NANOTECHNOLOGY IN

FORENSIC TOXICOLOGY
How the little guys are giving us the big picture
Bhavika Kapadia, 2023

University of Florida, College of Pharmacy

Master’s in Forensic Science
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I. Introduction
Nanotechnologies are rapidly expanding across scientific fields and have been applied to fields
like biomedical science, physics, and engineering. Its contribution to forensic science has
brought innovations and evolution in devices and analyzers in the lab and on the field. Forensic
science is the application of scientific principles and technology to the legal process, while
forensic toxicology is a subset of forensic science where toxicology and other disciplines like
analytical chemistry, pharmacology, and clinical chemistry aids in medico-legal cases. This
science can be used in cases of workplace drug screening or in sports for doping [13, 14]. It’s
also used in drug-facilitated crime investigations or crime scenes where there is suspected illicit
drug use. What encompasses all of these is the detection of the drug itself. The substance’s
presence needs to be confirmed and then identified in a manner that is scientifically sound and
will be able to be used in a courtroom as evidence.
Since the 19th century, forensic toxicology has improved dramatically. It started with an
investigation into toxins and observations of how the body reacted. Then devices and machinery
like the spectrophotometer were used as a way to quantize these toxins and analyze them. Over
the decades, the technology to analyze these substances has improved immensely, with
nanoparticles and nanotechnology leading the new wave of improvement. Nanoparticles are
simply materials that are so small that they measure in the nano scale, which is 1 billion times
smaller than 1 meter. When materials are reduced to such a small scale, two properties appear:
increased surface area and quantum physics. This causes the small particles to behave in ways
that are different than if it was larger (bulk) material [3,5]. Forensic toxicologists take advantage
of these new properties to aid in detection, analysis, and identification of toxic substances.
The current methods of toxicological analysis include mass spectrophotometry, liquid
chromatography, gas chromatography, immunoassays, chemiluminescence and more as
confirmatory testing. While those methods are great, they come with expensive equipment,
require a substantial amount of knowledge, training, and sample preparation; it is time-
consuming, not as widely available, and has room to be more accurate and reliable. The main use
of going smaller is detecting smaller. Trace evidence will become easier to detect because of the
increased sensitivity with the incorporation of nanoparticles. This can come in the form of new
types of sample preparation, new probes, and new sensors. This is especially helpful in evidence
specimens of complex or mixed media, like hair, urine, and blood. Crime scenes are rarely in
pristine condition for finding toxicological evidence in a straightforward manner. Thus,
pinpointing the target molecules by purifying, cleaning, and polishing are required for the
toxicological characterization and identification of substances.
The well-known methods of characterization with equipment like HPLC and GCMS are being
used in forensic laboratories all over the world. At the same time, improvements in these
methods have come along in the past decade or so. The following are explanations of just a
handful of the diverse ways nanoparticles have been experimented with in hopes of being applied
as potentially routine protocols in a forensic toxicology setting.
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A. Morphine and KH-570

