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Bruijns, B.; Tiggelaar, R.; Knotter, J.; Van Dam, A. Use of Lateral Flow Assays in Forensics. Encyclopedia. Available online: (accessed on 29 November 2023).
Bruijns B, Tiggelaar R, Knotter J, Van Dam A. Use of Lateral Flow Assays in Forensics. Encyclopedia. Available at: Accessed November 29, 2023.
Bruijns, Brigitte, Roald Tiggelaar, Jaap Knotter, Annemieke Van Dam. "Use of Lateral Flow Assays in Forensics" Encyclopedia, (accessed November 29, 2023).
Bruijns, B., Tiggelaar, R., Knotter, J., & Van Dam, A.(2023, July 18). Use of Lateral Flow Assays in Forensics. In Encyclopedia.
Bruijns, Brigitte, et al. "Use of Lateral Flow Assays in Forensics." Encyclopedia. Web. 18 July, 2023.
Use of Lateral Flow Assays in Forensics

Already for decades lateral flow assays (LFAs) are ‘common use’ devices in daily life. Also, for forensic use LFAs are developed, such as for the analysis of illicit drugs and DNA, but also for the detection of explosives and body fluid identification. Despite their advantages, including ease-of-use, LFAs are not yet frequently applied at a crime scene. 

lateral flow assays forensic investigation body fluid identification illicit drugs analysis

1. Lateral Flow Assays for Biological Forensic Applications

1.1. Body Fluid Identification

Within the forensic field, body fluid identification (BFI) is extremely important, as the origin of body fluid is indicative of the presence of DNA and additionally might help in reconstructing the events that took place at a crime scene, as well as for investigative leads [1][2]. Identification of body fluids is not an easy task, as their appearance can be similar to each other or other substances. Therefore, different presumptive and confirmatory methods are developed to identify human biological stains, including chemical, enzymatic, spectroscopic and microscopic methods [3][4][5]. LFAs can also be used as a rapid and specific tool to identify body fluid specific markers. The LFAs that are currently available for body fluid identification are mainly based on antibody-antigen interactions, whereby proteins are the target molecules. Protein-based interactions have the advantage of being highly selective and have high affinity with their target molecule. Several commercial LFA kits are available, such as the Rapid Stain Identification (RSID) tests from Independent Forensics, tests from SERATEC and BlueSTAR-forensics [6][7][8]. Most of these commercial tests focus on the identification of blood, semen, saliva and urine. These body fluids are frequently encountered at the crime scene, and specific biomarkers are identified that can be targeted using antibodies.
For the identification of blood, various commercial tests are available. For example, the RSID kits are immunochromatographic assays, using labeled antibodies that react to body-fluid-specific markers. For example, the RSID blood test uses anti-glycophorin A monoclonal antibodies to specifically detect the blood specific glycophorin A marker, a protein that is expressed by red blood cells [9]. To discriminate between peripheral blood and menstrual blood, SERATEC developed a LFA that is able to detect two different biomarkers in one multiplex LFA: hemoglobin and D-dimer. Hemoglobin is present in peripheral and menstrual blood, whereas D-dimer is only present in menstrual blood [10]
For the detection of saliva, various commercial LFAs are available, including the RSID saliva test and SERATEC Amylase test [11]. In both tests, anti-amylase is included to test for the presence of amylase A, which is an enzyme that is produced by the salivary glands, and will break down carbohydrates to maltose. The test has a sensitivity of about 1 ng of α-amylase, which corresponds to 1 µL of saliva. No cross-reactivity is found with saliva samples from other species [12].
LFA tests are also developed to indicate the presence of semen. Many different commercial tests are available including the RSID semen test, ABAcard-p30, BLUESTAR Identi-PSA and the PSA Semiquant test from SERATEC. The RSID test detects the presence of semenogelin, a protein involved in the formation of a gel matrix enclosing ejaculated sperm cells [13][14], whereas the ABAcard-p30 and the PSA Semiquant test detect prostate specific antigen (PSA), also called p30. In a study in which the RSID semen test was compared with the ABAcard-p30, minimal differences were found related to the sensitivity and the user-friendliness of the tests. However, samples obtained from vaginal swabs 24 h post-coital resulted in positive test results with the ABAcard-p30, whereas negative results were obtained with the RSID semen test [15]
To identify the presence of urine the RSID urine test is available, which detects the urine-marker Tamm-Horsfall protein. Positive test lines were obtained when urine was diluted 1:20. In the case of dried urine stains the identification can be difficult since the stain needs to be diluted in buffer, before it can be analyzed, which might result in a too diluted sample for analysis [16].

