Portable analytical platforms for forensic chemistry: A review

Accepted Manuscript Portable analytical platforms for forensic chemistry: a review William R. de Araujo, Thiago M.G. Cardoso, Raquel G. da Rocha, Mário H.P. Santana, Rodrigo A.A. Muñoz, Eduardo M. Richter, Thiago R.L.C. Paixão, Wendell K.T. Coltro PII:

S0003-2670(18)30779-7

DOI:

10.1016/j.aca.2018.06.014

Reference:

ACA 236029

To appear in:

Analytica Chimica Acta

Received Date: 3 February 2018 Revised Date:

18 May 2018

Accepted Date: 7 June 2018

Please cite this article as: W.R. de Araujo, T.M.G. Cardoso, R.G. da Rocha, M.H.P. Santana, R.A.A. Muñoz, E.M. Richter, T.R.L.C. Paixão, W.K.T. Coltro, Portable analytical platforms for forensic chemistry: a review, Analytica Chimica Acta (2018), doi: 10.1016/j.aca.2018.06.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Portable analytical platforms for forensic chemistry: a review William R. de Araujoa, Thiago M. G. Cardosob, Raquel G. da Rochac, Mário H. P. Santanad, Rodrigo A.A. Muñozc, Eduardo M. Richterc, Thiago R. L. C. Paixãoa,* and Wendell K. T. Coltrob,*

Instituto de Química, Universidade de São Paulo, 05508-000, São Paulo, São Paulo, Brazil

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Instituto de Química, Universidade Federal de Goiás, Campus Samambaia, 74690-900 Goiânia, Goiás, Brazil

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Instituto de Química, Universidade Federal de Uberlândia, 38400-902, Uberlândia, Minas Gerais, Brazil

Unidade Técnico-Científica, Superintendência Regional do Departamento de Polícia Federal em MG, 38408-680,

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Uberlândia, Minas Gerais, Brazil

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*Corresponding authors: [email protected] and [email protected]

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ACCEPTED MANUSCRIPT Abbreviation List

AgNP = Silver nanoparticles; APCI = atmospheric pressure chemical ionization; AuNP = Gold nanoparticles; 4A2NP = 4-amino-2-nitrophenol; BDD = Boron-doped diamond; BIA-SPE = Batch

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injection analysis for screen-printed electrodes; BIA-SWV = Batch Injection Analysis and Square Wave Voltammetry; CA = Chronoamperometry; CE = Capillary electrophoresis; CSI = Crime scene investigation; CV = Cyclic Voltammetry; C4D = capacitively coupled contactless

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conductivity detection; DART = direct analysis in real time; DESI = desorption electrospray ionization; DNT = 2,4-Dinitrotoluene; DPV = Differential Pulse Voltammetry; EIS =

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Electrochemical impedance spectroscopy; ESI = electrospray ionization; GCE = Glassy-carbon electrode; HMTD = Hexamethylene triperoxide diamine; HMX = Octogen; ILR = ignitable liquid residues; LOD = Limit of Detection; LR = Linear range; LSD = Lysergic acid diethylamine; LSV = Linear sweep voltammetry; LTP = low-temperature plasma; MA = Multiamperometry; MDQ = minimum quantity detectable, ME = Microchip electrophoresis; MS = mass spectrometry; MEKC

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= micellar electrokinetic chromatography; NB = Nitrobenzene; NIR = Near infrared; NPSs = New psychoactive substances; NR = Not reported; PA = Picric acid; PADs = Paper-based analytical

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devices; PON = Point-of-Need; µPADs = Microfluidic paper-based analytical devices; PCR = polymerase chain reaction; PETN = Pentaerythritol tetranitrate; RDX = Trimethylenetrinitramine;

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PRIED = Portable Raman improvised explosives detector; SPCE = Screen Printed Carbon Electrode; SPGE = Screen Printed Graphite Electrode; SERS = Surface-enhanced Raman spectroscopy; SPEs = Screen-printed electrodes; SS = swab spray; SWSV = Square Wave Stripping Voltammetry; SWV = Square Wave Voltammetry; TATP = Triacetone triperoxide; Tetryl = Trinitrophenylmethylnitramine; TNB = 1,3,5-Trinitrobenzene; TNT = Trinitrotoluene; UFCE = ultrafast capillary electrophoresis; VH = Vitreous humour; WoS = Web of Science.

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Abstract

This current review article focuses on recent contributions to on-site forensic investigations. Portable and potentially portable methods are presented and critically discussed about (bio)chemical trace analysis and studies performed outside the controlled laboratory environment to rapidly help in crime scene inquiries or forensic intelligence purposes. A wide range of approaches including

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electrochemical sensors, microchip electrophoresis, ambient ionization on portable mass spectrometers, handheld Raman and NIR instruments as well as and point-of-need devices, like paper-based platforms, for in-field analysis of latent evidences, controlled substances, drug screening, hazards, and others to assist in law enforcements and solving crime more efficiently are

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highlighted. The covered examples have successfully demonstrated the huge potential of portable devices for on-site applications. Future investigations should consider analytical validation to

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compete equality and even replace current gold standard methods.

Keywords: Forensics; on-site analysis; Point-of-care testing; Crime scene investigation; Drugs of

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abuse; Explosives.

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1. Introduction

The first hours of a crime scene investigation are usually of decisive importance for the police/forensic analysts to get more information and insights about the identity of potential suspects; and to obtain relevant facts and data. Considering this point, the real-time and on-site forensic investigations are extremely relevant to strongly increase the speed and efficacy of the criminal justice system, since it can prevent delays that naturally occur when evidence has to be

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dispatched to and analyzed by forensic laboratories [1], or enables the authorities to find the person until he/she leaves the crime scene or destroys evidence, which is very common. As an example, on-site devices could help to decrease the number sexual aggression suspects with a simple preliminary on-site DNA information on who is the most likely criminal resulting more focus work

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of the police authorities and avoid the necessity of 72 hours of lab analysis to call the suspects to collect more information or evidences.

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Portable sensors, chip-based systems and handheld instruments for in-field analysis of forensic interest samples have been growing up quickly in the last years, resulting in the development of presumptive tests to screening samples. In addition, the development of robust and validated methods capable of providing reliable analyses directly at the crime scene has also received considerable attention. The recent advances may be attributed to the advantages offered by miniaturized platforms including electrochemical sensors, paper-based analytical devices,

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microfluidic devices and portable instruments like mass, Raman and near-infrared (NIR) spectrometers. Some of them include sample-in-answer-out capability, reduced consumption of sample and reagents, low-cost per analysis and minimal instrumental training. The number of forensic analytical methods has been increasing considerably in the last

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decade. Based on the reports found in the Web of Science (WoS) database using the keywords “forensic analytical methods”, the number of publications has demonstrated a noticeable growing.

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When extended to almost two decades, the number of articles using the keyword “forensic chemistry” has revealed a continuous increase since 2000, as denoted in Figure 1A. However, the number of portable analytical devices for forensic purposes only starts to grow in the last ten years. In 2015 van Asten and collaborators [1] wrote a very interesting manuscript dealing with how technology could cause a paradigm shift in forensic institutes "The benefits of real-time, on-site forensic investigations are manifold and such technology has the potential to strongly increase the speed and efficacy of the criminal justice system". As displayed in Figure 1B, the number of records achieved in the WoS database using the keywords “Portable forensics” revealed a representative growth in the last ten years. Based on the total number of publications depicted in Figure 1A, it can

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be inferred that the percentage of articles on “portable forensics” (displayed in Figure 1B) in the period between 2013 and 2018 is higher than 50%. Considering the relevance for forensic chemistry, this review aims to cover the development of portable analytical devices in the last five years to enhance their impact for the on-site crime scene investigation. The review is divided into five main subheadings to separately discuss the contributions on electrochemical sensors, forensic oriented paper-based analytical devices,

NIR spectroscopy.

Number of publications

50

75

50

25

0

40

30

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Number of publications

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microchip electrophoresis, paper spray ionization and portable spectrometers including Raman and

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10

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2000 2002 2004 2006 2008 2010 2012 2014 2016 2018

2000 2002 2004 2006 2008 2010 2012 2014 2016 2018

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Figure 1. Number of publications over the last years found in the Web of Science database using the keywords (A) forensic chemistry and (B) portable forensics.

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2. Electrochemical sensors

Electrochemical methods have several key characteristics which are important requirements

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for on-site and in-situ measurements, such as low cost, rapidity, minimal sample manipulation, high sensitivity, adequate selectivity, easy miniaturization and the existence of commercially available battery-powered equipment [2]. However, electrochemical methods have also some disadvantages, such as difficulties in electrodes handling (usually three electrodes) and need for frequent calibration due to poor stability as a function of time (electrode poisoning and/or fouling problems) [3,4]. Therefore, when the objective is field sensing (outside standard laboratories), the replacement of conventional three-electrode electrochemical cells by easy-to-use sensor strips (e.g. screenprinted electrodes - SPEs) has been successfully carried out [5–7]. In these sensors, all electrodes (working, reference and counter) are printed on the same substrate (plastic, ceramic or silicon) forming an electrochemical cell capable of analyzing a single sample drop (~ 50 µL).

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2.1. Drugs of abuse 2.1.1. Illicit drugs

The attractive characteristics of SPEs (ease of handling and low cost) were explored for the development of portable electrochemical systems dedicated to forensic applications. Cocaine is an electroactive compound and one of the most used illegal drugs in the world, therefore, the use of

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SPEs for the development of electrochemical methods for rapid in situ analyses of this compound was reported in the literature [8–15].