HPLC is high performance liquid chromatography and has been used in forensic labs across the
globe. With nanotechnology, the sample preparation is what is improved. It has been established
that little sample volume is required for HPLC and is useful for non-trace evidence. However,
there are detection limits, as with any analytical instrument. Boojaria et al. used HPLC with a
photodiode array detector for their experimentation of using “silane modified magnetic
nanoparticles as a novel adsorbent for determination of morphine at trace levels in human hair
samples” [6]. Here, magnetic nanoparticles, Fe3O4, were synthesized via co-precipitation using
“iron oxides from aqueous Fe2+/Fe3+ salt solutions in the presence of a base with a high yield and
a relatively narrow size distribution” [6]. The magnetic nanoparticles were then coupled with 3-
(trimethoxysilyl) propyl methacrylate, also known as KH-570. The purpose of this coupling was
to reduce the aggregation of the Fe3O4 nanoparticles and to function as an adhesion promoter for
the analyte [11]. Now as a novel adsorbent, silane coupled Fe3O4 was used to preconcentrate the
samples of morphine after solid phase extraction from hair. Without preconcentration, the
extracted morphine might have been too diluted and below the limit of detection. The magnetic
property gave ease to the separation of analyte adsorbed onto the particles from the matrix, and
again during elution. They managed to preconcentrate to a factor of 208.69, with a relative
recovery of 87.62% after HPLC. In addition to a high yield, the retention time was less than 3
minutes, showcasing the speed of analysis with appropriate detection.
B. THC and Polydopamine
Cannabinoids have been a popular drug and have people on the fence of whether it should be a
schedule two drug or not. However, it is undeniable that natural and synthetic analogs of
cannabis can be purposed for more harm than health. Because of its widespread use,
understanding a population’s usage prompts analysis of wastewater [7]. Chen et al. have
proposed a method of determining the commonly observed trace amounts of psychoactive drugs
in wastewater using functionalized magnetic polydopamine nanoparticles. It is known that the
active component of cannabis is THC, and its metabolite is THC-COOH, which is excreted in
urine. Synthetic cannabinoids however do not have the same metabolite, even amongst
themselves. Because of the diverse metabolites, it is difficult to target one metabolite or one
characteristic of them to use as a general detector in wastewater. Generally, liquid or gas
chromatography used in tandem with mass spectroscopy is employed for analysis of wastewater.
The sample preparation is costly and labor intensive and the most commonly used technique is
solid phase extraction. An improved version is the use of micro solid phase extraction with
magnetic nanoparticles as adsorbents, as discussed earlier. Chen et al. have added polydopamine
(PDA) to the Fe3O4 nanoparticles as a “means of increasing the hydrophilicity and stability of
magnetic nanoparticles” [7]. In addition, a polymer, poly (methacrylic acid–co-ethylene glycol
dimethacrylate (abbreviated to poly (MAA-co-EGDMA)), provides helpful properties such as
being able to have “both hydrophobic and electrostatic interactions with basic compounds
including most illicit drugs” [7]. Fe3O4 nanoparticles functionalized by this polymer and coupled
with PDA were synthesized, for the first time, and used as a novel method for detecting cannabis
metabolites in wastewater. First, the characterization of this nanoparticle was conducted with
different analytical instrumentation. Once the nanoparticles are synthesized, a transmission
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electron microscope is used to visualize the shape and formation of the nanoparticles. To ensure
