Paper-Based Microfluidic Devices For Forensic Sciences: Development And Validation Of Innovative Tools

Laboratory analyses usually require sophisticated equipment and skilled personnel. However, very often, the use of simplified procedures and techniques in various working conditions can prove useful in solving several problems. Forensic applications represent an emblematic example of this requirement to simplify and solve various difficulties. Legal medicine requires both highly sophisticated techniques to solve analytical problems and simple techniques with fast procedures that allow analyses to be carried out outside the laboratory, in remote areas by unqualified personnel. The implementation of paper-based microfluidic analytical devices (PADs) for legal medicine presents an opportunity to tackle all the aforementioned limitations and requirements in the field of forensic science. It is also important to note that little to no sample preparation is required, with the ability to use many samples directly onto the PADs for the analysis of the desired analyte. PADs are very inexpensive to produce and ca

Paper-based Microfluidic Devices for Forensic Sciences: Development and Validation of Innovative Tools

Laboratory analyses usually require sophisticated equipment and skilled personnel. However, very often, the use of simplified procedures and techniques in various working conditions can prove useful in solving several problems. Forensic applications represent an emblematic example of this requirement to simplify and solve various difficulties. Legal medicine requires both highly sophisticated techniques to solve analytical problems and simple techniques with fast procedures that allow analyses to be carried out outside the laboratory, in remote areas by unqualified personnel. The implementation of paper-based microfluidic analytical devices (PADs) for legal medicine presents an opportunity to tackle all the aforementioned limitations and requirements in the field of forensic science. It is also important to note that little to no sample preparation is required, with the ability to use many samples directly onto the PADs for the analysis of the desired analyte. PADs are very inexpensive to produce and can be produced in resource deficient and remote areas. They are rapid, require minute sample volumes, are easy to use and do not require skilled technicians. The use of PADs for various forensic science applications has been well investigated by many research groups with several other areas to explore [1] . To date, a wide range of drugs can be identified and/or quantified by PADs. These include but not limited to, illicit drugs such as amphetamines, cocaine, morphine and codeine as well as the identification of substandard drugs, which usually occur when the amounts of active pharmaceutical ingredients (APIs) are altered in pharmaceutical drugs, in antibiotics and in herbal supplements which are not heavily regulated[2-5] .These devices have also been used for forensic serology, in the evaluation of gunshot residue and in the ground-breaking application of the estimated time of death by establishing the post-mortem interval, experiments which can all be performed at the scene of the crime [6-8]. In 2020, an innovative development in the implementation of PADs in forensic science was achieved as Azuaje-Haulde et al developed a device with the ability to identify the Y human amelogenin gene, identifying male from female human specimen [9]. PADs have also shown utility in the identification of agents of warfare like explosives and chemical warfare agents like nerve agents, which can be instrumental for military intelligence and defence whose aim is to acquire intel before, to protect civilians and after, to identify the components and if they are linked to a specific group or organization [10, 11]. Based on the application of these devices in other forensic contexts, the core of my research was the implementation of PADs in forensic science aspects which required point-of-need platforms to be more beneficial to doctors, forensic scientists and investigators. This PhD thesis involves: • The use of microfluidic devices to determine the estimated time since death. • The development of a paper-based microfluidic device as a preliminary screening test for drugs of abuse in urine. Additionally, as part of my research period abroad, an emphasis was placed on exploring different fabrication methods to create devices that were resistant to more chemically aggressive assays. The final project discussed is: • The development of a paper-based microfluidic device capable of conducting Recombinase Polymerase Amplification (RPA) for the detection of carbapenemases

Paper-based microfluidic devices: On-site tools for crime scene investigation

The use of paper-based microfluidics devices (μPADs) for designing rapid, easy, and on-site devices has undergone a dramatic increase in the last few years and the development of applications in the field of forensic science has had concomitant advances. The use of μPADs at the crime scene is finalized not only to obtain a rapid response but also to have crucial information on evidence collection. Several authors have proposed μPADs as alternative approaches to existing tests. These devices are designed to be used on-site without, or minimal, power sources. Furthermore, the technology provides prompt-readouts using a small sample volume, preserving important evidence for instrumental analysis. This review aims to provide a critical evaluation of developed μPADs. The document includes a brief overview on fabrication methods and on detection strategies, and it assesses advantages and limitations of applications related to forensic matters. A final section is devoted to future developments