Asturias-Arribas et al. [9] reported the use of a SPE produced with carbon ink mixed with cytochrome CYP450 2B4 (7% v/w) for the determination of cocaine in street samples. The performance of the developed biosensor has been successfully tested for the analysis of the purity of

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cocaine street samples. The capability of detection was estimated at 0.2 mmol L-1. In the following year, the same authors showed the voltammetric determination of cocaine in the presence of three

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different interferences (codeine, paracetamol and caffeine) using a carbon SPE functionalized with multiwalled carbon nanotubes [10]. The modified SPE enabled the analysis of cocaine street samples using ordinary least squares regressions to build the analytical curves. Balbino et al. [11] described a voltammetric method for the determination of cocaine and ∆9-tetrahydrocannabinol in seized samples using a portable potentiostat and commercial SPEs. The best results for the determination of cocaine were obtained with platinum SPE modified with a cobalt hexacyanoferrate

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film. On the other hand, the best results for the determination of ∆9-tetrahydrocannabinol were obtained with an unmodified carbon SPE. According to the authors, both voltammetric methods require small volume of samples and can be applied in routine analysis using portable systems. In

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another study, Ribeiro et al. [12] showed that a carbon SPE modified with a uranyl Schiff base film can be used for the voltammetric determination of cocaine. This drug was electrochemically deposited at -0.80 V (120 s) and exhibited a well-defined irreversible anodic peak at +0.85 V in the

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stripping scan. The limit of detection (LOD) was 110 µmol L-1. Recently, Jong et al. [13] presented an interesting strategy for on-site screening of cocaine and its most common cutting agents (phenacetin, caffeine, levamisole, lidocaine, paracetamol, procaine, benzocaine, diltiazem and hydroxyzine) in street samples using a wearable fingertip sensor. It was possible to demonstrate the feasibility of the proposed sensor as an electrochemical fingerprint approach in solutions and powder street samples. The direct analysis of suspicious powders was made possible by using a flexible gelatin hydrogel as a solid electrolyte. Based on their report, the new concept thus holds considerable promise as a portable screening method. Vidal et al. [14] demonstrated a multi-electrochemical competitive immunosensor for fast determination of unmetabolized cocaine in three different samples (urine, saliva and human serum). The proposed 6

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immunosensor was built on an array of eight carbon SPEs for simultaneous measurements of eight samples at once. The simplicity and cost-effectiveness were emphasized, once low amounts of reagents and a reusable sensor array can be used. In a recent study, Abnous et al. [15] designed an electrochemical aptasensor for the sensitive and selective detection of cocaine. A commercial gold SPE was modified with complimentary aptamers (strands and H-shaped structures), which act as an access gate for the redox probe (Fe(CN)64− / Fe(CN)63−). In the presence of cocaine, the physical

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obstacle for the access of redox probe to the surface of electrode decreases, and the increase in the current of the redox probe is proportional to the concentration of cocaine. The aptasensor was successfully used to detect cocaine in serum with a LOD of 0.27 nmol L-1. Chen and Lu [16] proposed the immobilization of ferrocene-labeled aptamer onto a carbon SPE modified with MnO2

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nanosheets for sensitive screening of cocaine. The sensor showed good specificity of the electrochemical detection of cocaine in the presence of other analytes (thrombin, morphine,

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diazepam and caffeine) as well as a very low LOD for cocaine (32 pM). In addition, other studies have also described the determination of adulterants in cocaine seized samples. Araujo and coworkers described an electroanalytical method for the quantification of aminopyrine in seized cocaine samples using platinum electrode [17]. Salles and colleagues developed a molecularly imprinted modified electrode to monitor phenacetin, one of the most commonly employed adulterant found in seized samples in Brazil [18].

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The electrochemical detection of cocaine was also proposed using conventional three electrode systems and conventional working electrodes, such as glassy carbon, gold, platinum, and boron-doped diamond (BDD) electrodes [19–27]. However, these methods cannot be considered as being truly “portable” due the difficulties with the cell and electrode handling procedure and

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consequent difficulty of sample exchanges (low practicality and low throughput). The study reported by Freitas et al. [24] should be considered an exception because the three electrodes were

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coupled to a batch injection analysis (BIA) system (robust portable system) [4,28]. This system allows around two hundred determinations without electrodes handling and without the need of additional solutions; only a small sample volume (50 – 150 µL) needs to be injected by analysis. In addition, all components that make up the system (electronic micropipette, potentiostat and tablet or laptop computer) are battery-powered, and therefore, ready for use in outside laboratory examinations. The portable system promoted the selective determination of cocaine and screening of the most common adulterants (benzocaine, caffeine, lidocaine, phenacetin, paracetamol, and procaine) in seized cocaine samples. The use of boron-doped diamond (wide electrochemical potential window in anodic region) as the working electrode enabled for the first time the electrochemical oxidation of cocaine in acid medium (0.1 mol L−1 H2SO4). 7

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As mentioned above, the positioning of all electrodes on the same strip facilitated the handling of SPEs for on-site analysis. However, these devices also have problems with stability over time due to phenomena such as poisoning and/or contamination of the electrode surface, similarly to other solid electrodes. Recently, an alternative to minimize this problem was proposed by Richter et al. [29] through the coupling of SPEs to BIA systems. In this approach (BIA-SPE), the analysis can be performed under hydrodynamic conditions (flow through condition) and using small

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volumes of samples (5 to 100 µL) with consequent decrease in electrode contamination and improvement in analytical throughput (> 60 injections h-1). Furthermore, the BIA-SPE system has some requirements of a portable system, such as easy-to-operate, high robustness, excellent cost effectiveness, and low-power requirements. An image (photo) of the compact batch injection

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analysis cell for screen-printed electrodes (BIA-SPE system) is shown in Figure 2.

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Figure 2. Compact portable BIA-SPE system for on-site analysis. (1) Battery-powered electronic micropipette; (2) BIA-SPE cell; (3) Battery-powered potentiostat; (4) Notebook showing examples of voltammograms recorded for a mixture containing phenacetin (PHE), caffeine (CAF), lidocaine (LID) and cocaine (COC).

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2.1.2. New psychoactive substances New psychoactive substances (NPSs) have appeared in the crime scene as supposedly legal drugs in replacement to illicit drugs or controlled substances, such as lysergic acid diethylamine (LSD). 2,5-dimethoxy-N-(2-methoxybenzyl) phenethylamine structures known as NBOMes, as well as their 2,5-dimethoxyphenyl phenethylamine derivatives (2C-X, where X can -Br, -Cl or -I substituent groups), also present hallucinogenic properties and have been found in seized blotting paper. Due to their electrochemical activity, these NPSs have received attention by analytical electrochemists [30–32]. The electrochemical oxidation on glassy-carbon electrode (GCE) was reported presenting a single main oxidation peak at +1.35 (vs. Ag/AgCl/saturated KCl) [30] while 8

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two oxidation processes were observed on a BDD electrode due to its large potential window in comparison with GCE [31]. Similarly to results obtained on the BDD electrode, the electrochemical oxidation of NBOMes on a carbon SPE presented two oxidation processes, leading to oxidation productions that adsorbed on the carbon surface that undergo electrochemical reduction in the reverse scan [32]. A similar electrochemical behavior was verified on the BDD electrode, except to the adsorption of the oxidation products, which make possible the continuous use of this electrode

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for NBOMe sensing [31]. LSD is not electroactive on carbon electrodes and therefore NBOMes can be identified selectively in blotting paper by voltammetry. Considering portable purposes, the use of a SPE is more attractive for NBOMe detection in seized blotting paper even with the adsorptive properties of NBOMes on such surfaces, as a single SPE strip can be replaced for a new

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measurement.

Another new NPS that imitates the effects of cocaine is "synthacaine", a slang term for

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synthetic cocaine, which is basically composed of methiopropamine. The electrochemical sensing of this NPS using graphite SPEs was demonstrated after chemical derivatization [33]. In addition, the combination between paramethoxyamphetamine and 3,4-methylenedioxymethamphetamine has also been found as ecstasy in seized drugs. These compounds can be oxidized on a graphite SPE and thus such a disposable platform can be applied for sensing of this NPS in field [34]. The electrochemical sensing of methamphetamine in human urine using a BDD electrode

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was reported by Švorc and coworkers [35]. An electrochemical irreversible oxidation process was observed at around +1.2 V (vs. Ag/AgCl/saturated KCl) and spiked human urine samples were analyzed, resulting in acceptable recovery values (93-98%). Other strategies were proposed for the determination of methamphetamine, such as the use of a SPE modified with gold nanoparticles on

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multi-walled carbon nanotubes coated by a Nafion film, on which the substance underwent electrochemical oxidation at +0.3 V (vs. pseudo-reference Ag electrode) [36]. In their study, the

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authors showed the possible voltammetric detection of the drug at a reduced oxidation potential but also demonstrated the use of electrochemical impedance spectroscopy (EIS) for the sub-nanomolar detection of the same drug. The voltammetric detection seems to be more attractive for on-site analysis, and the electrocatalytic activity of the modified surface resulted in gain of selectivity [36]. Another electrochemical strategy was based on an amperometric immunosensor formed on a gold electrode, which was modified sequentially with L-cystine, Prussian blue, gold nanoparticles and methamphetamine antibodies [37]. The use of a Prussian blue modified surface was due to its electrocatalytic activity to H2O2 that was selected to amplify the amperometric responses of the modified electrode [37]. The disadvantage of a biosensor is related to its shelf life that is highly dependent on the storage conditions to keep the biomolecule under optimal conditions for the selective detection. 9

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2.1.3. Synthetic cannabinoids The electrochemical detection of tetrahydrocannabinoids, commonly presented in marijuana, was reported [38,39]. The strategy applied for the sensitive detection of this hallucinogenic

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substance is adsorptive stripping square-wave voltammetry on GC or platinum electrodes. After 30 s accumulation on the electrode surface under stirring and application of a negative potential (-0.5 V vs Ag/AgCl/saturated KCl), subnanomolar detection limit was obtained on GCE. New synthetic cannabinoids emerged in the recreational drug market, known as "spike", "K2", etc., found in herbal

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mixtures, incense or room odorizers. The electrochemical sensing of indole- and indazole-based synthetic cannabinoids in seized street samples and artificial saliva was investigated in different working electrodes, including platinum, glassy-carbon and BDD electrodes [40]. The best

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performance for oxidation of these cannabinoids were obtained on platinum and BDD electrodes in acetonitrile, with good agreement with chromatographic techniques. The use of SPEs to monitor cannabinoids urges investigation due to their promising application for on-site analyses of saliva and seized samples. For example, a bismuth-film SPE was applied for the determination of synthetic cathinones, a new group of NPSs, in seized street samples, with good agreement with

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liquid chromatographic analyses [41]. The electrochemical oxidation of this new group of NPSs on graphite SPE was demonstrated and thus it is another strategy for on-site monitoring of this drug [42].