structurally that the coupling occurred, fourier-transfer infrared spectroscopy was performed, and
all indicative peaks showed on the spectra. Finally, X-ray photoelectron spectroscopy (XPS) was
done to confirm that the environment of the carbon and oxygen atoms was appropriate by
measuring the binding energies of the surface composition. Afterwards, the micro solid phase
extraction was done with the nanoparticles and, when compared to other studies that did not use
nanoparticles, it is clear that this method is quicker (20 minutes versus 30-45 minutes), more
sensitive (LOQ 0.1-1.0 versus above 0.4), and more efficient by using less absorbent amount
(mg) per sample volume (ml) (100 ml/10 mg versus 50 ml/60 mg, 20 ml/150 mg or 100 ml/150
mg). Although this paper was for wastewater, the methods proposed can easily be applied in a
forensic toxicology setting for urine samples collected for drug screening.
C. Methamphetamine & MDMA with Gold Nanorods and D-SERS
Raman spectroscopy is based on the scattering of light from a laser (the incident light) after
interacting with a sample. The laser makes the electrons in the sample go to an excited state, thus
producing photon emission. The difference in energy levels of the molecular bonds from the
incident light and the scattered light is what is detected and is unique to every molecule [9].
Although helpful in the identification of samples, its low sensitivity and high cost makes it
cumbersome even with portable versions now available. Surface enhanced Raman Spectroscopy
(SERS) has some solutions. SERS makes use of colloids, which are mixtures with nanoparticles
less than 1000 nm in size being evenly distributed in solution [8]. The typical nanoparticles used
are metals like silver, copper, and gold, which enhance the sensitivity and electromagnetic fields
by 10,000-fold, thus proving useful in ultra trace evidence analysis. Though SERS has its
advantages, it still has trouble with analysis through complex media. Dong et al. have developed
a new novel, rapid method of drug detection using dynamic surface-enhanced Raman
spectroscopy (D-SERS), with methamphetamine and MDMA as their target analyte. In
combination with gold nanorods as the colloid, a portable Raman spectrometer, and a
sophisticated algorithm for classification called support vector machines (SVM), signal
detections now have “gigantic enhancements” [10] and high reproducibility. In understanding the
role of nanoparticles in the enhancing power of D-SERS, state translation nanoparticles from a
wet droplet of colloid to a dry state is what drives the distinct geometry of with less gaps (fully
coated surface) and sharp Raman peaks. As the solvent of the colloid dries, the capillary actions
and surface tension help to create a fairly even layer on the substrate (silicon or glass wafer) that
can be placed into the Raman spectrometer. During the formation of this uniform setting, a
beneficial three-phase solid/liquid/air interface is created, which enhances Raman “of at least 2
orders of magnitude larger than that of dried substrates but also provides reproducible and stable
SERS signals for at least 100 s” [10]. Further enhancement comes from the formation of the
nanoparticles coupled with mPEG-SH, a polymer that prevents aggregation and induces self-
assembly of the nanorods alongside the capillary action during solvent evaporation. For the
actual sample analysis of methamphetamine in urine, Dong et al. basically skipped the sample
preparation step typically seen in other analytical methods by placing the drop of the urine-
nanoparticle mixture directly onto a glass slide (Figure 1) and observed the evaporation and real-
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time continuous recording of the Raman spectra.

