How new nanotechnologies are changing the opioid analysis scenery? A comparison with classical analytical methods

Abstract Opioids such as heroin, fentanyl, raw opium, and morphine have become a serious threat to the world population in the recent past, due to their increasing use and abuse. The detection of these drugs in biological samples is usually carried out by spectroscopic and/or chromatographic techniques, but the need for quick, sensitive, selective, and low-cost new analytical tools has pushed the development of new methods based on selective nanosensors, able to meet these requirements. Modern sensors, which utilize “next-generation” technologies like nanotechnology, have revolutionized drug detection methods, due to easiness of use, their low cost, and their high sensitivity and reliability, allowing the detection of opioids at trace levels in raw, pharmaceutical, and biological samples (e.g. blood, urine, saliva, and other biological fluids). The peculiar characteristics of these sensors not only have allowed on-site analyses (in the field, at the crime scene, etc.) but also they are nowadays replacing the gold standard analytical methods in the laboratory, even if a proper method validation is still required. This paper reviews advances in the field of nanotechnology and nanosensors for the detection of commonly abused opioids both prescribed (i.e. codeine and morphine) and illegal narcotics (i.e. heroin and fentanyl analogues).


Introduction
Papaver somniferum L. produces milky juice which is converted to dark brown resinous material after drying and is called raw opium (Figure 1), consisting of more than 30 alkaloids, resins, fatty acids, sterols, polysaccharides, triterpenoid alcohols, porphyroxine, meconic acid, and plant debris.Narcotic alkaloids of opium poppy are synthesized in specialized vesicles called "laticifers" which are found mainly in the stem.When some parts of the plant, especially the unripe pod, are incised, the milky latex oozes out, which is transported to the point of injury by laticifers and phloem tissues [1].Out of 30 alkaloids, five are the main compounds and are classified into two categories, namely isoquinoline alkaloids (noscapine and papaverine) and phenanthrene alkaloids (codeine, morphine, and thebaine).Opium is not only used for illicit purposes (e.g. the synthesis of heroin from morphine) but also has legitimate applications.It is used for medicinal purposes, and for cooking, e.g. the seeds are used in bakery products (oil extracted from seeds) [2,3] and in past, the oil obtained from opium was used in varnishes and paints [4,5].
Opium is classified as a narcotic analgesic and is the only source of natural opioids.Since their origin, opioids are classified into three major categories, i.e. natural, semisynthetic,