Flunitrazepam, a potent sedative for treatment of insomnia, and other substances of similar

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properties has been purposely added to alcoholic beverages for drug facilitated assaults [43]. The electrochemical sensing of this substance in non-alcoholic and alcoholic beverages using graphite

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SPEs was reported as a fast screening method to identify if the drug is present in suspected spiked drinks. Atropine is another example of drug that has been purposely added into beverages for poisoning and its electrochemical sensing was reported using graphite SPE based on its electrochemical oxidation [44]. These examples indicate that a simple voltammetric scan using a SPE immersed in the suspected beverage can give relevant information on the presence of sedative substances. Portable electrochemical systems have also been proposed for doping analysis. The electrochemical sensing of bumetanide, a banned substance for athletes, in urine samples was described using a GCE modified with reduced graphene-oxide [45]. Depending on the electrochemical profile of the prohibitive substance, electrochemical sensing on SPEs can give valuable information on such molecules in biological fluids. 10

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2.2. Explosives

An electroactive molecule that presents unique voltammetric profile is the explosive

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trinitrotoluene (TNT) and for this reason several reports have devoted to the electroanalysis of TNT and other nitroaromatic explosives, as recently reviewed [46]. The use of carbon SPEs was demonstrated [47,48] for the identification and determination of a wide range of nitro-explosive due to the different electrochemical profiles observed for the different explosives. Therefore, any solid

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suspicious material can be simply diluted in supporting electrolyte and a SPE strip can identify the presence of TNT or other nitro-explosive by a simple voltammetric scan towards the negative

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potential, leading to the electrochemical reduction of the nitro groups occurs (6 proton and 6 electrons for each nitro group) to the respective amino groups, and then towards the reverse oxidation direction resulting in the formation of hydroxylamine groups [46]. Junqueira and coworkers developed a flow-injection analysis method coupled to a copper electrode for the detection of picric acid (PA) and demonstrated the potential use of a portable and disposable device for in-field applications [49].

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Triacetone triperoxide (TATP) and hexamethylene triperoxide diamine (HMTD) are peroxide explosives used in more recent terrorist attacks due to the facile obtaining of reagents for their production. One simple strategy to amperometrically detect TATP or HMTD is based on the conversion of both peroxide-explosives into H2O2 by simple UV irradiation or acid treatment [50].

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The second treatment is more feasible for portable applications considering the simple mixing of the suspicious powder with an acid solution to generate H2O2 that can be selectively detected on a

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Prussian-blue modified carbon SPE, as the Prussian-blue modifier is a well-known electrocatalyst for the electrochemical reduction of H2O2 also called as artificial peroxidase [51].

2.3. Gunshot residues

Another relevant contribution of electrochemical sensing for forensic applications is related to the analysis of gunshot metal residues. SPEs and gold microelectrodes have been proposed for the discrimination of gunshot residues [52,53] An interesting approach reported for sampling and detection of gunshot residues was presented by O’Mahony et al. [52]. They proposed the use of microfabricated carbon sensor strips for abrasive sampling of gunshot residue from the hands of a 11

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suspected person followed by electrochemical sensing in adequate electrolyte. The single voltammetric scan identifies trace concentrations of copper, lead and antimony. A similar idea was proposed for sampling and detection of gunshot residues and trace explosives by printing the collector and SPE sensing platform on a wearable fingertip sensor containing an ionogel electrolyte layer [54]. This completely portable wearable device which does not require sample preparation and liquid electrolyte for further voltammetric experiment is very

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promising for on-site applications in crime scene investigation. The electric contacts of such wearable sensors were not mentioned in this paper. Table 1 summarizes the analytical characteristics of the electrochemical methods using SPEs (portable features) reported for the

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detection of species with forensic interest.

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Table 1. Comparison of portable electrochemical methods employed for determination of analytes with of forensic interest detailing the working electrode as well as the achieved values for the linear range (LR) and limit of detection (LOD). Analyte Technique Working Electrode LR / µmol LOD / µmol L-1 Ref. L-1 Cocaine SWV SPCE modified with 0.001 – 0.001 [8] graphene/ AuNP 0.500 Cocaine CA SPCE mixed with 200 – 1200 20 [9] cytochrome P450 2B4 Cocaine SWV MWCNT-SPCE 0.55 – 155 NR [10] 28.8

[11]

250 – 3720

110

[12]

NR

2

[13]

NR

0.001 (urine), 0.0003 (saliva) 0.000228

[14] [15]

0.000032

[16]

0.89

[24]

23.4

[32]

26.2

[32]

0.0003

[36]

224

[42]

148.3

[42]

1.5x103

[43]

18.4

[44]

NR

NR

[47]

SPCE

NR

NR

[48]

SPCE - KAu(CN)2

NR

NR

[52]

Cocaine

LSV

Cocaine

SWV

Platinum SPE Co2[Fe(CN)6] film SPCE - UO2(4MeOSalen)(H2O)]·H2O films SPCE on nitrile finger

Cocaine

MA

Eight-electrodes SPCE

Cocaine

DPV

Cocaine

SWV

Gold SPE - H-shape aptamers SPCE - MnO2 nanosheets

Cocaine 25I-NBOMe

BIA – SWV DPV

25B-NBOMe

DPV

Methamphetamine

EIS

4’-methylmethcathinone Methcathinone

CV

Bi-film SPE

CV

Bi-film SPE

Flunitrazepam

CV

SPGE

Atropine

CV

SPGE

23.4 – 187.2 26.2 – 209.6 0.00115 0.0269 90.3 – 1974 191.1 – 1225 3.2x 103 – 3.0 x106 5 – 50

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SPCE

CV SWSV

TNT, DNT, RDX, NG TNT, RDX and PETN Gunshot residues

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CV

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119 – 568

Cocaine

BDD

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SPCE

SPCE

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MWCNT-SPCE – AuNP

0.0003 – 0.015 0.0001 – 0.020 19.8 – 98.8

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3. Forensic Oriented Paper-based Analytical Devices

Paper platforms have proven their potentiality and versatility for the development of chemical sensors over the last decade [55]. Paper has many advantages for the manufacture of portable sensors for analyses directly in the field, such as: low cost, large abundance, lightweight and flexibility to transport, disposability, biocompatibility, and many others [55–60]. The structure and physical characteristics of the cellulose enable a natural and spontaneous fluidic transport of

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sample and reagents. This attractive feature enables the creation of paper devices capable of pumping, mixing, separating, and preconcentrating solutions without any extra equipment or power source, allowing the complete integration of a routine laboratory process into a simple paper device [59,60].

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The use of paper-based analytical devices (PADs) for in-field analysis requires the coupling with detectors that can also be portable and miniaturized, as well as inexpensive, enabling a quick

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and easy interpretation of the result (even by unskilled users) to remain attractive for applications in resource-limited settings. Thus, electrochemical, colorimetric and luminescence detectors have been highlighted as the main methods for on-site analysis, especially when associated with the use of smartphones (as simple and widespread readout technology) [57,58,60]. In this section of the review, we will focus on forensic applications using portable paper platforms using different approaches and detection methods, for which we will divide this section

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into the following subheadings: analysis of explosives and warfare agents, detection of drugs of abuse and related applications, analysis of evidence and crime scene investigation (CSI), and other applications. At this section, and due to the lack of any review about paper-based devices and

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forensic application, the timeline window used was 10 years.

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3.1. Explosive analysis

The development of portable methods that allow forensic analysis directly at the point of interest/need with good sensitivity and accuracy is extremely desirable. In this sense, the detection of explosives is of great relevance to tactical and humanitarian demining, forensic criminal investigation ("counter-terrorism" activities and homeland security), as well as remediation of explosives manufacturing sites [57,61,62]. Paper-based devices present potential utility for security-screening of explosives in specimens like luggage or bags in the port regions, for example [63]. Moreover, in a terrorist incident they are useful to screen a large number of individuals or surfaces prior to confirmatory

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laboratory analysis [63], or even, efficiently way to monitor an inappropriate disposal of explosives in soil to detect the presence of environmental hazards directly at the point-of-need (PON) [57,64]. Colorimetric methods for identification and quantification of these hazardous compounds in paper platforms are attractive, since visual detection/inspection is simple, quick and can be performed without the need for extra apparatus [65,66]. A simple change in color or intensity may indicate alertness for critical decision, since it is not expected to civilians carrying, transporting, or

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manipulating any amount of explosives (prohibited by law) [57]. So, a binary "yes" or "no" response to the presence of these compounds quickly and accurate are extremely relevant independent of quantification. Salles and collaborators [57] demonstrated the use of an array of 3 colorimetric spots on filter paper to detect and discriminate 5 explosives (2 peroxi explosives and 3

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nitro compounds) using PCA as chemometric approach. Peters and coworkers developed two microfluidic paper-based analytical devices (µPADs) with five lines (channels) on chromatography

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paper to multiplex colorimetric detection of some improvised explosives and military explosives. Each channel was designed to contain colorimetric reagents capable of reacting with one or more explosive compounds resulting in a specific colorimetric reaction [65]. Luminescent methods are powerful techniques to be coupled on PADs due to the simplicity and excellent sensitivity alloying low LOD's even for in-field analysis [60,65–72], as it can be noted in Table 2. Lu and collaborators [67] presented the fabrication of fluorescent film on paper that

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provides the detection of low-ppm-level of TNT with visible signal change in only 5 s. The authors demonstrated a practical application in real water samples including domestic and river water. Blanes and coworkers [62] developed a fluorescence quenching of pyrene on paper devices that under ultra-violet (UV) illumination can detect until ten different organic explosives using a

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prototype of a portable battery operated instrument to in-field explosive screening. Electrochemical methods are commonly used to detect and quantify explosives, mainly nitro

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compounds due to the possibility to monitor the reduction of the nitro moiety under different electrode materials on paper [61,68,69]. De Araujo and Paixão [68] demonstrated a simple and low cost approach to fabricate silver electrodes on conventional office paper for several applications, among them the detection of picric acid, a military explosive. Wang and coworkers [70] reported the rapid in situ detection of 2,4-dinitrotoluene (DNT) solids on various substrates (e.g., plant leaves, gloves and metal knives) using a sandwiched electrochemical paper-based sensor achieving detection limits lower than 0.33 ng/mm2. Other kind of technique employed in PADs is the Raman spectroscopy, since portable instruments are already commercially available (Table 2). The use of metal nanoparticles, mainly AgNP and AuNP, provides a large enhancement factor to SERS method. Raza and collaborators [71] fabricated a AgNP decorated agar film coated filter paper to SERS test stripes by a simple and 15

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practical approach. These stripes showed high sensitivity for TNT, detecting concentrations lower than 10-9 mol L-1. Wang and coworkers [63] fabricated a silver nanoparticle paper sensor by inkjetprinting and applied for the detection of odor released from the crystalline explosives in the open environment. The authors demonstrated the practical utility of the method for the instant detection of explosive particulate residues in various matrices, like clothing, leather, envelopes, and soil. Table 2 summarizes different methods and their detectability levels for on-site monitoring of

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explosives using PADs.

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Table 2. Comparison of detection methods for different kind of explosives, the concentration range and LOD achieved using portable PADs.

Colorimetric Colorimetric Colorimetric Colorimetric Electrochemical Electrochemical Electrochemical

Ref.