Simultaneous analysis of potassium and ammonium ions in the vitreous humour by capillary electrophoresis and their integrated use to infer the post mortem interval ({PMI})

Post-mortem changes of ions in the body fluids have been proposed as an objective tool for inferring the time of death. In particular, the post-mortem increase of potassium concentrations in the vitreous humour has gained great attention in the literature. On the other hand, ammonium, another ion released in post-mortem processes, has received much less attention, potentially due to unresolved analytical issues using current clinical chemistry methods. This paper presents an application of a new analytical approach based on capillary electrophoresis providing the simultaneous analysis of potassium and ammonium ions in the vitreous humour. In addition, to assess the consistency of the post-mortem increase of ammonium concentrations in the vitreous humour, the determination of this ion in the vitreous humour of the two eyes of the same body at the same post-mortem interval has been verified. Vitreous humour was collected from 33 medico-legal cases where the time of death was known exactly. Prior to analysis, all samples were diluted 1:20 with a 40 μg/mL solution of BaCl2 (internal standard). In the study of the variability of the ammonium concentration between the two eyes, no statistically significant differences were found, supporting the hypothesis of an even post-mortem increase of the ion concentrations in this particular biological fluid. Significant correlations of potassium and ammonium ions with the post-mortem interval were found, with r2 of 0.75 and 0.70, respectively

Thanatochemistry at the crime scene: a microfluidic paper-based device for ammonium analysis in the vitreous humor

Most of the on-site approaches for inferring of the post-mortem interval are still based on observative data from the direct body inspection, whereas, objective and quantitative analyses, such as potassium in the vitreous humor, are require laboratory instrumentation and skilled personnel. The present paper presents a simple and low cost analytical method suitable for use at the crime scene for inferring the time since death. The method uses a microfluidic paper-based device (\u3bcPAD) for the determination of ammonium in the vitreous humor (VH) based on the selective interaction between the ammonium and the Nessler's reagent. The color change was measured in terms of "RGB distance" by using a simple and free smartphone application. The optimized device showed a limit of detection of 0.4\u202fmmol\u202fL-1, with between days precision less than 9.3% expressed as relative standard deviation, and accuracy between days from 94.5% to 104.5%. The selectivity of the Nessler's reaction was tested towards the main vitreous humor compounds, and no significant interferences were found. This paper-based analytical device was successfully used for the determination of ammonium ion in VH samples from forensic autopsies. The results obtained with the proposed method, although for a limited number of cases (n\u202f=\u202f25), showed a close correlation with the data obtained with an instrumental analysis based on capillary electrophoresis. Moreover, in order to make the evaluation of results as simple as possible, a direct correlation between the color intensity, expressed as RGB distance, and the post-mortem interval was studied and a significant correlation was found (R2\u202f>\u202f0.78). In conclusion, the present preliminary study showes that the proposed device could be an additional tool to the traditional methods for a more accurate, although still presumptive, estimation of the time of death directly at the crime scene

A 3D Origami Paper-Based Microfluidics Device for Creatine Analysis in Urine: A Disposable Tool for Identifying Urine Sample Adulteration by Dilution