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FORENSIC SCIENCES RESEARCH and synthetic.Codeine and morphine are natural opioids obtained from opium resin.Semisynthetic opioids are synthesized from natural opioids, like diacetylmorphine (heroin) which is the acetylated product of morphine [6].Hydrocodone and oxycodone are also semisynthetic opioids.Fentanyls (whose pain-relieving capacity is 100 times greater than that of heroin) are synthesized in laboratories and classified as synthetic opioids.The lethal dose of fentanyl in humans is 2 mg.Non-pharmaceutical fentanyls (NPFs) are synthesized in clandestine laboratories and supplied to the drug market.Sufentanil, alfentanil, remifentanil, and carfentanil are the most common fentanyl analogues.Opioids have been used as pain relievers, antitussives, and sedatives.Opioids (e.g.codeine, morphine, oxycodone, heroin, dextromethorphan, pentazocine, dextrorphan, and norpethidine) affect the μand k-opioid receptors and decrease pain sensation by inhibiting the release of neurotransmitters [7].A list of the most common opioids, their use, and legal status is reported in Table 1.Due to their psychotropic effects, opioids are widely abused, hence causing serious health issues.The oral ingestion of raw opium in high dose cause myocardial suppression [8].About 209 million people around the globe used opioids in 2022 [9].Since several people abuse these illicit drugs, the identification and quantitative determination of drugs are becoming more important.Nowadays, several advanced analytical techniques, such as gas and high-performance liquid chromatography (GC and HPLC, respectively), very often hyphenated with mass spectrometer (MS) detector, nuclear magnetic resonance (NMR) spectroscopy, capillary electrophoresis (CE), Raman spectroscopy, and Fourier transform infrared spectroscopy (FTIR), are being used for the analytical determination of opioids [10][11][12][13][14][15][16].For forensic opioid analysis, different samples, i.e. biological (blood, urine, hair, nail, and exhumed body), pharmaceutical (tablets, injections), and street drug samples (street powder, clandestine tablets) are submitted and analyzed routinely [12,[17][18][19][20][21][22][23][24][25][26][27][28][29].
Nanomaterial-based techniques have been employed for the determination of opiates, offering innovative approaches with high sensitivity and versatility.In this review, new analytical methods based on nanomaterials used as biochemical sensors or electrochemical sensors for qualitative and quantitative analysis of opioids have been discussed and compared with classical analytical determinations.Chemical sensors based on nanomaterials have applications in the determination of opioids in a variety of samples [30][31][32].Carbon nanotubes (CNTs), like single-walled CNTs (SWCNTs) or multiwalled CNTs (MWCNTs) [33,34], graphenes [35], tungsten oxide nanoparticles [36], palladium nanoparticles [37], platinum nanoparticles [38], gold nanoparticles (AuNPs) [39], and other modified nanomaterials have been used as sensors for opioids and their extraction from biological samples [31,40].CNTs are well-known for their good biocompatibility and electrical sensitivity, and their applications in biosensing are numerous [41,42].CNTs have been widely used in sensor preparation for the analysis of electro-active drugs [43].When deposited on metal electrodes, SWCNTs, MWCNTs, and CNTs can be used as chemical sensors.Graphene, composed of layers of tightly bound carbon atoms (honeycomb network), nanomaterial is also used as a chemical sensor or in the construction of modified electrodes [44].Various types of electrodes, i.e. glassy carbon electrode [45], carbon paste electrode [46], indium tin oxide electrode [47], Pt electrode [48], and carbon ceramic electrode [34] are combined with amperometry, voltammetry, or impedance spectrometer for drug analysis.The chemical sensors have several advantages over the other techniques, i.e. low cost, portability, shorter time of analysis, high sensitivity, and low limits of detection.These advantages make chemical sensors a very attractive material for future research.

Colourimetric assays
Colour testing assays are based on the generation of colour by specific chemical/biochemical reactions between target analytes and reagents.Therefore, colour tests produce simple visual results (i.e.change in colour), and complex instrumentation is not required for the analysis.Colour tests are rapid detection tools for qualitative determinations and can be used to evaluate the presence or absence of drugs in a test sample, outside the controlled laboratory environment.Nevertheless, false positive and false negative results represent a major concern for analysts during drug analysis, since the presence of interfering compounds in biological samples may negatively affect the analytical results.For this reason, colour tests are generally used in combination with other techniques (e.g.GC-MS, LC-MS, or FTIR).A schematic overview of the main tests used for presumptive screening of opioids in suspected drug samples, namely, Marquis, Froehde, Meck, nitric acid, Oliver, Mandelin, Liberman, and AuNPs tests [50,11,25,51,52], is given in Table 2.

Spectroscopic methods
Spectroscopic techniques are frequently applied in forensic investigations for the analytical determination of controlled substances.However, according to the protocols of the Scientific Working Group for the analysis of seized DRUGs (SWGDRUG), chromatographic techniques have to be employed in addition to spectroscopic techniques (i.e.UV, IR, FTIR, Raman Spectroscopy, and Mass Spectrometry) [16,58,59].Spectroscopic instruments are generally userfriendly and require a very minimum amount of sample.Recently, portable spectroscopic instruments have been developed, which make these techniques ideal for field testing and on-spot analysis [60].A portable ultraviolet Raman Spectroscopy device, developed by Hopkins et al. [61], was successfully applied for the determination of heroin [61,62].By terahertz spectroscopy, it is possible to obtain the structural and chemical information of substances concealed within packaging (e.g.cardboard, plastic, and paper).Due to this feature, terahertz spectroscopy can also be used for on-spot street drug identification.Terahertz time-domain spectroscopy (THz-TDS) shows characteristic peaks at 1.42 and 3.94 THz for heroin [63].In addition to field testing of drugs, spectroscopic techniques have been applied for component analysis and drug profiling.FTIR-ATR has been used for the qualitative and quantitative determination of morphine and three commercial variants of thebaine in poppy extracts [64].Surface-enhanced Raman spectroscopy [65,66], diffuse reflectance near-infrared spectroscopy (DR-NIR) [67], and near-IR Raman spectroscopy [68,69] have been also reported for the analytical determination of natural and synthetic opioids.