NR

0.4 x 10-3 – 1.0 x 10-1 mg

[57]

NR

2.0 x 10-4 – 1.0 x 10-3 mg

[57]

1 – 200 ppm

7.5x10-6 – 1.5x 10-5 mg

[64]

Nitro compounds (PA, NB, 4A2NP) Peroxi explosives (HMTD, TATP) TNT, TNB, Tetryl Improvised explosives (ClO-, ClO4-, NO3-, NO2-, urea nitrate) Military explosives (TNT, RDX, HMX, PETN) TNT PA PA TNT DNT

NR NR 0 – 1000 ppm

2.64 x10-3 – 2.1 x 10-2 mg

[65]

1.3 x 10-3 – 7.9 x10-3 mg

[65]

2

NR

[66]

3

NR

[61]

3

6.9 ppm

[68]

1.1 x 10 – 4.6 x 10 ppm 1

4.6 x 10 – 2.5 x 10 ppm -4

-3

1.0 x 10 – 1.0 x 10 mg 0.33 – 65.1 ng mm

-2

-4

1.0 x 10 mg

[69]

-2

[70]

0.33 ng mm

2.27 x 10-3 – 1.81 ppm

7.5 x 10-4 ppm

[72]

NR

5 x 10-6 mg

[73]

0 – 45.4 ppm

11.3 ppm

[74]

Nitro aromatics

NR

2.0 x 10-4 – 9.0 x 10-4 mg

[62]

Fluorescence

Nitramines

NR

2 x 10-4 – 8.0 x 10-4 mg

[62]

Fluorescence

Nitrate ester

NR

8 x 10-4 mg

[62]

Fluorescence

TNT

0 – 120 ppm

NR

[67]

TNT

1.1 x 10-5 – 1.1 x 102 ppm

1.1 x 10-5 ppm

[75]

Fluorescence

TNT, DNT, PA

1.0 x 10-5 – 2.0 x 10-4 mg

5 x 10-6 – 2.0 x 10-5 mg

[76]

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Electrochemical Ratiometric Fluorescence Ratiometric Fluorescence Fluorescence and colorimetric Fluorescence

LOD

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Colorimetric

Concentration range

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Colorimetric

Explosive/sample

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Detection method

Fluorescence

PA

2.3 x 10-6 – 2.3 x 101 ppm

7.1 x 10-6 ppm

[77]

Fluorescence

8 nitro compounds

NR

1.4 x 10-3 – 5.6 x 10-3 mg

[78]

Fluorescence

TNT

2.3 x 10-4 – 5.6 x 10-1 ppm

9.6 x 10-3 ppm

[79]

TNT

2.3 x 10 – 2.3 x 10 ppm

Phosphorescence SERS SERS

TNT

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TNT

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Fluorescence

TNT

TNT TNT

-4

-5

-8

-1

-3

3

2.3 x 10 – 2.3 x 10 ppm 2.3 x 10 – 2.3 x 10 ppm

-4

[80]

-9

[63]

-3

[71]

2.3 x 10 ppm 2.5 x 10 ppm 2.3 x 10 ppm

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Overall, a critical point for the potential use of the PADs or any portable method is the assessment of robustness of its analytical response to adversity regarding the complexity of the sample and environment for on-site analysis. From the practical point of view, the composition and purity of the explosives are different from lab-based studies and they may contain contaminants, particulate materials and strong odors. In general, different environmental conditions (temperature, humidity, luminosity, wind and etc) may lead to misinterpretation of the result. So, researchers need

expected in real scenarios. 3.2. Chemical and Biological Warfare Agents

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pay attention to carry out the experiments beyond the proof-of-concept, i.e., closer to what might be

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Other relevant topic to forensic field concerns the analysis of chemical and biological warfare agents. Chemical warfare agents (CWAs) are very toxic synthetic chemicals commonly

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dispersed as a gas, liquid, aerosol or as agents adsorbed to particles to become a powder. These CWAs can cause lethal or incapacitating effects on humans in a war. Amongst the variety of CWAs, nerve agents and vesicants have been weaponized in large quantities and used in military conflicts and in terrorist activities as well [81]. These nerve agents could be lethal within minutes if inhaled due to their ability to irreversibly inhibit the acetylcholinesterase (AChE), a critical central nervous system enzyme [82].

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In a warzone or conflict area, the ability to detect toxic chemicals and explosives is imperative. In order to help this scenario, Pardasani and coworkers [81] described a very interesting microfluidic paper based analytical device (µPAD) for on-site detection of nerve and vesicant agents. The naked-eye detection of analytes was based on their reactions with rhodamine

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hydroxamate and para-nitrobenzyl pyridine, producing red and blue colours, respectively. Under optimized conditions, it was possible to obtain a LOD of 2.5 mmol L-1 to nerve agents like Sarin,

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and around 100 µmol L-1 to vesicant agents like Sulfur mustard. Cinti and coworkers [83] developed an integrated paper-based screen-printed electrochemical biosensor device able to quantify nerve agents. The authors used Paraoxon as nerve agent simulant and they reported the detection of this compound down to 3 µg L-1. River and wastewater samples were spiked with Paraoxon and good recoveries were obtained, demonstrating the potential use in military field (i.e. for nerve agent detection) and civil sector (i.e. organophosphorus pesticide detection). In the biological weapons scenario, Crooks and coworkers [84] demonstrated a PAD to detect ricin. The electrochemical sensor is based on the quantitative detection of silver nanoparticle labels linked to a magnetic microbead support via a ricin sandwiched immunoassay. The authors achieved concentrations as low as 34 pM. Additionally, the assay is robust, even in the presence of 18

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100-fold excess of hoax materials. Table 3 summarizes both chemical and biological warfare agents detected by portable methods based on paper analytical platforms. Table 3. Comparison of detection methods for chemical and biological warfare agents and their concentration range and limit of detection using portable PADs. Concentration range / -1

LOD / mmol L-1

Ref.

mmol L

Fluorescence

Diethyl chlorophosphate

0 – 0.2

0.21

[82]

Fluorescence

Diethyl chlorophosphate

0 – 10

0.015

[85]

Colorimetric

Sarin

20 – 100 and 100-500

7.5

[81]

Colorimetric

Soman

NR

2.5

[81]

Colorimetric

Cyclosarin

NR

2.5

[81]

Colorimetric

O-Ethyl S-diisopropylaminoethyl

NR

3.0

[81]

Colorimetric

Methylphosphonothiolate

10 –75

NR

[81]

Colorimetric

Sulfur mustard

NR

0.1

[81]

Colorimetric

Nitrogen mustard

NR

1

[81]

Colorimetric

Oxygen mustard

NR

0.25

Colorimetric

Paraoxon Aflatoxin B1

Electrochemical

Paraoxon

Electrochemical

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Colorimetric

Ricin

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Chemical or biological

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Detection method

[81]

1 x 10

-4

[86]

0 – 10

3 x 10

-5

[86]

0 – 3.63 x 10-4

1.1 x 10-5

[83]

3.4 x 10-8 – 2.1 x 10-5

3.4 x 10-8

[84]

0 – 0.1

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3.3. Detection of drugs of abuse, cutting-agents and licit drugs

The development of in-field drug analyzers could help the police intelligence in the control

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of the drug traffic, as well as, could help in the development of portable anti-doping devices to obtain fast responses trying to avoid the laborious analysis of a large amount of samples by the official methods considered by other law enforcements. Hence, this section will discuss two approaches associated to the direct detection of drug of abuse and the detection of cutting-agents in drug of abuse samples to understand the drug trafficking. One of the first portable approaches suggesting the use of lab-on-paper device was proposed by Krüger and coworkers in 2009 [84] to detect cocaine based on lateral flow immunochromatographic assay coupled with fluorescence technique as optical readout device. The authors reported a decrease of 10 times in the LOD when compared to the cocaine strip test (300 ng mL-1, www.smarttestdiag.com). The authors used a paper-based detection strip placed between 19

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organic light-emitting diode and a CCD minicamera equipped with band-pass 670 nm filter to extract the analytical information. On the paper substrate was immobilized DyLight649 fluorochrome, instead of an immuno-compound, and the cocaine detection was performed by suppression of the fluorescence compound. Based on the author’s proposition, this method could employ portable instrumentation for detection of cocaine in sweat of professional drivers in the PON. However, there is no discussion in the manuscript about the interfering species or the

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necessity to measure cocaine metabolites, considering that cocaine is metabolized inside the body to benzoylecgonine and other compounds, and consequently they should be found in urine as reported in the literature [87].

The lateral-flow principle was used 4 years after the pioneering work to demonstrate the

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potential of paper-based surface containing a surface-enhanced Raman inkjet-printed material (silver nanoparticles) to detect heroin (9 ng) and cocaine (15 ng) [88]. SERS detection will be

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discussed later in this review. This low cost portable application opened a forensic field to be explored using SERS detection on paper portable devices. In 2014, Erho and coworkers [86] inkjetprinted Anti-morphine Fab M1 and anti-human F(ab′)2 to detect morphine on paper substrate also using the lateral-flow principle. Hence, during the lateral-flow, morphine was captured by the immobilized anti-morphine Fab and the immunocomplex formed was recognized by the goldconjugated anti-immunocomplex Fab indicating the presence of morphine by a color readout. The

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authors achieved a very low LOD for morphine (ca. 1 ng mL-1). This article was the first one reported in the literature using a immunocomplex and a paper-based device to detect a forensic target to test drugs of abuse in diluted oral fluid.

The use of wax as hydrophobic barrier was proposed by Carrilho et al. in 2009 [89] as one

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of the landmark for the renaissance of paper as microfluidic platform for analytical applications. However, only in 2015 portable paper-based analytical devices fabricated using wax printing were

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proposed by McCord and coworkers [90], as shown in Figure 3. The developed device was able to detect presumptive drugs (cocaine, opiates, ketamine, and various phenethyl amines) based on colorimetric measurements using a smartphone with a good applicability and acceptable interfering test.

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Figure 3. Demonstration of the µPAD developed to detect seized drugs: Top - Blank Sample; Bottom- a positive result for morphine. Each lane of the device is labeled with the name and color at which each analyte should appear. Lane 1 ephedrine (Eph), metamphetamine (MA) and MDMA; Lane 2 cocaine (Coc), codeine (Cod), ketamine (Ket) and thebaine (The); Lane 3 codeine (Cod), metamphetamine (MA), MDMA and morphine (Morp); Lane 4 ketamine (Ket) and morphine (Morp); Lane 5 amphetamine (Amp); Lane 6 morphine (Morp) and MDMA. Reprinted from ref. [90], with permission.

A very clever idea combining smartphone detection and aptamer recognition (anticocaine

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aptamer) was used to recognize and quantify cocaine based on luminescence of upconversion nanoparticles [82]. The cocaine concentration was indicated by the quenching of the upconversion gold nanoparticle functionalized with poly(ethylenimine) detecting low concentration of cocaine in human saliva (50 nM) in-field and suggesting a universal platform for field drug testing in future. Additionally, Table 4 summarizes all the PADs used to detect drugs of abuse reported up to now.

