloaded from : https://e-space.mmu.ac.uk/621849/ Version : Accepted Version Publisher : Elsevier Usage rights : Creative Commons : Attribution-Noncommercial-No Deriva- Substrat. 137–144.
Shamirian, A., Ghai, A., & Snee, P. T. (2015). QD-based FRET probes at a glance. Sensors (Switzerland), 15(6), 13028–13051. https://doi.org/10.3390/s150613028
Shigeev, S. (2007). Severity of opiate intoxication to gender and age. Soudní Lékarství / Casopis Sekce Soudního Lékarstvi Cs. Lékarské Spolecnosti J. Ev. Purkyne, 52(1), 21–24.
Shin, M. J., & Shin, J. S. (2020). A molecularly imprinted polymer undergoing a color change depending on the concentration of bisphenol A. Microchimica Acta, 187(1). https://doi.org/10.1007/s00604-019-4050-0
Smith McWilliams, A. D., Ergülen, S., Ogle, M. M., De Los Reyes, C. A., Pasquali, M., & Martí, A. A. (2020). Fluorescent surfactants from common dyes – Rhodamine B and Eosin y. Pure and Applied Chemistry, 92(2), 265–274. https://doi.org/10.1515/pac-2019-0219
Suzaei, F. M., Daryanavard, S. M., Abdel-Rehim, A., Bassyouni, F., & Abdel-Rehim, M. (2023). Recent molecularly imprinted polymers applications in bioanalysis. In Chemical Papers (Vol. 77, Issue 2). Versita. https://doi.org/10.1007/s11696-022-02488-3
Tao, X., Peng, Y., & Liu, J. (2020). Nanomaterial-based fluorescent biosensors for veterinary drug detection in foods. Journal of Food and Drug Analysis, 28(4), 575–594. https://doi.org/10.38212/2224-6614.1267
Tsunghsueh WU, C. C. (n.d.). WO2015006720A1 Colorimetric method and kit to detect illicit drugs.
Wharton, R. E., Casbohm, J., Hoffmaster, R., Brewer, B. N., Finn, M. G., & Johnson, R. C. (2021). Detection of 30 Fentanyl Analogs by Commercial Immunoassay Kits. Journal of Analytical Toxicology, 45(2), 111–116. https://doi.org/10.1093/jat/bkaa181
Wu, Y., Ali, M. R. K., Chen, K., Fang, N., & El-Sayed, M. A. (2019). Gold nanoparticles in biological optical imaging. Nano Today, 24, 120–140. https://doi.org/10.1016/j.nantod.2018.12.006
Yılmaz, E., Garipcan, B., Patra, H. K., & Uzun, L. (2017). Molecular imprinting applications in forensic science. Sensors (Switzerland), 17(4), 1–24. https://doi.org/10.3390/s17040691