Chromatographic methods
Chromatography is the most used and widely accepted analytical technique amongst the forensic community.GC-MS [11,12,20,70], or tandem mass spectrometry (GC-MS-MS) [18], and flame ionization detection (GC-FID) [71] have been reported for the determination of illicit drugs in human-origin biological samples.A quick, economical and eco-friendly method by dispersive liquid-liquid microextraction (DLLME) coupled with injection port silylationgas chromatography-mass spectrometry (IPS-GCMS) was developed and validated by Jain et al. [72] for the determination of morphine in illicit opium, using bis(trimethylsilyl) acetamide for the derivatization process of morphine.For the analysis of non-volatile and higher molecular weight drugs, liquid chromatography coupled to mass spectrometry (LC-MS) [ 17,23,73] and ion mobility tandem mass spectrometry (LC-TIMS-MS) [74] has been employed.Recently, UHPLC-MS-MS has been reported for the determination of synthetic opioids including fentanyl in hair samples [75].Lui et al. [76] proposed an ultra-performance liquid chromatography method coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF) for the characterization of opium samples from different geographical sources.Mass spectrometry-based chromatographic methods allow accurate qualitative and quantitative multi-residual determinations of drugs at the trace level in a variety of samples, providing high analysis selectivity and sensitivity.Nevertheless, GC-MS and LC-MS methods are generally time-consuming and labour-intensive and require sophisticated equipment for exclusive laboratory-level use [77,78].As an alternative to mass spectrometry, GC coupled with vacuum ultraviolet spectrometry (VUV) has been used for the determination of opioids (i.e.morphine, heroin, and fentanyl).The specificity of VUV spectra was evaluated by using principal component analysis and discriminant analysis (DA) statistical techniques, then the GC-VUV method was successfully applied for the analysis of street heroin samples with 100% accuracy of the DA model [79].A list of the most common methods used for the determination of opioids/opioids is reported in Table 3.