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Table 4. Comparison of portable PADs methods for drug of abuse detection and their concentration range, LOD (or MQD). Drug/sample

Concentration range / µg µL-1

LOD / µg µL-1

Ref.

Fluorescence

Cocaine / Sweat (proposition)

1.5 x 10-5 – 3.0 x 10-5

3.0 x 10-5

[91]

Colorimetric

Morphine

0 – 1.0 x 10-4

1.0 x 10-6

[92]

Colorimetric

Cocaine, opiates, ketamine, and various phenethyl amines

Morphine: 12.5 – 100 MDMA: 25 – 100 MA: 12.5 – 50 Amphetamine: 12.5 – 75 Ketamine: 25 – 75 Ephedrine: 6.25 – 25 Cocaine: 6.25 – 25

3.2* 8.7* 5.6* 1.2* 4.1* 2.4* 1.4*

[90]

Luminescence

Cocaine

NR

3.03 x 10-6# 1.5 x 10-5

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*MDQ = Minimum quantity detectable # in aqueous solution in human saliva

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Detection method

[83]

Forensic paper-based devices are also helping to understand drug tracking based on the

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chemical profiling of the street cocaine, i.e. quantification of cutting agents like benzocaine, phenacetin, caffeine, lidocaine, aminopyrine, levamisole, hydrazine, procaine and diltiazem [93]. In this scenario, Paixão’s group proposed two paper-based devices for quantification of procaine [94]

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and phenacetin [95]. In the first approach, authors proposed a colorimetric paper-based device coupling an electrochemical pretreatment and colorimetric detection of procaine without the interfering of common cutting-agents found in cocaine sample and quantifying low quantities of

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procaine in the integrated device (LOD = 0.9 µmol L–1). In the second approach, authors proposed a simple approach to quantify phenacetin in cocaine samples based on a colorimetric detection and office paper-based device achieving a LOD equal to 3.5 µg mL-1 for phenacetin. Both strategies are interesting of the forensic point of view to extract a “chemical fingerprint” of the composition of the seized drugs to aid police intelligence and anti-traffic strategies in field. Licit drugs like ethanol have also been detected on PADs. This kind of application is related to the necessity to detect adulterations in alcoholic beverages or to propose in-field law enforcement applications, like the ethanol detection in blood/breath to decrease the number of road accidents, and forensic investigations. Hence, Zaman and Wu [96] proposed a portable electrochemical paperbased device for ethanol detection in blood using a commercial glucometer. Authors used a paper 22

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patterned with wax, a graphite 3-electrode cell designed by stencil-printing on paper and alcohol dehydrogenase to achieve selectivity. Using this simple approach, the authors proposed a clever and low-cost way to measure ethanol using a portable device measuring concentrations of 2 mmol L–1 (approximately 0.01 % (v/v)). The blood limit of ethanol in blood alcohol in US is 0.08 % (v/v). In 2017, Arduini and coworkers [97] proposed the use of an office-paper modified with Carbon Black, Prussian Blue nanoparticles (PBNPs) and alcohol oxidase (AOx) to monitor electrochemically

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ethanol in beers, which could be helpful to detect adulteration and characteristics of beverages achieving a LOD of 0.52 mM. Most recently, Cardoso et al. [98] demonstrated a simple approach to detect adulterations in alcoholic beverages. In their study, the authors used a low cost colorimetric paper-based device (< US$ 0.02 per sample) to colorimetrically detect adulterations in whisky

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samples counterfeited with caramel.

In the same context of the alcoholic beverages, it has been growing the use of illegal

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compounds added in beverages to put the victim unconsciousness for the practice of crimes. Hence, the detection of these drugs is significant to handle clear of the crimes with these victims. Based on that, Pundir and coworkers [99] proposed an electrochemical microfluidic PAD for sensing ketamine (normally spiked at alcoholic drinks). They used a hybrid based electrochemical microfluidic paper-based analytical device achieving a very low LOD (0.001 nmol mL-1). The PAD was modified with zeolites nanoflakes and graphene-oxide nanocrystals to achieve large surface

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area, tunable surface charge, chemical stability, good electrical conductivity, strong mechanical strength and high thermal conductivity. The device was fabricated using a stencil-painting process and wax printing. The stencils process was used for the fabrication of the two carbon ink electrodes,

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and each one was modified with the nanomaterials by drop deposition.

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3.4. Analysis of evidences and crime scene investigation (CSI)

The detection of latent traces (physical and chemical evidences) is an important aspect of crime scene investigation to solution of the cases. So, in this part of the review, we highlight some aspects of (bio)chemical analysis using paper-based devices that, in our judgment, have potential application in the forensic crime solution. Forensic DNA analysis from biological specimens (saliva, blood, hair, sperm,…) found at the crime scene, usually, is a key to the conviction or exoneration of suspects and the identification of victims of crimes, accidents, etc. All DNA exhibit variability both among and within species, therefore, any biological material associated with a legal process contains information about its source [100]. 23

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Conventional DNA testing is based on methods usually limited to the laboratory. For example, the polymerase chain reaction (PCR) is the most commonly used method for viral load detection. Similarly, the flow cytometry-based sperm chromatin structure assay is the gold standard for sperm DNA integrity assessment [101]. However, DNA analysis is very important for fast screening of suspects who may be involved in a crime; especially when time is of the essence or when large numbers of samples need to be quickly processed. These situations created the need for

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rapid screening of DNA samples. While conventional DNA typing methods afford the best biometric information that provide identity, kinship and geographic origin, they are not fast enough to allow the identification of a suspect’s DNA in real time [102].

In this scenario, paper-based devices appear as an important tool to simplify, make quicker

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and often allow analyses directly in the field of these forensic evidences, supplanting the need for intensive laboratory procedures. Over the last 10 years from the resumption of the use of paper as

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platform to analytical procedures by the Whitesides group [55], many studies have been devoted to the identification/analysis of DNA in PADs, whether with the use of colorimetry [103,104], electrochemistry [105], chemiluminescence [106] and many other methods [101,107–112]. Most of the papers are devoted to a more clinical or genetic bias, but the method or concept could be easily adapted to assist in forensic problem solving.

The examination of bloodstains found at crime scenes is extremely relevant [113], and

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another important analysis in this matrix, besides the DNA, is the blood type, since it enables a first screening of possible suspects. Similarly, there are some studies in the literature regarding the use of PADs for point-of-care identification of the ABO system and even Rh factor of blood samples [114–117]. Garnier et al. [117] described an assay for detection of blood group quickly by

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application of blood to a filter paper pre-soaked with antibodies. The ABO blood group could be detected by observing a plasma separation band from the initial blood droplet that appears with

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agglutinated blood. Later, the same group presented a similar assay for blood typing using 3 µL of sample and performed the validation of the method with 100 different samples demonstrating the viability of the method for rapid analyses in critical situations such as checking the compatibility of the group before the blood transfusion or any situations where no access to laboratory facilities is available [116]. Guan and collaborators [118] developed a simple paper-based blood typing device with barcode-like design that is rapidly read by smartphones. Users can obtain all eight ABO/RhD blood types as text messages on the screen of the smartphone, without the need of further interpretation, which enhanced a practical on-site application. Other interesting forensic-oriented sensors that we can highlight is the determination of the cause of death [119] and the estimation of post-mortem interval [120] using PADs. In the first work, the authors developed a colorimetric approach to detect benzodiazepine alprazolam intoxication in 24

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real blood and vitreous humour (VH) samples with a LOD of 10 ng mL

-1

[119]. In the second

study, Garcia et al. [120] demonstrated an elegant approach to fast estimation of post-mortem interval based on the colorimetric determination of Fe2+ concentration levels in human VH samples using simple wax printed paper-based microzone array and microfluidic devices.

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3.5. Other applications

As previously mentioned, adulterated alcoholic beverages present forensic interest due to possible understanding of a crime practice to a victim. However, non-alcoholic beverages are also relevant from the point of view of the forensic field to understand some other frauds practiced to

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consumers like poisoning and other diseases. In this context, PADs are also used to detect milk adulteration. Some counterfeits used milk adulteration to meet demand and/or increase their profits.

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The MilkPADs, as reported in the literature, was proposed by Liberman and coworkers [121] to detect adulteration in milk samples. In their report, the authors successfully demonstrated the ability of MilkPADs to detect common frauds in this kind of sample like the addition of water, sugar, urea, and starch. The proposed colorimetric paper-based device allowed the detection of starch (0.005% w/v), urea (in excess of 70 mg dL−1), and the sugars (in excess of 0.1 mmol L-1) with a sensitivity and specificity greater than 90% at concentrations that are characteristically found in milk

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adulterated by some counterfeits. The proposed device could be an interesting tool from the perspective of a forensic view to screen adulterated milk in less than 40 min. In the same sense of forensic adulteration and/or falsification of samples, there are studies addressed to the development of portable methods for screening antimalarial drugs and antibiotics,

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for example, Remcho and his group [122] developed a paper-based colorimetric assay for detection of counterfeit Artesunate antimalarial drugs. The authors pointed out that the method is rapid,

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simple, and enables the end user to test the authenticity of the drug. This simple detection technique could benefit government officials and local pharmacies to combat the growing problem of drug counterfeiting. Weaver and Lieberman [123] demonstrated an interesting approach for presumptive testing of very low quality antimalarial medications by colorimetric paper test cards. The paper devices comprise 12 lanes delimited by hydrophobic barriers and different reagents already deposited in these lanes. The screening of various antimalarial medications (chloroquine, doxycycline, quinine, sulfadoxine, pyrimethamine, primaquine) and some fillers usually found in low-quality pharmaceuticals were indicated by color patterns produced on the test cards. The Lieberman’s group used the same approach to fast in-field screening of pharmaceutical drugs containing beta lactam antibiotics or antituberculosis drugs [124]. The devices were able to detect active pharmaceutical ingredients like ampicillin, amoxicillin, rifampicin, isoniazid, ethambutol, 25

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and pyrazinamide and also to screen acetaminophen and chloroquine found in counterfeit pharmaceuticals. Other advantage to be highlighted is the possibility to detect binders and fillers such as chalk, talc, and starch not revealed by traditional chromatographic methods. However, some drawbacks of these test cards include the need to compare with standard results of authentic samples and the fact that different brands may contain different excipients, which can alter the colorimetric pattern. The schematic procedure for analysis of pharmaceuticals using this color test

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cards are presented in the Figure 4.

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Figure 4. Testing a pharmaceutical with a paper analytical device (PAD). A crushed tablet or the content of a capsule is applied to the card. The card is stood upright in water, which wicks up the individual lanes to activate the color tests. After 3 minutes, the card is removed and laid flat, and a photograph is recorded in 3– 5 minutes. Differences between the image of the result and images of test cards run with authentic pharmaceuticals are used to identify suspicious samples. Reprinted from ref. [118], with permission.