Figure 1 Diagram of D-SERS and SVM procedure

300 urine samples from Science Island Hospital, Hefei, China were simulated for the comparison
of conventional SERS (C-SERS) and D-SERS. When the C-SERS results were compared to the
results of D-SERS, D-SERS had about 11% more specificity, sensitivity, and accuracy each.
When combined with SVM for classification, and thus identification, methamphetamine at levels
as low as 0.1 ppm were able to be detected and identified. The detection time was about 2
minutes, and the sample volume could be as low as 2 microliters. Similar experiments were done
with MDMA, and similar specificity, sensitivity, and accuracy was observed. To further validate
this new method, real urine samples from the Institute of Pharmacology and Toxicology,
Academy of Military Medical Sciences, Beijing, China, were analyzed. The speed, efficiency,
accuracy, and sensitivity all showed how simple and quick this method was. Though
nanoparticles had been used in C-SERS, the development of D-SERS with nanoparticles coupled
to the polymer shows how helpful and versatile nanoparticles are to the evolution of drug
analysis.
D. Caffeine and Silver Nitrate
Alharbi et al. also used conventional SERS in their analysis of the 2 major metabolites of
caffeine, theobromine and paraxanthine. The differences between this study and the previous one
is that 2 analytes are attempted to be analyzed simultaneously and different nanoparticles needed
to be characterized before settling on the best singular one. Caffeine is metabolized into 3
metabolites: paraxanthine (80%), theobromine (12 %), and theophylline (7%). Theophylline was
disregarded in this study since it was deemed too difficult to detect. (Maybe with D-SERS, it
would have been possible). Next came the generation of colloids, of which there were three:
silver citrate, silver borohydride, and gold citrate. While all three were assessed by UV-visible
spectroscopy for proper formation, when assessed for reproducibility, only silver citrate showed
good results. This means that when the tertiary mixture was to be analyzed, no one signal would
overpower the other two. With silver borohydride colloids, theobromine dominated the SERS
spectra, and with gold citrate, paraxanthine was the dominant signal. Once silver nitrate was
deemed the best nanoparticle to use, they optimized the pH of the analyte-sol mixture, chose the
optimal time span of how long the analyte-sol mixture was to be mixed, the best aggregating
agent, optimized the amount of aggregating agent to add, and the optimal time span for
aggregation. The optimization of adding the tertiary mixture to the colloid to create the analyte-
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sol mixture was done by allowing the two parts to associate for 60 minutes while spectra was
collected every 5 minutes. They observed that “theobromine and paraxanthine associated almost
instantaneously with the silver surface, whilst there was slower association for caffeine, which
started to plateau after 10 min” [2]. This shows that the sample preparation step in this case is
simple and short-lasting if the nano particles are created and stored beforehand. For the pH of the
analyte-sol mixture, Raman spectra was done for each pH between 2 and 12 for all three
analytes, where the best signal was compared between all three. A pH of 8 was concluded to be
the best one. Based on research, Alharbi et al. chose 1.0 mol/dm3 NaCl as the aggregating agent
because it produced the most reproducible results. The time span of adding the aggregation agent
was optimized when measurements were taken every 30 seconds and the resulting spectra were
identical. Thus the instantaneous spectra right after addition was done for actual analysis. This
shows how this step does not lengthen the time of the entire process. When it came to analysis of
simulated samples, serial dilutions were made to concentrations as low as 10-7 mol/dm3, which is
within the bounds of real metabolite levels after coffee consumption. The results were first
predicted using analysis tools like principal component analysis (PCA) and artificial neural
network (ANN). These are ‘trained’ for quantification of the analyte after (or in tandem with)
detection. The simultaneous results were “excellent” [2], and the researchers assess that this
technique can be used in more complex media like blood and urine.
E. Morphine and MOFs
Morphine is a commonly abused drug for its pain-relieving effects. Its metabolic pathway can
start via oral consumption, intravenously, intramuscularly, rectally, epidurally, subcutaneously,
and intrathecally, though the usual routes of administrations are intravenous and intramuscular
injection. After reaching its peak effect within an hour, about 90% of it is excreted as its
metabolites, with 10% of that being the unmetabolized morphine. Morphine is electronegative
and thus plays an important role in creating hydrogen bonds with chromium(III) metal-organic
framework nanoparticles [1]. This is what Alhaddad and Sheta worked with as a way to detect
morphine in biological real samples. Another aspect that is different from the previous articles
discussed is that detection can be seen using the optical chemosensor as well as with the naked
eye due to a color change, which is something
usually associated with preliminary drug testing.
Metal organic frameworks (MOFs) are hollow
structures, as opposed to particles, which have a
much greater internal and external surface area. The
hollowness provides advantages like enhanced
surface-to-volume ratio, low density, and higher
loading capacities [4]. The structure is also like a
cage, and thus provides a porous surface as well.
Typically, there are metal atoms bound in a uniform
pattern held together by organic ligands. The
metallic portions (nodes) and ligands can be chosen
specifically for whichever analyte is the target.
Alhaddad and Sheta chose morphine as their target Figure 2 Synthsized Cr(III)-MOF-NP formed from
CrCl3•6H2O and organic nano linkers (NL)
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and used chromium MOFs (Cr(III)-MOF-NPs) as their sensor probe (Figure 2). As mentioned,
the Cr(III)-MOF-NPs can hydrogen bond with the electronegative morphine, and that bonding
causes a blueshift when measuring photoluminescence. Before Cr(III)-MOF-NPs can function as
a probe for morphine, it is first synthesized and characterized by various methods such as field-
emission scanning electron microscopy/energy-dispersive X-ray spectroscopy (FE-SEM/EDX)
(Figure 3a-b) and high-resolution transmission electron microscopy (TEM) (Figure 3c-d).

c d

Figure 3 (a-b) FE-SEM images of the chromium MOF [Cr(III)- MOF-NPs] at different magnifications (c-d) TEM images of the Cr(III)-
MOF-NPs at different magnifications