Electrochemical sensors based on nanomaterials
Nanomaterial-modified electrochemical sensors have been used for the detection of opioids.The use of nanomaterials for the synthesis of electrochemical drug sensors attracted the interest of scientists because they can be employed for rapid and on-site qualitative determination of illicit drugs in different substrates [80].There are numerous advantages to using electrochemical sensors due to their sensitivity, selectivity, specificity, and easiness of use, even with complex biological media, like human urine and pharmaceutical samples [41].Electrochemical sensors are classified into three categories on the bases of their sensing mechanism: amperometric, conductometric, and potentiometric.Usually, electrode material is specifically developed for a target drug.Electrochemical sensors show a good linear range response and high selectivity, but they suffer from a slow response.Ensafi et al. [38] developed an electrochemical sensor based on Pt nanoparticles supported on porous silicon flour for the simultaneous determination of morphine and codeine, using adsorptive stripping voltammetry.Detection limits of 30.0 and 20.0 nmol/L were achieved for morphine and codeine, respectively.The electrochemical determination of morphine was achieved by properly modifying carbon paste electrodes [81].Differential pulse voltammetry (DPV) was applied to a carbon paste electrode modified with multiwall carbon nanotubes (MWCNTs) for drug determinations.Cyclic voltammetry, chronoamperometry, and electrochemical impedance spectroscopy were used by Mokhtari et al. [103] with modified carbon paste electrode with vinylferrocene/MWCNTs to study the response as a function of morphine presence in biological and pharmaceutical samples.Gold nanoparticles, deposited on nafion, can be used for modification of carbon paste electrodes to detect morphine in urine samples using DPV [104].A carbon ceramic electrode was modified by SWCNTs for the simultaneous detection of codeine and caffeine using DPV [34].Atta et al. [105] applied a modified gold electrode with gold nanoparticles over poly(3,4-ethylenedioxythiophene) for the determination of morphine in urine and pharmaceutical tablets, using sodium dodecyl sulphate (SDS) as a surfactant.A carbon glassy electrode modified by reduced MWCNTs-doped graphene oxide was developed for the analysis of morphine.The electrochemical response of morphine was investigated using pH 4.5 phosphate buffer solution by cyclic voltammetry and linear sweep voltammetry [106].A similar electrochemical sensor was developed by Babaei et al. [43]: they modified a glassy carbon electrode by MWCNTs/chitosan for the detection of dopamine and morphine in human blood and urine.Cyclic voltammetry, DPV, and chronoamperometry were used for the analysis of drugs using the above-mentioned sensor, obtaining a sensitive amperometric sensor for morphine detection, which exhibited high sensitivity, stability, and long life.Heroin, morphine, and its metabolite morphine-3-glucuronide can be also detected by amperometric devices, properly synthesized for this scope [45,47,[107][108][109].Tey et al. [6] synthesized a disposable device for onsite drug screening based on a liquid-gated carbon nanotube field effect transistor (LG-CNTFET), for the detection of 6-acetylmorphine (a heroin metabolite).
The detection limit of LG-CNTFET has been improved by incorporating gold nanoparticles into morphine antibodies.El-Naby and Kamel AH [110] developed a potentiometric assay method for the determination of dextromethorphan in pharmaceutical preparations and illicit drug market samples.The membrane-based polymer, composed of molecularly imprinted polymer (MIP) with methacrylic acid (MAA) and acrylonitrile (AN) acting as functional monomers, was developed, and the relevant biosensor characteristics have been fully validated, showing short response time, high  sensitivity, selectivity, stability, and accuracy.The limit of detection (LOD) of MIP/MAA and MIP/AN sensors were 1.9 × 10 −6 and 1.0 × 10 −6 mol/L, respectively.Metal-organic frameworks (MOFs) and their composites have emerged as pivotal players in the development of electrochemical sensors dedicated to opioid detection.Renowned for their rapid production, cost-effectiveness, high sensitivity, and remarkable low detection limits, these materials represent a paradigm shift in sensor improvement.Their intrinsic potential in facilitating real-time drug concentration measurement, a critical aspect for tailoring treatment dosages to uphold therapeutic thresholds, is particularly important in the clinical application of opioids.Furthermore, the integration of microfluidic chips with electrochemical sensors can enhance detection capabilities [111].
Razavi et al. [112] developed a CuONPs/MWCNTs/carbon paste modified electrode for tramadol analysis.Employing eco-friendly synthesis, cupric oxide nanoparticles (CuONPs) were crafted using Origanum majorana extract, revealing perfect structures, as verified through X-ray diffraction (XRD), SEM, and FTIR.Voltammetric methods highlighted the electrode's precision, demonstrating exceptional selectivity for tramadol and establishing accurate linear calibration curves.Cutting-edge techniques based on B3LYP/LanL2DZ quantum method were used to calculate the energy characteristics of the nanocomposites.CuONPs/CNT demonstrated effectiveness in detecting Tramadol in actual samples with a recovery rate of 96.0%-104.3%.
A novel electrochemical platform, integrating a covalent organic framework (COF) and reduced graphene oxide (rGO), was successfully devised for the highly sensitive detection of fentanyl and alfentanil in human serum.The COF nanomaterials, exhibiting a distinctive chrysanthemum morphology, significantly increase electrochemical catalytic reactions, enhancing the overall sensor performance.Concurrently, the introduction of reduced graphene oxide increased the sensitivity by facilitating electron transfer.Under optimized conditions, the developed electrode presents a linear detection range of 0.02-7.26μmol/L for alfentanil and 0.10-6.54μmol/L for fentanyl, with impressive detection limits of 6.7 and 33.0 nmol/L, respectively.The sensor showed outstanding selectivity, reproducibility, and stability, positioning it as a viable tool for the reliable monitoring of fentanyl and alfentanil in human serum samples [113].
Fentanyl and its analogues (powerful synthetic opioids and the primary cause of drug overdose deaths in the United States) pose a critical challenge for their analytical determination.Canoura et al. [115] developed a series of innovative aptamer-based assays and electrochemical sensors, which offer reliable, accurate, rapid, and cost-effective means to detect fentanyl-based compounds.These sensors show no cross-reactivity with other illicit drugs or substances, even in interferent-laden mixtures containing only 1% fentanyl.The potential for routine use extends to medical professionals, law enforcement agencies, and the general public, facilitating swift and precise identification of fentanyl.
In recent developments within nanomaterials-based electrochemical sensors, significant progresses have been made in the analytical determination of tramadol, a centrally acting analgesic utilized for treating various pain conditions.The application of distinct electrochemical techniques, including DPV, amperometry, and SWV, has yielded noteworthy outcomes in the realm of tramadol detection by using MWCNTs-AuNPs, CoO-CNTs, and graphitic carbon nitride nanomaterials.Notably, these sensors exhibited a broad spectrum of detection limits and linear ranges, underscoring their versatility across applications.The integration of metal nanoparticles like AuNPs and metal oxide NPs, exemplified by Co 3 O 4 NPs and CuONPs, has substantially enhanced the sensitivity of tramadol detection.Additionally, the utilization of diverse composites such as Nafion/CTAB-Au and Yb 2 O 3 -SPE underscores the variety in sensor fabrication approaches.Overall, these sensors demonstrated promising sensitivity over a wide concentration range, highlighting their potential for practical applications in detecting tramadol and emphasizing the pivotal role of nanomaterials in advancing electrochemical sensing technologies for pharmaceutical analyses [116].