The attendees will be receive detailed information on the use of a novel tech to control the integrity of urine samples using paper-based microfluidics technology. In the present case this technique will be applied to the analysis of creatinine, the most common analyte to control urine adulteration by dilution. Because of the low cost and portability of the developed devices, this method has a potential application out-side the specialized laboratories and particularly to control urine integrity immediately at the site of collection in doctor’s offices, in small laboratories and in occupational medicine centers. The relevance of the problem of sample tampering is well-known in forensic toxicology and sample dilution is the most used method to cheat toxicological controls. The prevalence of this phenomenon with a long history in analytical toxicology, is reported also in recent papers [1–3]. Among the criteria to assess urine integrity by ex-cluding dilution, the quantification of creatinine probably represents the most popular method. Although the majority of analytical methods offer adequate sensitivity and specificity, at present creatinine anal-ysis requires laboratory instrumentation which hinders the possibility of direct test of urine at the collection site. This hinders the immediate interaction with the urine donor, which could be important to prevent claims of post-collection counterfeiting. Since the first introduction by Whitesides et al. [4], the use of paper-based microfluidic devices (µPADs) for the development of chemical sensors has been extensively reported [5]. Among several approaches for producing µPADs, the use of commercial wax printers proved to be inexpensive and straightforward in the fabrication of the device. The procedure is based on two steps: (i) patterning chromatography paper into hydrophilic channels by fabricating hydrophobic barriers, and (ii) addition of the reagent to the hydrophilic portion of the paper sup-port. The sample is driven through the reagent zone as results of the wicking capacity of the paper without ex-ternal assistance. This approach does not require highly qualified personnel nor expensive instrumentation. Al-so, it can be performed on-site, enabling a prompt analytical response also in less-equipped environments. The advantages of µPADs have provided forensic science with reliable tools to face different forensic issues. On the grounds of the above considerations, the goal of the present work was to develop a low-cost device able to provide a rapid and sensitive colorimetric detection of creatinine in urine samples. This presentation will provide details of the developed procedure which was conceived as a first-line screening potentially to be con-firmed with laboratory instrumentation. The proposed microfluidic devices were designed as a three-dimensional origami pattern. The test was based on three specific reactions for the detection of creatinine using picric acid, 3,5-dinitrobenzoic acid and Nessler’s reagent. The urine sample is transferred without any treatment directly onto the hydrophilic portion of the paper, and colorimetric reactions are developed in few minutes. The color change is measured in terms of "RGB dis-tance" by using a simple and free software for smartphone cameras. The device was also validated for quantita-tive determinations in terms of accuracy and precision. The optimized method was tested on real urine samples (n=53) using as reference a clinical chemistry method performed on immunoassay instrument. In conclusion, the perspective usefulness of paper-based microfluidics as a low-cost and easy to use technique in forensic toxicology will be presented with a specific focus on its possibilities of on-site analysis to prevent urine adulteration. Keywords Microfluidic paper-based devices (µPADs); Urine adulteration; forensic toxicology References [1] B. Holden, E.A. Guice, An Investigation of Normal Urine with a Creatinine Concentration Under the Cutoff of 20 mg/dL for Specimen Validity Testing in a Toxicology Laboratory , J. Forensic Sci. 59 (2014) 806–810. doi:10.1111/1556-4029.12386. [2] S.Y. Lin, H.H. Lee, J.F. Lee, B.H. Chen, Urine specimen validity test for drug abuse testing in workplace and court settings, J. Food Drug Anal. 26 (2018) 380–384. doi:10.1016/j.jfda.2017.01.001. [3] T. Arndt, Urine-creatinine concentration as a marker of urine dilution: Reflections using a cohort of 45,000 samples, Forensic Sci. Int. 186 (2009) 48–51. doi:10.1016/j.forsciint.2009.01.010. [4] A.W. Martinez, S.T. Phillips, M.J. Butte, G.M. Whitesides, Patterned paper as a platform for inexpensive, low-volume, portable bioassays., Angew. Chem. Int. Ed. Engl. 46 (2007) 1318–20. doi:10.1002/anie.200603817. [5] D.M. Cate, J.A. Adkins, J. Mettakoonpitak, C.S. Henry, Recent developments in paper-based microfluidic devices, Anal Chem. 87 (2015) 19–41. doi:10.1021/ac503968p

An origami microfluidic paper device for on-site assessment of urine tampering. First use of Nessler's reagent for the colorimetric determination of creatinine

The relevance of the problem of urine tampering is well-known in forensic toxicology, with sample dilution being the most used method to cheat toxicological controls. Among the criteria to assess urine integrity, the quantification of creatinine probably represents the most popular method. The present paper presents a simple and low-cost analytical device for on-site creatinine determination as first-line screening for urine dilution. The proposed microfluidic devices were designed as a three-dimensional origami pattern. The device included three colorimetric reactions based on picric acid (PA-based reagent), 3,5-dinitrobenzoic acid (DNBA-based reagent), and Nessler’s reagent. The last one, to the best of our knowledge, has never been used before for creatinine determination. In order to assure the highest ease and economy of operation, the color detection and data processing were performed using a built-in smartphone camera and the associated software. The optimized device showed a detection limit of 0.02 g/L. The proposed method was used for the qualitative screening for urine dilution of 48 samples, showing a diagnostic sensitivity and specificity for PA-based, DNBA-based and Nessler’s reagent of 83.3%-80.0%, 72.2%-70.0%, and 100.0%-93.3% respectively, versus reference enzymatic method adopting a cut-off of 0.2 g/L. In conclusion, the present preliminary study shows that the proposed device could be a useful tool for on-site screening for urine tampering at the time of sample collection for toxicological testing