Other possible forensic oriented paper-based sensor is devoted to analysis of dyes used to adulterate food and drugs around the world. Usually, these substances provide adverse effects to human health, so their use as food additives is prohibited. However, due to their wide availability

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and low cost, these dyes have been illegally used to improve the product color and to mask illicit behavior of undeclared premarket extraction of effective compounds [120]. Li and coworkers used

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commercially available filter paper functionalized with silver nanoparticles as flexible wiper to collect dye molecules. These substances were detected by SERS on the surfaces of medicinal herbs with detection limits ranging from 10−6 to 5×10−8 g mL-1. It is worth noting that these levels are lower than the minimum concentrations needed to visibly dye the “colorless” herbs [125]. Raza and Saha [126] also described the use of Raman spectroscopy to detect trace levels of colorants. The authors developed a silver-doped agarose gel disk that exhibits property of quenching fluorescence and enhancing Raman signals for the analysis of rhodamine 6G and crystal violet dye, relevant synthetic dyes of forensic interest since these are found in many textiles, cosmetics, food items, writing and printing inks.

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ME devices together with portable electrophoresis systems have emerged as promising platforms for rapid screening and on-site monitoring. When integrated with multiple processing steps including extraction, purification, amplification and detection, ME systems are powerful tools for forensic applications, especially due to their sample-in-answer-out capabilities. Consequently, these

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portable platforms appear as powerful and promising alternatives to be used for crime scene investigations. In the last five years, different authors have successfully described the use of ME

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and integrated devices for forensic genotyping, separation and detection of drugs, explosives and adulterations in alcoholic beverages. The highlighted reports are summarized in this subheading.

4.1. Forensic genotyping

Forensic genotyping is a key step for human identification making possible to extract

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individual molecular fingerprints and genetic markers, which may be used to distinguish one person from another. Short tandem repeat (STR) analysis is the gold standard method for this purpose and it involves multiple steps associated to sample collection, DNA extraction and purification, amplification and electrophoretic separation [109,128,129]. The pioneering studies on STR analysis

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using portable chip-based analyzer were reported by Mathies group [130]. Due to the attractive features offered by microfluidic devices, all the mentioned functionalities are feasible to be

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connected each other towards portable and truly micro total analysis systems (µTAS) [131]. McCord group reported excellent articles on the development of portable platforms for forensic genotyping. In 2013, a rapid thermal cycling procedure combined with a direct amplification from a FTA® paper punch was reported by Aboud and coworkers [128]. This strategy provided a high-speed amplification of a 7-locus multiplex with no extraction step. STR analysis was performed on a Bioanalyzer 2100 system making it possible to obtain a complete separation within 2 min. The proposed technique could be perfectly implemented for using at borders or police stations as well as mass disaster sites once it allows the rapid processing of STR genotype within 25 min. The same group reported in 2015 [132] ultrafast STR separations in less than 80 s with precision of 0.09-0.21 bp over the entire size range. The proof-of-concept was successfully 27

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demonstrated using an allelic ladder from 6 STR loci and amelogenin as sex marker. Recently, the creation of a four locus Y-STR multiplex to screen crime scene samples for male DNA was described by Gibson-Daw and coworkers [102] employing rapid PCR and chip-based separations on a Bioanalyzer 2100 system. These procedures revealed to be helpful for screening suspect and crime scene samples. Kim et al. [133] developed a fully integrated slidable and valveless microsystem for forensic

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genotyping. The device was designed to perform solid phase DNA extraction (SPE), micropolymerase chain reaction (µPCR) and electrophoretic separation coupled with a portable genetic analyzer (see Figure 5). All the analytical functionalities were serially connected by moving a slidable chip fabricated on glass wafer. The proposed platform demonstrated great potential to

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identify amelogenin and four mini Y STR loci from the human whole blood in 60 min using 1 µL of sample. Han et al. [109] described a fully integrated microfluidic device for STR analysis. The

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integrated device consisted of two plastic DNA extraction and amplification chips and one glass capillary array electrophoresis chip. Discs of PDMS and filter paper were inserted between the middle and bottom layers to act as elastic films for microvalve actuation, and a capture phase for DNA extraction, respectively. In their report, buccal swab and venous blood samples were analyzed and all the alleles were resolved and typed, generating a valid STR profile. The total analysis time

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was ca. 2 h.

Figure 5. Presentation of a digital image of a slidable SPE-µPCR-µCE platform for forensic genotyping. Steps labeled as (1), (2), (3) and (4) represent the solid phase DNA extraction, PCR mixture loading, PCR and CE procedures, respectively. The inset photograph displays the entire and portable instrumentation comprising a miniature hardware, a fluorescence detector and a laptop computer. Reprinted from ref [133], with permission.

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ACCEPTED MANUSCRIPT In addition to the mentioned reports, Thompson and coworkers [129] presented a rapid, inexpensive and portable DNA analysis tool based on centrifugal microfluidics for human identification in forensic investigations. The microfluidic platform was based on multiple polyestertoner fluidic layers, a cyclic olefin copolymer (COC) separation channel architecture and integrated gold leaf electrodes. The proposed platform enabled the full reagent loading and delivery by

COC channels provided resolution of 2 base.

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4.2. Drugs, explosives and beverages

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centrifugal force and produced a 6-plex STR profile in less than 5 minutes. The separation step on

Lloyd and coworkers [134] developed a methodology based on an Agilent 2100 Bioanalyzer

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for the rapid screening and comparative analysis of synthetic cathinones commonly found in illicit tablet seizures in New Zealand. Four cathinones were successfully separated and detected within 1 min. The use of commercial ME devices demonstrated to be a promising and practical alternative to a relevant problem in forensic drug analysis. In comparison with conventional techniques, the presence of tablet components and their relative amounts were determined on ME devices in a costeffective and timely manner.

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A simple method for the identification of four substituted amphetamines employing a portable capillary electrophoresis (CE) integrated with capacitively coupled contactless conductivity detection (C4D) was reported by Nguyen and coworkers [135]. The authors successfully detected these compounds in different seized illicit club-drug tablets and urine samples

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collected from different suspected users. The achieved results were comparable to those obtained by GC-MS. Evans et al. [136] have also used a portable CE system equipped with a dual C4D to

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investigate the ionic profile of pharmaceutical samples and precursors of two illicit drugs. Cationic and anionic species were separated within 6 min with baseline resolution. The developed method revealed great potential for the identification or differentiation of unknown tablets, and real samples found in illicit drug manufacture scenarios. Rollman and Moini [137] described a portable ultrafast capillary electrophoresis (UFCE) method for chiral analysis of drugs as amphetamine, cathinone, nor-mephedrone and pregabalin within 2 min. The performance of the proposed method was reported using a non-portable MS detector. However, the UFCE device can be interfaced to portable ESI-MS instruments. Ueland and colleagues [78] reported the analysis of trinitrobenzene, 1,3-dinitrobenzene, 2,4,6-trinitrotoluene, methyl-2,4,6-trinitrophenylnitramine, 3,4-dinitrotoluene, 2-amino-4,6-dinitrotoluene, 4-amino-2,629

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dinitrotolune and 2,4-dinitrotoluene in soil samples using commercial ME devices coupled with laser-induced fluorescence detection. Interestingly, prior to ME analysis, the authors used wax printed µPADs to extract explosives from soil. The extracted target compounds were separated in their neutral forms by micellar electrokinetic chromatography (MEKC) on ME devices. Sáiz and coworkers [138] explored a portable CE-C4D system for the rapid determination of scopolamine (a drug with hallucinogenic effects) in alcoholic drinks and moisturizing cream. The

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optimized method allowed the detection of scopolamine in six different beverages within ca. 200 s. In 2016, Rezende et al. [139] reported the use of ME devices coupled with C4D to investigate the authenticity of seized whiskey samples. The authors used commercial glass devices for monitoring negatively charged inorganic species in original and seized samples. Based on the presence of Cl−,

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F−, SO42− and NO2− in different amounts, the authenticity of seized whiskeys was compared to the profile recorded for original samples. Considering the peak intensities recorded for Cl− and F−, 90%

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of the analyzed samples were correctly identified as adulterated.

5. Ambient Ionization on Portable Mass Spectrometers

In the last years, mass spectrometry (MS) has become a powerful tool for forensic analysis due to its great ability to identify many target analytes based on mass to charge ratio. Most of recent instrumental advances has been dedicated to the development of portable systems to be used in

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airports, seaports, police operations and borders inspections for scanning suspected illegal actions. Different authors have demonstrated forensic applications using several ambient ionization sources including direct analysis in real time (DART) [140,141], desorption electrospray ionization

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(DESI) [142–145], electrospray ionization (ESI) [144], atmospheric pressure chemical ionization (APCI) [143,145], leaf spray [146], low-temperature plasma (LTP) [147], swab spray (SS) [148] and paper spray ionization (PSI) [144]. This latter has emerged as one of the fastest techniques with

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ability to provide a forensic screening. In the last five years, many publications have been reported describing applications of PSI associated with abuse drugs [142–144, 147,149–152], pen inks [147,153–156], alcoholic beverages [157,158], questioned documents [147], banknotes [147,159], perfumes [160] and abused prescription drugs [144]. Although PSI has successfully demonstrated great potential for forensic investigations, most of the reports found in the literature employed benchtop instruments, which are bulky, expensive, and heavy, thus making difficult their use for onsite applications. In this way, miniature MS systems have been recently developed enabling their use for studies in the field. One of the commercially available examples is the FLIR Systems AI-MS 1.2 cylindrical ion trap (CIT) mass spectrometer. This instrument presents a modest size (ca. 60 cm 30

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long × 50 cm width × 38 cm height) and weight (44 kg). This system was explored by O`Leary et al. [143] to monitor clandestine synthesis of methamphetamine. The same group used a reference library to perform the identification of 25 positive controls, 4 negative controls and 3 authentic evidences of samples with forensically relevant analytes [144]. The results showed that portable MS with automated reference library provides rapid and accurate chemical identification. Even presenting reduced resolution, the portable system provided correct information for all analyzed

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samples. The analysis protocol developed in this equipment was analytically validated to provide high throughput forensic evidence screening [145]. The authors reported that the system reliability was relatively unaffected when investigating low complexity samples.