1.2. DNA

Another application of LFAs for forensic usage related to biological samples involves the detection of unique nucleic acid sequences. Upon hybridization of reporter and capture probes with the target sequence, the nucleic acid sequence of interest can be detected [17]. This technique can be used for the detection of, among others, spores and (human) DNA analysis.
Anthrax spores can cause an infection, depending on contact and/or inhalation, to the skin, lungs and intestinal. Also, fever, chest pain and shortness of breath can occur after inhalation. These spores became of forensic interest after the spreading of anthrax letters in 2001, in which anthrax was used as a bioweapon. Hartley et al. [17] developed a lateral flow test for the detection of a mRNA sequence of the anthrax toxin activator gene, present in B. anthracis (anthrax) spores. After RNA extraction and nucleic acid sequence-based amplification (NASBA) of the RNA the sample is placed on the lateral flow assay. 
Zasada et al. [18] compared three different isothermal nucleic acids amplification methods, i.e., loop-mediated isothermal amplification (LAMP), thermophilic helicase-dependent isothermal amplification (tHDA) and recombinase polymerase amplification (RPA), to address their sensitivity when combined with lateral flow dipstick analysis. Three different pathogens, B. anthracis, F. tularensis (tularemia) and Y. pestis (plague) were included in this assay. Different reaction times were needed for the three DNA amplification methods, varying from 60, 90 and 30 min, respectively for LAMP, tHDA and RPA. All three methods could be combined with lateral flow dipstick analysis, resulting in positive detection of the pathogen of interest. However, the authors indicate that there is a risk of false-positive results in the case of pathogens with high genetic similarities to non-pathogenic species, which might be due to the isothermal amplification method and this should be further investigated.
Prior to the lateral flow detection of male DNA Kubo et al. [19] used LAMP to amplify the target of interest. The time-to-result was around 60 min and 10 pg of male DNA could be detected. An assimilating probe and a biotin-labeled primer are used in the LAMP assay. The dual-labeled amplicons are subsequently captured, whereafter the fluorescent signal can be detected.

2. Lateral Flow Assays for Chemical Forensic Applications

2.1. Illicit Drugs

Nowadays many techniques are available for the analysis of illicit drugs, such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and Raman spectrometry. However, these conventional analytical laboratory techniques are relatively bulky and require trained operators. The use of indicative tests is a valuable, cheap and time-consuming alternative [20].
For an indication of the presence or absence of illicit drugs in a sample usually presumptive colorimetric tests are used. These tests are used at the crime scene as rapid screening methods, but are also used in the lab for this purpose. Examples of such color tests are the Marquis test (morphine, 3,4-methylenedioxymethamphetamine (MDMA) and (met)amphetamine), the Scott test (cocaine), Simon’s test (methamphetamine and MDMA) and the Mandelin reagent (among others, ketamine, cocaine, MDMA and morphine). The exact structure of the complexes that are generated upon the reaction of the test reagents and the illicit drugs are in many cases not known [21][22].
As reported by Noviana et al. [23] tests based on a microfluidic paper-based analytical device (µPAD) are nowadays routinely used for the analysis of illicit drugs. Musile et al. [24] made a µPAD in which the unknown sample is split up into six different lanes, which can detect different types of compounds based on conventional color tests within 5 min. With this test multiplexed detection is possible of various (classes of) illicit drugs, such as cocaine, ketamine and morphine.
Angelini et al. [25] tested commercially available lateral flow strips for the detection of fentanyl (derivatives). The LOD of the test strips for fentanyl and norfentanyl was found to be 0.25 µg/mL and 0.05 µg/mL, respectively. The strips could be scored after 5–10 min. With the commercial Cozart RapiScan System (CRS) (also known as the Drug Detection System) also illicit drugs in oral fluids can be detected, such as (meth)amphetamine, MDMA and 3,4-methylenedioxy-N-ethylamphetamine (MDEA). Wilson et al. [26] concluded that the CRS is suitable for screening purposes, although the sensitivity and selectivity are lower compared to GC-MS. A total of 121 oral fluid samples tested positive (methamphe-tamine and MDMA) with the CRS, whereas 230 samples contained amphetamines (MDEA), 3,4-methylenedioxyamphetamine (MDA), MDMA or amphetamine according to confirmatory GC-MS.
Taranova et al. [27] designed a lateral flow test for the simultaneous detection of morphine, amphetamine, methamphetamine and BZE based on a microarray. The microarray consisted of 8 × 3–4 rows yielding 24 or 32 detection spots. Since Taranova et al. [27] used a competitive assay, the color intensity of the spots decreases (up to complete disappearance) for increasing concentrations of the analyte. 