To confirm the structure was synthesized correctly, mass spectra was done, and it showed results
that aligned completely with the proposed fragmentation. To confirm that those fragments were
actually the desired atoms and bonds, fourier transform infrared spectroscopy was done. When
the spectra of Cr(III)-MOF-NP was compared to one of the more well studied MOFs MIL-101-
Cr-MOF/CoAl-1@MIL(Cr), two different bands showed, thus indicative of “the
chelation/complexation of the chromium ion with NL across the N and O atoms” [1]. UV-Visible
spectra showed band gaps that were indicative of the ligand/metal charge transferring transition,
thus showing that linkage has occurred. X-ray photoelectron spectroscopy (XPS) data analysis
scans showed not only the composition but the purity, since the only main peaks were for carbon,
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oxygen, nitrogen, and chromium. After thermal analysis and analysis of its magnetic properties,
the final method of characterization was with photoluminescence (PL). Using auto survey mode,
wavelengths between 220 and 550 nm were scanned for excitation and wavelengths between 240
and 900 nm were scanned for emission. The excitation peak was at 416 nm, and afterwards the
emission peak was at 593 nm. When tested as a spectrofluorimetric chemosensor for morphine,
three remarkable things happened: the intensity of Cr(III)-MOF-NP was enhanced with the
addition of morphine; an evident blue shift from 593 nm to 566nm was recorded on the PL
spectra; the visual color change from brownish yellow to yellow was observed in the cuvette
(Figure 4a). The recorded peaks and color change happened within minutes, thus showcasing the
speed of detection and quantification. There were bold differences in PL spectra across many
concentrations of morphine, as low as 0.1 nanomolar. The actual limit of detection was
determined to be 0.167 nanomolar. Lastly, the detection of morphine was evaluated when in the

Figure 4 (left) Photoluminescence scan showing the Cr(III)-MOF-NP excitation peak and the Cr(III)-MOF-NP with morphine
excitation peak with a blueshift. (left inset) Photo shows the visual color change after morphine is added to the Cr(III)-MOF-NPs.
(right) Photoluminescence histogram (right inset) showing the enhanced intensity of Cr(III)-MOF-NPs towards morphine against
interfering analytes.

presence of other analytes, and the intensity was significant (Figure 4b).
Now that the functionality of the MOFs has been established, simulated serum and urine samples
were evaluated. Each sample was spiked with different concentrations of morphine, 0.5, 5, 50,
and 100 nanomolar, and each sample analysis was conducted three times for reproducibility. The
percent recovery for each sample was an astounding 96% and above, specifically between 97.04-
99.15% for serum samples and between 96.1-98.74 % for urine samples. The percent-error
between recorded concentration and actual concentration was low, between 0.954-1.029%. In
conclusion, using Cr(III)-MOF-NPs as a sensor probe for detecting morphine at concentrations
as low as 0.1 nano molar has been shown to be an accurate, fast, repeatable, sensitive, and
efficient method.
F. Codeine Sulphate and AuNPs
It has been previously mentioned that portable Raman spectroscopy is a useful tool for detection
and analysis of samples inside the laboratory, and with samples like blood and urine. Outside in
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the field however, where samples