Hall et al. [142] described the trace-level detection of chemicals related to desomorphine

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production, which is the active principle in the drug known as “Krokodil”. Brkić et al. [161] developed a portable non-scanning mass spectrometer in the configuration of linear ion trap for the

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detection of cocaine simulants (methyl benzoate), TNT (2-nitrotoluene) and sarin (dimethyl methylphosphonate). Other example of portable MS (weighing less than 18 kg) for forensic application was presented by Giannoukos and coworkers [162]. In their report, the monitoring of male human chemical signatures and odor signatures emitted from threat or hazardous substances was successfully demonstrated.

Wiley et al. [147] built a handheld, wireless low-temperature plasma (LTP) probe as

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ambient ionization source and successfully demonstrated the direct finger analysis of methamphetamine. The handheld source exhibited similar or slightly better analytical performance, when compared to the analysis performed on a benchtop MS system. The probe was coupled in a miniature MS (weight of ca. 10 kg) developed around 10 years ago [163]. Hendricks et al. [164]

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described the autonomous in situ analysis and real-time chemical detection of illicit drugs, explosives, and pharmaceuticals using a backpack miniature (see Figure 6) mass spectrometer

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(weight of ca. 15 kg). DART and portable MS were explored by Brown and coworkers [141] for a rapid identification of designer drugs on-site. The authors used a compact MS system (30 cm × 43 cm × 50 cm) weighting ca. 35 kg. The proposed method offered potential for drug analysis and only very closely related isomers were not distinguished. In 2016, Bernier et al. [140] performed a comparative study using a benchtop highresolution MS equipment and a portable low-resolution single quadrupole instrument. Both instruments were equipped with a DART ionization source. In their study, anti-malarial tablets were used as target due to its high falsification incidence in Africa. The authors did not observe any noticeable difference between the data recorded with both instruments. In 2017 Bruno et al. [165] reported a study using portable PSI-MS to detect trace of drugs and medicines in the crime place. The target analytes were fentanyl, synthetic drugs (2C-I, mephedrone and α-PVP), heroin, cocaine, 31

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codeine, guaifenesin and nicotine. The samples were collected using a paper swab in different surfaces of common use like, for example, cup, car steering wheel and seat belt, glass and identification card. The obtained results showed that PSI-MS can be used as a powerful tool to

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routine analysis in law enforcement activities.

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Figure 6. Example of (a) backpack miniature mass spectrometer for in situ applications including (b) cocaine detection. Reprinted from ref [164], with permission.

In addition to the mentioned examples, it important to highlight two excellent reports from Verbeck group. In 2015, Mach et al. [166] described the mounting of a portable MS system in a vehicle to detect residues associated with the methamphetamine synthesis by clandestine

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laboratories. Chemical information was extracted and mapped versus longitude and latitude positions supplied by global position systems (GPS). In 2016, Giannoukos et al. [167] reported a review article showing a wide range of technologies able to identity and detect volatile organic

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compounds, chemical signatures and threat compounds. As described in this section, ambient ionization techniques have demonstrated great feasibility for on-site forensic investigations. Table 5 summarizes the LOD for different analytes detected in

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samples of forensic interest using several ionization sources.

5.1. Portable GC/MS Instrumentation

In 2005, Laughlin et al. [168] reported the developed and use of miniature MS with atmospheric pressure ionization (API) to detect chemical warfare agents and medicines. This pioneering report enabled the use of this system for applications in the PON like airport inspection and narcotics operations due to, mainly, its small dimensions (46 × 50 × 38 cm), weight (38 kg) and short power consumption (210 W). The LOD´s achieved for warfare agents and medicines were < 1.24 ppb and < 100 µM, respectively. In addition to above-mentioned instrumentation, several companies like 32

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Agilent Technologies, CDS Analytical, D-tect Systems, Bruker Corporation, Scintrex Trace Corp., FLIR Systems Inc, Smiths Detection Inc., Torion Technologies, Inficon and Perkin Elmer have developed portable gas chromatography (GC) systems equipped with MS, demonstrating high potential to be used in forensic studies. In the last years, diverse other GC/MS systems were successfully developed. In 2015, Visotin and Lennard [169]reported a method to detect ignitable liquid residues (ILR) at a fire scene using a

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portable GC/MS, named as TRIDION-9®. In their study, the authors successfully detected ILR in a total of 38 simulated samples. In 2016, Leary et al. [170] showed an interesting overview about the current state of portable GC/MS instrumentation. In their report, different application examples associated with the detection of counterfeit drugs, explosive residues and ignitable liquid residues

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were successfully demonstrated. The developed methodologies offered fast results (< 3 min) and offered a huge potential to be used in the PON by emergency responders, military and law-

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enforcement officers or organizations.

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Table 5. Presentation of the different ionization sources for the forensic analysis of potential target analytes and their respective LOD values. Ionization Sample Analyte LOD Ref. source Acetone, Butanone, Diethyl Flammable 0.5-2.0 ppm Ether, Isopropanol [145] APCI Solvents 1:100 dilution Turpentine Amitraz, Atrazine, Buprofezin DEET, Diphenylamine, Drugs, Ethoxyquin, Isofenphospesticides and 0.001–300 ng [147] LTP methyl, Isoproturol, Malathion, explosives Parathion-ethyl, Terbuthylazine RDX, TNT, PETN and Tetryl Organic gunshot Methyl-centralite and Ethyl50 ng [148] SS residue centralite Desomorphine 0.50-150 ng Street Drug [142] DESI Codeine 0.90–350 ng Cocaine, Codeine, Drugs Methamphetamine, 2C-B 200–1250 ng [145] DESI 25B-NBOME Desmorphine 2.0–8.0 ng Drugs [142] PSI Codeine 3.0–10 ng Cocaine, Codeine, Drugs Methamphetamine, 2C-B 5.0–345 ng [145] PSI 25B-NBOME MDA, MDMA, MDEA, mCPP, Drugs 0.17–1.0 ppb [146] PSI MA, Cocaine, LSD and DOB Cocaine, Levamisole and Drugs 6.51-351.08 µg mL-1 [150] ESI Lidocaine 33

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Drugs

PSI

0.04-12 ng mL-1

[152]

6. Raman and NIR Spectroscopy Raman and NIR spectroscopy can be considered two of the most widely explored techniques

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in forensic chemistry. When coupled with chemometric tools, these analytical platforms become extremely powerful to generate fingerprinting and library of compounds. In addition, instrumental advances in both Raman and NIR techniques contributed to the miniaturization of common benchtop equipment enabling their use for on-site applications. Furthermore, a remarkable

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advantage of these techniques is the possibility to carry out non-destructive analysis. In general, NIR and Raman techniques have been extensively used for forensic studies involving drug and

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explosive analysis, beverages screening, blood, gemstone and mineral analysis. The main examples found in the literature are summarized in Table 6 or presented in the next subheadings.

Table 6. Comparison of portable IR spectroscopy, SERS and RAMAN instruments for the analysis of samples of forensic interest including the LOD for key analytes. Sample

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Technique

Analyte

LOD

Ref.

Saliva and drugs

Cocaine

500 ppm

[171]

SERS

Saliva and fingerprints

Cotinine Benzoylecgonine

8.8 ppb 29 ppb

[172]

Drugs

JWH-018 JWH-073 JWH-081 JWH-122

18 - 51 ppb

[173]

SERS

Drugs

Heroin Cocaine

9 ng 15 ng

[88]

RAMAN

Alcoholic beverages

Flunitrazepam

<0.01% w/v

[174]

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SERS

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IR spectroscopy

6.1. Drugs and explosives

34

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Wägli et al. [171] presented a portable microsystem to detect cocaine in human saliva. Basically, the microfluidic platform included a droplet-based liquid-liquid extraction to transfer cocaine from saliva to the infrared transparent organic solvent. The optofluidic system was able to analyze cocaine in undiluted saliva, making this portable platform quite suitable for point-of-care testing. Ali et al. [175] successfully demonstrated the analysis of abuse drugs like cocaine and

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amphetamines by Raman in association with principal component analysis. This tool revealed great potential to be applied in seaports and airports. The feasibility of SERS instruments for forensic applications was also explored by other authors to detect trace levels of drug-related biomarkers in saliva [172], to quantify caffeine and its two major metabolites in tertiary mixtures without need of

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analytical separation [176], to analyze synthetic cannabinoids in simulated urine samples [173], to screen ricin toxin on letter papers [16] and to investigate the subtyping of avian influenza viruses

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[177].

Oliveira and coworkers [178] reported the analysis of flunitrazepam in necrophagous insects by NIR spectroscopy and variable selection techniques. The identification of flunitrazepam in Chrysomya megacephala larvae, puparia and adult was applied as an alternative to determine the time of death. In addition, this strategy has also contributed to the qualitative identification of abuse drugs present in the corpse.

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Tsujikawa et al. [179] developed a library search-based screening system for 3,4methylenedioxymethamphetamine (MDMA) in ecstasy tablets using a NIR spectrometer. The authors reported that ca. 80% of the tablets were correctly classified as MDMA-positive. Due to

of MDMA tablets.

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acceptable discrimination percentage, the proposed system can be a useful tool for on-site screening

Hopkins and coworkers [180] used a man-portable Raman improvised explosives detector

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(PRIED) to provide a rapid, standoff detection of chemicals with high potential to be used as targets for forensic investigations. The PRIED was assembled in a backpack containing the power supply, chiller, computer, spectrometer and detector (see Figure 7A). This portable instrument allowed the identification of unknown materials from distances between 1 and 10 m. The weight of this portable system was ca. 14 kg. The proposed system was used to detect heroin, cocaine and excipients, as denoted in Figure 7B. Nuntawong and coworkers [181] showed the detection of perchlorate anions in industrialgrade emulsion explosive using portable SERS. The proposed technique offered high sensitivity allowing the detection of minimal trace amount of perchlorate in five explosive samples with short operating time. 35

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Figure 7. Presentation of (a) backpack PRIED for the detection of (b) cocaine and (c) heroine in samples of the investigated alcoholic beverages. Reprinted from ref [180], with permission.

6.2. Beverages

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Kwiatkowski and coworkers [182] explored the surface-enhanced Raman spectroscopy (SERS) to detect denatonium benzoate (Bitrex) remnants in noncommercial alcoholic beverages. Bitrex is a bitter chemical compound often used as marker to illegal alcoholic beverages fabricated using technical alcohol. In general, the state income tax for the technical alcohol is cheaper than genuine alcoholic beverages and, for this reason, many countries have lost large amount of state

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income tax due to the adulterations. The authors successfully demonstrated that SERS can be a powerful portable technique to correctly recognize genuine and suspected samples. Raman spectroscopy was explored by Ali and coworkers [174] to detect rape drug (flunitrazepam) in alcoholic beverages including vodka, gin and rum. This kind of drug is known as

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“forget pill” and it is often used for induction of general anesthesia. The authors demonstrated that this technique can be efficiently used for in situ identification of rape drugs in beverages based on

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the monitoring of the symmetric NO2 stretch at 1335 cm-1.