2.2. Biowarfare

The rapid and secure detection of biowarfare agents or biotoxins is critical for forensic investigators. For example, ricin, botulin and aflatoxin can be used as potential agents of warfare or terrorist attack. Such agents and toxins are usually detected by an antibody-antigen reaction in an LFA [28].
Hodge et al. [29] tested commercially available test strips for ricin. The strips could be read out by the unaided eye or by the use of the Rapid BioAlert Reader. For both 3 and 6 ng ricin the average time-to-read was found to be 19 min. The LOD for this assay was about 0.54 ng, which is equal to about 3.6 ng/mL.
B. anthracis, the causative agent of anthrax, can be detected with a LFA based on antibody-antigen detection. Wang et al. [30] developed a LFA by which 400 pure spores could be detected within 30 min. As detection probe super-paramagnetic particles, a method to improve the sensitivity of the LFA, were used.
Aflatoxin B1 (AFB1) can be detected by a dipstick type of LFA as developed by Shim et al. [31]. The detection principle is based on an aptamer and DNA probes, with a Cy5 coupled dye for fluorescent read out. The LOD was found to be 0.1 ng/mL AFB1 in buffer and 0.3 ng/g AFB1 for corn samples. The time-to-result of this LFA is 30 min.

2.3. Explosives

When explosives are involved at a crime scene it is of utmost importance to collect fast information about the unknown substances. Since conventional on-site instrumental techniques, such as ion mobility spectrometry and infrared spectroscopy, can be relatively large and not easily operatable by, e.g., military or law enforcement, the use of (indicative) paper-based lateral flow devices has emerged [32].
Lateral flow assays for the detection of explosives are mainly based on either a colorimetric reaction or an antibody-antigen reaction. For the latter modified antibodies are present on the lateral flow strip that can bind the specific target molecule; a type of explosive in this case [33][34]. Similar to illicit drug analysis, explosive compounds can also be detected via a colorimetric reaction. Examples of these color tests are the Nessler reagent (2,4,6-trinitrotuluene (TNT), ammonium ions), Peroxides reagent (peroxides), Chloride reagent (chloride) and Griess reagents (cyclotrimethylenetrinitramine (RDX), pentaery-thritol tetranitrate (PETN), nitrate ions) [35].
TNT is one of the most used explosives. Romolo et al. [36] tested three immunochemical assays for the detection of TNT: (1) an indirect competitive ELISA with chemiluminescent detection (CL-ELISA), (2) a colorimetric lateral flow immunoassay (LFIA) and (3) a chemiluminescent lateral flow immunoassay (CL-LFIA). The first test was a laboratory test based on a microplate setup for comparison with the two LFIA tests. For the CL-ELISA trinitrobenzene and ovalbumin conjugates were used, whereby horseradish peroxidase (HRP) and luminol were used for detection, for the CL-LFIA anti-TNT antibodies were used with HRP as detection molecule, and for the LFIA test anti-TNT antibodies were implemented with colloidal gold as detection molecule. 
Chaboud et al. [37] developed a µPAD for the colorimetric detection of metallic salts (lead, barium, antimony, iron, aluminum and zinc) present in primer residues and pyrotechnic low explosive devices. This device is based on earlier research on the development of a µPAD to detect inorganic explosives (e.g., flash powder and ammonium nitrate) and a µPAD to detect military explosives (e.g., TNT and urea nitrate). The first device was able to detect, among others, nitrate, nitrite and ammonium in combination with deionized water as solvent. TNT, RDX, hydrogen peroxide and urea nitrate could be detected by the second µPAD using 50%/50% acetone/water as solvent. Both devices utilized a colorimetric reaction of the explosive with the spotted reagent(s) with time-to-result within 5 min [38]

3. Future

Due to their advantages—viz. ease-of-use, relative low-cost as well as sufficient sensitivity and sensitivity—LFAs are nowadays mainly used for screening purposes for BFI, illicit drug analysis and detection of explosives and biowarfare. In order to become applicable for more forensic applications, the time-to-result of LFAs has to be reduced: an outcome within 10 min is required at a scene of interest (e.g., a crime scene). Moreover, it seems essential that such LFAs are able to perform multiplex analyses. Overall, LFAs have been partially implemented in forensic case work and can be of additional use to extract important information directly at the crime scene. However, more research is needed to improve current LFAs and make the transition to commercialization and implementation.


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