like bone, bone marrow, and soil
can be present, it usually does not
happen. That is until Lodha et al.
[15] used gold nanoparticles as a
probe for the colorimetric
detection of codeine sulphate with
a smartphone accompanying it.
These gold nanoparticles (AuNPs)
have been known to have good
surface adsorption of electron-rich
ligands with the metal atoms’
surface being electron-deficient.
When in a stable state, the AuNPs Figure 5 Schematic illustration of the progression of aggregation between
codeine sulphate with citrate coated gold nanoparticles
are surrounded by these electron
rich ligands like citrate that are weakly bound and do not aggregate. For the purposes of this
experiment, aggregation was desired, thus codeine sulphate was added and replaced the citrate
because of stronger coordination (binding). This prompts aggregation, as seen in Figure 5.
Characterization of the AuNP-Codeine sulphate colloid was done by surface plasmon resonance.
When compared to just the AuNPs with citrate, there was a change of absorption maxima after
codeine sulphate was added, from 520 nm to 582 nm. Along with that, there was also a stark
color change from red to purple/wine red. Then, the colloid’s selectivity was tested by observing
the absorption change at 520 nm while in the presence of other substances like morphine,
phenylephrine, phenobarbital, diazepam, lorazepam, and clonazepam. The relative absorbance
changed significantly only for codeine sulphate. Further characterization was done by evaluating
the molecular linker-based aggregation via dynamic light scattering (DLS) and visualizing them
via TEM. DLS histograms showed that as the concentration of codeine sulphate added to the
AuNP mixture increased, the size of
the aggregated particles also
increased. TEM images confirm the
lack of aggregation without codeine
sulphate and the successful
aggregation after the addition of
codeine sulphate (Figure 6). For
the quantification of the sample, an
iPhone5S was used to take a photo Figure 6 TEM micrograph of AuNPs (left) and AuNPs with 0.1 mM codeine
sulphate (right)
of the sample inside of a safety
cabinet with the “no flash” setting. Then the photo was imported to adobe photoshop where the
red, green, blue, and RGB was counted and calculated. Samples with different concentrations of
codeine sulphate ranging from 1 to 10 micro molar were photographed and uploaded for RGB
counting. It was observed that the color intensity had a linear relationship with codeine sulphate
concentration. After the presence of the nanoparticles are ligands were confirmed, the ligand
exchange reaction was investigated by FT-IR spectroscopy and Sei-MS spectroscopy. FT-IR
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spectra showed the difference between AuNP with citrate and AuNPs with codeine sulphate. The
main difference was the loss of the -S=O peak at 2599 cm-1 and peak shifts of the following: C-N
from 1143 cm-1 to 1196 cm-1, SO3- from 1041 cm-1 to 1080 cm-1, and O=S=O stretching in
SO3H from 1378 cm-1 to 1438 cm-1. ESI-MS showed a molecular ion peak at m/z = 893, “which
undoubtedly suggests the formation of AuNPs-cod complex in the mixture” [15]. As with others,
the pH was optimized as well, and the AuNPs had the greatest stability at pH 7.5. Finally, the
samples were tested, and the selectivity of this probe was evaluated by detecting the drug
content in bone, marrow, and soil. Each simulated sample was prepared by administering acute
and chronic doses of codeine sulphate to mice, that were then sacrificed. The sacrificed animal
was then buried in soil. Then the samples of bone marrow, bone, and soil were collected, and the
target analyte was extracted, isolated, and prepared until it was in a dry format. Then the
detection of codeine sulphate was performed using UV-Vis and the phone camera. The results
showed a recovery percentage between 98% to 102.4% across all three sample types. It was
concluded that this citrate-coated gold nanoprobe has the potential to be used as a “rapid,
ultrasensitive, on site semi quantitative field test for the analysis of narcotics/codeine sulphate
from post-mortem blood, skeletal remains and soil” [15].
G. Drug Detection from Latent Fingerprints
Latent fingerprints are created when the finger touches a surface and the sweat that is naturally
produced on the fingertip is transferred to the surface. The sweat leaves patterns like ridges,
swirls, loops, and whorls that, in combination with second and third level detail, make an
individual’s fingerprint unique. The contents of the sweat can also tell of a person’s consumption
of drugs. Hazarika et al. [12] have found a way to detect drugs and their metabolites in the
fingerprint residue via magnetic powder and fluorescent dye that is visualized using a
stereomicroscope. Magnetic powder has been used to visualize the latent fingerprint since the
1960s. With a magnetic brush, the magnetic powder is dusted over the print, where the sweat or
residue from the print makes the powder stick to it, while any remaining powder is magnetically
collected by the brush, creating a very little to no waste of the powder. The powder enhances the
ridge patterns and other characteristics of the print and can thus be analyzed for comparison. In
this research, the magnetic particles are coated with protein A/G. This was bought separately
from a manufacturer, so this part of the sample preparation was not necessary. These coated
beads were 1µm-10µm in diameter. So although not in the scope of nanotechnology, the
experiments showed results that could only be bettered if nanoparticles were used. These coated
antibody magnetic particles (AMP) have been studied for their versatility, ease of application,
and specificity. These AMPs were functionalized by adding antibodies that have specific binding
to target analytes. They are placed on top of the latent fingerprint, which is on a glass slide, and
incubated for 30 minutes. Then the excess particles are removed using a magnet before adding
Alexa Fluor, a fluorescent dye that will bind as a secondary antibody and be incubated for
another 30 minutes. Finally, the tagged fingerprint was visualized using a stereomicroscope. The
analytes studied for detection in this paper were THC, methadone, 2-ethylidene-1,5-dimethyl-
3,3,-diphenylpyrrolidine (EDDP) (the major metabolite of methadone), and benzoylecgonine
(BZE) (the major metabolite of cocaine). It was important for the researchers to aim for detecting
the metabolites since the whole drug being present could be a result of accidental
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touch/exposure. The metabolite being