Nano-biosensors
Nano-biosensors are an effective tool for the analysis of drugs, due to their selectivity which is usually greater than those of traditional biosensors.They convert biological response to electrical or optical output response through a bio-receptor that identifies the analyte and a transducer, which converts the signal to an electrical or optical output.Nanobiosensors for the analyses of illicit drugs are of great interest, not only in forensic investigations but also in monitoring drug abuse in treatment centres and workplaces, due to their unmatched properties (high selectivity, reproducibility, stability, sensitivity, affordability, linearity of response, low cost, and easy functioning).Nano-biosensor can be subdivided, depending on the bio-receptor and the transducer used, into immunosensors, genosensors, aptasensors, and electrochemical biosensors [117-121, 122, 123].Genosensors obtained by recombinant DNA-based techniques have been used recently to produce antibodies for morphine-3-glucuronide (a metabolite of heroin).For example, an SPR-based inhibition immunoassay using anti-morphine-3-glucuronide recombinant scFv (single chain fragment variable) antibodies in Escherichia coli has been reported [124].The response of antibody binding to the surface of the chip was inversely proportional to the amount of free drug in urine.Similarly, it was found that AuNPs labelled scFv antibodies could be used for the detection of morphine [125].The developed recombinant antibodies bind morphine and monoacetylmorphine (MAM) very specifically.Doublestranded DNA (ds-DNA) immobilized on gold electrodes modified with mercapto-benzaldehyde was employed for sensing morphine [126].The electrostatic interaction of morphine with ds-DNA was studied by DPV.Another biosensor was prepared on a pencil graphite electrode (PGE) modified with MWCNTs dispersed in poly(diallyldimethylammonium) (PDDA) [127].In this case, the biosensor sensitivity is assured by conformational changes produced by codeine and morphine on ds-DNA, which provides variations on the electrochemical oxidation signals for both compounds.Differential pulse (DP) voltammograms of codeine and morphine showed depressed currents in the presence of DNA shifting to more negative and positive potentials, respectively.The electrostatic attraction for codeine and the intercalation for morphine permits clear discrimination between the peaks for both analytes.Voltammetric analysis, with an accumulation time of 5 min, provided a linear dependence between 0.05 and 40 mg/mL for codeine and 0.05 and 42 mg/mL for morphine, with detection limits of 0.041 and 0.043 mg/mL, respectively.This biosensor was successfully used for the analysis of blood, urine samples, and pharmaceutical preparations.Shao et al. [128] developed an exceptionally sensitive norfentanyl sensor, using a semiconductor-enriched single-walled carbon nanotube (sc-SWCNT) field-effect transistor modified with norfentanyl antibodies.Motivated by the pervasive global opioid crisis (particularly the impact of synthetic opioids like fentanyl), the study strives to meet the pressing demand for a portable analytical tool.Various sensor configurations have been explored, covering a direct coupling strategy and an approach incorporating gold nanoparticles (AuNP), with sensitivity optimization through the integration of a "reduced" antibody.The sensor exhibited notable sensitivity, achieving a 2.0 fg/mL LOD for norfentanyl in synthetic urine samples.Nanomaterials possess unique characteristics, such as high surface area, high conductivity, enhanced electron transfer rate, and ease of modification, which enable diverse applications, including sample pretreatment and biosensor establishment, facilitating the monitoring of illicit drugs in diverse complex matrices, including biological, pharmaceutical, and environmental samples [121,129,130].Furthermore, the integration of nanomaterials in electrochemical sensors is advantageous, particularly for the detection of drugs present at low concentrations in body fluids.These sensors are also beneficial for on-site screening, as they enable non-invasive sample collection (allowing, as an example, drug detection in saliva samples), not requiring invasive blood sample collection [130].Nanomaterial-based aptamer sensors have been used for the analytical determination of illicit drugs, showing higher sensitivity, and sensitivity than traditional methods.In addition, nanomaterial-based sensors require minimal sample pretreatment, have fast response times, and show potential for on-site analysis [131].These sensors require minimal sample pretreatment, enabling fast and efficient analysis of complex matrices such as human urine, blood serum, and saliva [132].They typically require less sample consumption (in some cases ∼100 μL) contributing to potential cost savings in terms of sample usage; moreover, the utilization of nanomaterials provides superior detection efficiency, with limits of detection in the range of pg/mL and ng/mL [129].Nanomaterial-based sensors are affordable, easy to use, and portable, making them suitable for a wide range of applications, from health monitoring to more complex settings, such as border customs or aviation industries [132].On the other hand, these devices showed some limitations due to their drug specificity, i.e. it is necessary to use different sensors to detect different drugs [133].The development of nanosensors can be complex, requiring expertise in nanotechnology and nanosensor design.This complexity may limit the widespread adoption of nanosensors for opiate analytical determinations, especially in settings with limited resources [121].Whilst nanosensors offer high sensitivity and selectivity for the detection of opioids, ensuring specificity for different opiates can be challenging.Therefore, the development of nanosensors with high selectivity for various opiates remains an ongoing area of research [134].