6.3. Blood analysis

In 2017, two research groups independently described the use of portable Raman spectroscopy for blood analysis. Sikirzhytskaya coworkers [183] successfully showed the ability of this technique to determine the gender using a bloodstain. The association of the genetic algorithm coupled with artificial neuron network provided accuracy of ca. 98%. Fujihara and coworkers [184] reported the possibility of using Raman spectroscopy at a crime scene. In their report, the blood identification and discrimination between human and non-human blood was demonstrated using 36

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bloodstains from 11 species including human, mouse, rat, cow, horse, sheep, rabbit, dog, and chicken. The proposed technique was able to analyze a bloodstain sample of at least 3 months old.

6.4. Gemstone and minerals

Raman spectroscopy has been also explored for gemstone and mineral analysis. Ali et al.

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[185] reported the possibility to investigate the authenticity of gemstone using laser excitation at 785 and 1064 nm. In their study, this non-destructive analysis was effective to discriminate between genuine and fake lapis lazuli specimens, which are commonly employed to fabricate complex jewelry and furniture. Lin and coworkers [186] evaluated three portable or handheld Raman

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spectrometers to analyze uranium in industrial and laboratory samples. Although each spectrometer uses different laser wavelengths, the performance of all the three instruments was quite similar, thus

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demonstrating great potential to be used in nuclear forensic applications.

7. Conclusions, comparison and future steps

Overall, a wide range of analytical methods are applicable to real-time forensic analysis directly at the crime scene. Efforts have been made to miniaturize and modify well-established

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techniques that are usually limited to lab bench into portable analytical method as well as their coupling with portable and low-cost platforms such as paper. This review has summarized and critically discussed the advances on portable analytical platforms for forensic investigations reported in the last years and we can recap the reported

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portable methods reviewed here in order to indicate potential future approaches: (1) detection of potentially explosive compounds can be carried out directly or indirectly (after derivatization

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protocol) by electrochemical techniques with good sensitivity and rapid response (seconds range qualitative analysis (voltammetric recording) or a few minutes - quantitative determinations (calibration)). Colorimetric methods are interesting to identify, since for screening purposes, no matter the quantity, the carrying of this type of material is prohibited. However, in all cases, it is essential to evaluate carefully potential interferences, since it is crucial to avoid false negatives (dangerousness) and very few false positives are also desirable (wrong judgments); (2) In the scenario of drugs of abuse and related applications, there is a wide range of approaches using paperbased devices that generally provide results similar to electrochemical and microchips electrophoresis methods (throughput and sensitivity), except for analyzes of new psychoactive substances (NPS) that few studies were already addressed to those, being an important field to develop due the risk to public health and a challenge to drug policy. In this sense, the constant 37

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development of portable mass spectrometers and NIR analyzers provides a powerful way to identify these substances. These truly portable instruments have offered infinite advantages for forensic applications including rapid, sensitive and accurate analysis of abuse drugs, banknotes, questioned documents, and signatures as well as the real possibility to use them at airports, seaports and police operations at borders between neighboring countries. Paper-based sensors still have lower sensitivity and selectivity when compared to classical

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instrumental detectors. However, it has advantages such as easy multiplexed detection, either by using spot tests or by delimiting microfluidic paper channels that split the sample into several detection zones (different chromophores) allowing the identification of several species simultaneously. Validations of the portable methods are critical for acceptance in forensic cases by

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court and this should be considered during the development of methods. Another aspect that we believe to increase the versatility of portable devices is the coupling of wireless data systems for

and allow rapid decision making.

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data acquisition and transmission and simpler readout interfaces for operators to reduce subjectivity

At least but not less important, in the last years forensic DNA (or forensic genotyping) lab bench analysis has been fully automated using rapid PCR and STR, and rapid PCR could provide information in less than 90 minutes instead of 4 hours of conventional test, but the costs are higher than the convention DNA protocols [10.1098/rstb.2014.0252] and a full 15-locus (a fixed position

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on a chromosome) STR profiles could be generated in a laboratory setting in less than an hour [10.1002/elps.201400179]. In this field, faster analysis has been reported and the direction of this field shows that more time have to be spent with how improve the understanding genotypes results, as well as, how to deal with complex DNA mixtures from more than two different individuals

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[10.1098/rstb.2014.0252], showing that more time need to be spent with understanding the measurement extracted than with the development of the new analytical method.

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The portable genotyping methods reported in this review are more focus on miniaturization for in-field application with short-time analysis and the initial stage the analytical steps where the researchers and trying to show paper-based analytical approaches outside of a typical laboratory environment without to sacrifice quality and maintained, or enhanced, the speed of the proposed forensic portable method with a low-cost approach. Interesting results, but in initial stage, related to the identification of ABO blood system and to Rh factor are proposed and they could be useful for in-field analysis and detect individuals based on this information as pre-screening sample until an validated forensic method is used for law-enforcements. At this point and an important north for future portable method in this field, and others reviewed here, need to be highlighted in validation to compete equality with the gold standard methods used nowadays. 38

ACCEPTED MANUSCRIPT Acknowledgments

The authors are grateful for the Brazilian agencies’ CAPES (Grants Number 3359/2014, 3363/2014, Pro-Forenses Edital 25/2014), CNPq (307333/2014-0; 307271/2017-0; 308140/2016-8), FAPESP (Grant Numbers: 2017/10522-5 and 2016/16477-9, FAPEMIG (CEX - APQ-02118-15), and

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FAPEG.

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Highlights • Portable platforms have emerged as powerful tools for forensic applications.

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• Electrochemical sensors offer good sensitivity for abuse drugs and explosives.

• Paper-based devices have revealed desirable performance for point-of-cate testing. • NIR and RAMAN instruments have allowed fast screening at the point-of-need.

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• Portable MS instruments have exhibited good performance for on-site forensic applications.

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• Electrophoresis chips have provided excellent ability for STR genotyping.

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Biographies

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William R. de Araujo (Post-Doctoral Researcher): He received both bachelor's degrees in Science and Technology and in Chemistry from the Federal University of ABC (UFABC) in 2011. In 2016, he concluded the PhD in the Institute of Chemistry at University of São Paulo under the supervision of Dr. Thiago Regis Longo Cesar da Paixão. He is a postdoctoral fellow in the group of Professor Lucio Angnes at the same university. His fields of interest include, but not limited to development of portable chemical and electrochemical sensors, electroanalysis, new materials applied to (bio)sensors and forensic samples analysis.

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Thiago M. G. Cardoso received his BSc (2012) and MSc (2014) in Chemistry at the Federal University of Goiás. Currently, he is a third-year Ph.D student in Chemistry at the same University. From 2015 to 2016, he was a visiting scholar at the Colorado State University (USA) under the supervision of Professor Charles S. Henry. His main research interests involve instrumental development, paper-based microfluidic devices, clinical assays, forensic chemical analysis, colorimetric and electrochemical detection.

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Raquel Gomes da Rocha received is graduated in Chemistry from the Federal University of Uberlândia, Uberlândia, Brazil (2017). She is currently pursuing her master at the Institute of Chemistry of the Federal University of Uberlândia (Uberlândia) under the guidance of Prof. Eduardo Richter on forensic electrochemistry.

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Mario H. P. Santana (Senior Forensic Expert): He received a BSc (1997), MSc (2000) and PhD (2005), followed by a postdoctoral fellowship between 2005 and 2007 working with electrochemical and advanced oxidation processes at the University of São Paulo and Federal University of Uberlandia. He was then appointed as a Forensic Analyst in Brazilian Federal Police, working in Forensic Chemistry Laboratory and Crime Scene Group. In 2010, he was selected for working in the headquarter's Federal Forensic Chemistry Laboratory where we stayed for almost three years; since then, he manages the Forensic Unit of Federal Police in Uberlandia. His fields of interest include forensic analysis of drugs, pesticides and counterfeit products and development of new chemical sensors and portable devices for forensic applications. Rodrigo A. A. Munoz is graduated in Chemistry from the University of São Paulo, Brazil (2002), and received his Ph.D. in Analytical Chemistry from the same university in 2006. He completed a postdoctoral research at the Arizona State University (USA) during 2006–2007 and a postdoctoral research at the University of São Paulo during 2007–2008. He is currently Associate Professor of Chemistry at the Federal University of Uberlândia, Brazil. His current research interests focus on the development of

ACCEPTED MANUSCRIPT improved analytical methods and sensors emphasizing the use of modified electrodes associated with flow-injection and batch-injection analyses.

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Eduardo M. Richter is graduated in Chemistry from the University of Santa Cruz do Sul, Brazil (1994), and received his master’s (2000) and Ph.D. degree (2004) in Analytical Chemistry from the University of São Paulo, Brazil. He completed a postdoctoral research at the University of São Paulo, Brazil during 2005. He is currently Associate Professor of the Institute of Chemistry at the Federal University of Uberlândia, Brazil. His current research interests focus on the development of new analytical methods using capillary electrophoresis with conductometric detection and flowinjection and batch-injection analyses with amperometric detection.

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Thiago R.L.C. Paixão (Associate Professor): He received a BSc (2001), MSc (2004) and PhD (2007), followed by a postdoctoral fellowship between 2008 and 2009) working with electronic tongue devices at the Institute of Chemistry of the University of São Paulo. He was then appointed as an Assistant Professor at the University Federal of ABC where he stayed for two years. In 2011, he was hired as an assistant professor at the University of São Paulo and promoted to Associate Professor in 2016. In the beginning of 2018, he was nominated as affiliate member of the Brazilian Academy of Science as a young promising researcher. His fields of interest include chemical sensors, paper-based devices and electronic tongues aiming at forensic and clinical applications.

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Wendell K. T. Coltro obtained his BSc in Chemistry from the State University of Maringá (2002). He received his MSc (2004) and Ph.D. (2008) in Analytical Chemistry from the University of São Paulo (in the Institute of Chemistry at São Carlos. In 2006, he was a visiting scholar at The University of Kansas (USA) under the supervision of Professor Sue Lunte. He is currently Associate Professor of Chemistry at the Federal University of Goiás, Brazil. In the beginning of 2018, he was nominated as affiliate member of the Brazilian Academy of Science as a young researcher. His research interests involve the development of electrophoresis chips, electrochemical sensors, toner- and paper-based devices as well as 3D printed microfluidic chips for applications in bioanalytical and forensic chemistry.

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Author’s Photos

Thiago M. G. Cardoso

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Raquel G. da Rocha

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William R. de Araujo

Eduardo M. Richter

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Rodrigo A. A. Munoz

Mario H. P. Santana

Thiago R.L.C. Paixão

Wendell K. T. Coltro