present shows that the drug was consumed
by the person in some form. For the
detection of THC from a volunteer drug
user, anti-THC AMPs were used, and the
images show the color change (brightfield
image) and the fluorescence under a
stereomicroscope. The change in color
from the gray of the powder to yellow
brown after addition of AMPs and Alexa d) e)

Fluor 488 was observed. Under

fluorescence, and magnification, it indicated
a positive drug detection as well as details of
the fingerprints, such as ridges, bifurcations,
termination points, and even sweat pores
(shown as dark spots in Figure 7c). This was
exciting to observe since it showed that the
drug was already detected with the naked Figure 7 Brightfield images of a fingerprint a) before and b) after the
eye before being seen with fluorescence. In addition of AMPs and Alexa Fluor 488. c) The fluoresence image at a
higher magnification of the fingerprint after incubation of the
control experiments, the brightfield images aforementioned reagents. d) Brightfield and e) fluorescence images
of a volunteer who did not have THC in of a fingerprint after anitBZE AMPs and Alexa Fluor 546 incubation.
their system show no color change other
than another version of gray after incubation. For methadone and EDDP detection,
antimethadone-AMPs and antiEDDP-AMPs were used, respectively. Fingerprints were collected
from volunteers at a drug treatment facility. For methadone, the same method was used as it was
used for THC. For EDDP, Alexa Fluor 546 was used, showing red fluorescence instead of green.
In their control experiments, the same results of no detection were observed as it was for THC
control. For BZE, it was anticipated that, since the half-life of BZE is about 7.5 hours, detection
of predicted low concentrations would be a challenge. However, though the color change and
fluorescence signal were dim in comparison to the previous analytes’ signals, the Brightfield
images were clear enough for fingerprint analysis and fluorescence. In conclusion, using the
AMPs made detecting drugs with brightfield microscopy showed to be useful and successful. It
also brings about the idea of not having to use microscopy to see fluorescence since the color
change can be seen with a white light source. With further studies, this has the potential to be
implemented as a confirmatory test for on-spot drug detection.
H. Cocaine and Quantum Dots
Nanoparticles can be functionalized to detect specific drugs as a sensor, but Zhang and Johnson
used quantum dots instead as a nano sensor for cocaine. “Quantum dots are novel semiconductor
nanocrystals with the significant advantages of narrow, tunable, and highly stable
photoluminescence over organic fluorephore” [16]. Quantum dots coupled with aptamers that
can specifically recognize cocaine increase detectability significantly. Aptamers have been used
previously as single-stranded RNA or DNA that have ligands that can bind to small molecules
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like cells, but not yet at the nanoscale, such as toxins and drug metabolites. The functionalization
of the surface of quantum dots with aptamers that can recognize cocaine put this type of
detection into the single-molecule type, which is exciting for its potential in other applications as
it is specific for the molecule rather than an average of what is detected. The detection signals
come through the form of fluorescence resonance energy transfer (FRET), which quantum dots
have high compatibility with. Previously it
was reported that single-QD-based FRET
detected DNA and real-time RNA-peptide
interaction. Zhang and Johnson hypothesized
that this could be used as a nano sensor for
cocaine even in trace amounts. The quantum
dot used was 605QD, which reaches an
excitation maximum at 605 nm when
reflected by a 488nm laser beam. 605QDs
were functionalized by streptavidin, which is
what the aptamer-complexes are bound to in
the form of hybrid oligonucleotides (ONTs).
The aptamers were sandwiched between
three different ONTs for two variants of
detection: signal off and signal on. For signal
off, the hybrid sandwich ONT was formed as
such: 5’ - Cy5 ONT (a fluorescent dye tag) -
biotinylated ONT - cocaine aptamer – 3’, Figure 8 Diagram of a) signal-off and b) signal-on single QD-
where the cocaine aptamer can be seen as a based aptameric sensor for cocaine detection
‘base strand’ that binds to the other two shorter single-strands of the ONTs, leaving an active
‘tail’ for cocaine to bind to. As shown in Figure 8a, the ONT hybrid complex is bound to the
quantum dot (QD) via streptavidin, which is represented by the ‘spokes’. The ONTs are written
out, where the biotinylated portion is highlighted in green, the Cy5 tag is represented by the red
dot followed by its nucleotides written out, and the cocaine aptamer portion is written in red.
When not in the presence of cocaine, the QD will transfer energy to Cy5 by FRET and excite the
protein in order for it to cause an excitation signal. This was done specifically because Cy5 does
not get excited at 488 nm, thus ridding the chances of an accidental excitation signal as a sign of
non-detection. When in the presence of cocaine, a conformational change in the aptamer occurs,
causing the detachment of the Cy5 portion of the hybrid sandwich. The lack of Cy5 fluorescence
indicates the presence of cocaine, hence naming this variation signal-off. In a similar fashion, the
signal-on variant works where, without cocaine, Cy5 cannot be excited because a third ONT,
Iowa black RQ, serves as a quencher, represented as the black dot in Figure 8b. In the presence
of cocaine, the conformational change in the aptamer occurs, causing the detachment of the Iowa
black RQ portion of the hybrid sandwich. This causes the Cy5 fluorescence to be in its ‘on’ state.
The sample preparation step was for simulated samples, and pure cocaine from Sigma-Aldrich
was incubated with the ONT sandwich hybrids. Then it was subjected to single-molecule
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detection, where an argon laser, two dichroic mirrors, an