Supramolecular sensors
Supramolecular chemistry is a relatively young branch of science.In supramolecular sensors, the sensor molecule is selected based on the size, shape, and charge of the drug molecule.Sensor molecules contain binding sites, which are highly specific for drugs.The analyte molecule binds with the sensor at a specific binding site due to non-covalent forces, such as hydrogen bonding or Van der Waal forces.As a result of these attractive forces between the sensor and drug molecule, a supramolecular complex is produced.The stability of this complex depends on the strength of these noncovalent forces.The formation of supramolecular complexes can generate colourimetric, fluorescent, or electrochemical signals.Due to their high specificity, supramolecular sensors (Figure 2) are conveniently used in analytical chemistry for the determination of a variety of substances in complex samples.Acyclic cucurbituril (aCB) has been used and reported as a sensor molecule for the quantitative analysis of three opioids, i.e. morphine, heroin, and oxycodone, together with their metabolites normorphine, morphine-3-glucuronide, morphine-6-glucuronide, 6-monoacetylmorphine, noroxycodone, and oxymorphone [159].aCB is composed of four glycoluril units that terminate on both ends with naphthalene fluorophore units.The cucurbituril opening cavity is hydrophobic and flexible, and its diameter depends on the connectivity between naphthalene rings and glycoluril units.Due to its flexibility, the cavity is adaptable to the shape of the opioid molecule.Intermolecular naphthalene acts as a chromophore, producing the desired fluorescence in the presence of the analyte.This sensor has been successfully used using a new three-way calibration model for quantitative drug analysis in urine samples.A list of the most common nanomaterials/nanobiosensors used for the determination of opioids/opioids is reported in Table 4.

Conclusions
Classical and nanomaterial-based techniques are used for the analytical determination of opiates, each with their own advantages and limitations.Classical methods include colourimetric, spectroscopic, and chromatographic analysis, whereas nanomaterial-based techniques involve the use of nanoparticles-based biosensors and electrochemical sensors.Classical methods for detecting opiate have been widely used and are generally considered to be cost-effective, compared to nanomaterial-based methods which often involve the use of specialized nanomaterials and advanced technology, generally expensive to develop and implement.Electrochemical, nano, and supramolecular sensors are becoming essential tools for the detection of drugs due to their fast availability, reliability, and portability.Various techniques like conductometry, amperometry, voltammetry, and chronoamperometry are very versatile depending on the electrode type.These techniques provide high sensitivity and continuous monitoring of drugs, making them great tools for determining opioid intakes.The most used nanomaterials for electrodes are CNTs in MWCNT and glass wall CNT; however, platinum-based nanoparticles and AuNP show the lowest LODs.Moreover, the usage of nafion tubes has other advantages in the fabrication of biosensors for the opioid detection in biological fluids.Analysis time may vary between classical and nanomaterialbased techniques for the determination of opiates.Classical methods generally have relatively rapid analysis times, making them suitable for high-throughput screening; nevertheless, these methods require complex pre-sample treatment for drug extraction and concentration prior to analysis.On the other hand, although sample processing before analysis is not necessary for nanomaterial-based techniques, longer analysis times may be required compared to traditional methods, due to the complexity of the sensor fabrication, functionalization, and integration of nanomaterials into sensors, especially during the optimization phases, and the detection process.
In conclusion, both classical and nanomaterial-based techniques play important roles in the analytical determination of opiates.Classical techniques offer well-established methods, whereas nanomaterial-based techniques provide innovative tools for sensitive and continuous monitoring of opiates.New sensors can be considered promising technical devices, complementary to classical analytical systems, enabling fast, easy, and on-site drug detection, in specific preliminary analyses which need to be confirmed, at the moment, by classical methods.Despite the progress of an extensive spectrum of nanosensors over the last two decades, the future purpose of low-cost, high-throughput, multiplexed clinical diagnostic Lab-on-a-Chip instruments has yet to be fulfilled.Overall, the major advantages of nanoparticles in opioid detection are lower LOD, and portability; furthermore, nanosensors are particularly suitable for QuEChERS-based methods, representing a promising technology for the near future.Finally, it should be underlined that most of these new sensors contain materials and heavy metals that could be hazardous for human health and other living organisms.Therefore, safe handling along with data on the toxicity of nanomaterials is highly required.

Figure 2
Figure 2 Chemical structures of supramolecular sensors (S1-S3) used for the analysis of opioids and their metabolites.

Table 1 .
List of the most common opioids and their use.

Table 2 .
Colour tests used for preliminary opioid screenings.

Table 3 .
List of drug abuse analyses carried out by classical techniques, with the indication of analyzed opioids/opioids, matrices, limit of detections (LODs), and limit of quantitations (LOQs).

Table 4 .
List of drug abuse analyses carried out by electrochemical techniques, with the indication of nanomaterials/nanobiosensors, matrices, and limit of detections (LODs).