objective lens, a syringe pump, a pinhole, a focusing lens, two
band-pass filters, two avalanche photodiodes, and a digital
counter played their role in the detection analysis of the
quantum dots, fluorescence of Cy5, and interpretation of
cocaine concentrations. As seen in Figure 9, the linearity of the
cocaine-to-Cy5 fluorescence is pleasing, indicating a strong
relationship between the analyte and the signal-off variation of
the QD-based aptameric sensor. Similar, yet not as linear, Figure 9 A graph showing the number of
results were shown for the signal-on variation, with Cy5 burst Cy5 burst counts decreasing as the
concentration of cocaine increases to
counts increasing as cocaine concentrations increased. Zhang show the success of the signal-off single
and Johnson successfully displayed the feasibility, ease, and QD-based aptameric sensor.
variation of quantum dots being used as a nano sensor for cocaine. The one downside is the
relatively high limit of detection, reported to be 0.5 micromolar, thus not enough for trace
evidence that falls into the nanomolar range. But this was already an improvement when
compared to colorimetric methods involving nanoparticles.

II. Conclusion
Nanotechnologies are versatile, tunable, adaptable, and powerful when it comes to detecting
substances, whether it be illicit drugs or macro molecules like DNA and cells. They can come in
the form of particles, chips, probes, quantum dots, and more. The use of nanotechnology means a
quicker, more reliable, and inexpensive way to analyze, detect, quantify, and evaluate our target
analytes. Though this is being hailed as a vast improvement in the world of analytical chemistry,
these applications can easily be implemented into forensic laboratories.

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