Anal Chem. 2017 Apr 4;89(7):4314-4319. doi: 10.1021/acs.analchem.7b00510.

Coomassie Brilliant Blue G-250 Dye: An Application for Forensic Fingerprint Analysis.

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Despite great advances in forensic science over the years, fingerprint analysis has mainly stalled at simple pictorial comparison and matching for the past 110 years. The ultimate setback in fingerprint analysis is that if a matching fingerprint is not saved in a database or if the person of interest is not physically present for comparison, the use of the print is limited, despite having the image stored in a database for future use. The same is true for DNA. In our previous work, we have demonstrated that fingerprint, both whole and partial, can be used for more than just traditional analysis and identification which can often take days or weeks for a match to be confirmed. In two other methods, we have been able to successfully identify biological sex from fingerprints using both enzymatic assays and chemical assays. Both of these assays rely on all 23 amino acids that have+ been identified in sweat. However, despite the success of these methods, we would ideally like to have a method that relies on only one amino acid that corresponds to only one originator attribute. Multi-analyte assays that target a larger number of amino acids are not completely reliable because more than one attribute can ultimately effect the output of the assay. This convolutes the intentions of the assay altogether since it would be difficult to identify which attribute is ultimately responsible for causing the difference in the assay’s response.

 

 

Scheme 1. Bradford chemical assay – containing Coomassie Brilliant Blue G-250 dye, methanol, and phosphoric acid – for the detection of six amino acids from fingerprint content using authentic fingerprint samples.

 

As a result, we decided to begin the transition toward single amino acid analysis in fingerprint samples. To do so, it was important that we first identify ways to minimize the number of amino acid targets, while not compromising the sensitivity or selectivity of the concept that has already been established. Ultimately, this led to the use of the Bradford assay (Scheme 1). In the field of biochemistry, the Bradford Reagent is traditionally used for dying protein gels and quantifying protein concentrations. Once we identified that the Bradford assay was a possible option, it was important to be sure that the assay could target the components of proteins, rather than just proteins as a whole. To our benefit, when looking further into the chemistry behind the Bradford Reagent, it was determined that it reacts with two specific groups of amino acids in particular – those with basic side chains (arginine, histidine, and lysine) as well as those with aromatic groups (tyrosine, tryptophan, and phenylalanine). Additionally, in terms of long term use for law enforcement, this method would prove to be beneficial in that it is simple to use and straightforward to understand. In addition to having appropriate targets for our studies, the Bradford assay is time-efficient, simple, and cost-effective. These qualities are especially intriguing to the forensic science and law enforcement communities.

 

 

Figure 1. (A) Absorbance of blue-colored complex generated from the reaction between the Bradford reagent and authentic fingerprint samples from 530 nm to 630 nm with a 5 nm step. The signals produced correspond to the production of the blue-colored complex. The red traces correspond to the authentic female fingerprint samples and the blue traces correspond to the authentic male fingerprint samples. (B) Box and whisker plot of the absorbance at 595 nm for authentic fingerprints. The outline of the boxes represent the range of values from the 25th to the 75th percentile and the ends of the whiskers represent 5th and the 95the percentile of values. The median value for each group is denoted by the horizontal line within each box and the three dots are the maximum, mean, and minimum values, respectively.

 

 

Figure 2. Trade-off between sensitivity and specificity is shown as a receiver operating characteristic (ROC) curve. Area under the ROC curve (AUC) is 99%, which is the probability for the presented assay to correctly distinguish between males and females based on the amino acids’ concentrations. The optimum cut off point was chosen with a sensitivity of 92% and specificity of 96%. Random choice is denoted by the gray diagonal line.

 

 

The Bradford chemical assay described above has proven to be reliable and reproducible for the purpose of distinguishing between fingerprint samples obtained from both males and females. Furthermore, a reliable sample extraction protocol was employed for the extraction of the necessary substrates, including arginine, histidine, lysine, tyrosine, tryptophan, and phenylalanine, from real fingerprint samples collected from five male and five female volunteers (five thumbprints from each of the volunteers).

 

The results from the analysis of authentic fingerprint samples demonstrated the ability of the chemical assay to differentiate between male and female fingerprint samples based solely on the significant difference in absorbance intensities (Fig. 1.). The durability of this chemical assay and extraction process was successfully determined following the statistical analysis (ROC/AUC) of the 50 authentic fingerprint samples; it was determined that the chemical assay had a 99% ability to correctly identifying the biological sex of the fingerprint originator from the amino acid content that was extracted from authentic fingerprints (Fig. 2.). Furthermore, the extraction protocol was successfully utilized, along with a Bradford assay, to remove fingerprints from multiple surfaces and identify the biological sex of the originator regardless of the surface the fingerprint was removed from (Fig. 3).

 

 

Figure 3. (A) Average absorbance of blue-colored complex generated form the reaction between the Bradford reagent and authentic fingerprint samples (n=5 for each sex) that were taken from five different surfaces. The absorbance was measured from 530 nm to 630 nm with a 5 nm step. The signals produced correspond to the production of the blue-colored complex. The red traces correspond to the authentic female fingerprint samples and the blue traces correspond to the authentic male fingerprint samples. The abbreviation PEF represents the polyethylene film. (B) Bar diagram of the average absorbance for each surface and each sex at 595 nm. The red traces correspond to the authentic female fingerprint samples and the blue traces correspond to the authentic male fingerprint samples. Error bars are included in the figure to demonstrate the efficacy of transferring the fingerprint from the respective surface to the polyethylene film and subsequently performing the extraction. The abbreviation PEF represents the polyethylene film.

 

In addition to providing another option for the analysis of authentic fingerprints, the most important observation is that despite significantly reducing the number of amino acid targets from 23 to 6, the Bradford assay is still able to identify biological sex from the fingerprint content with superior accuracy. Bradford assay serves as the first example of being able to identify originator characteristics, such as biological sex shown here, by targeting a limited number of amino acids. Ultimately, since multi-analyte assays are not entirely reliable because more than one attribute can contribute to the change in analyte(s) levels, we hope to use the trend seen between the ninhydrin assay and the Bradford assay in order to develop additional chemical assays that continue to narrow down the analyte pool. Furthermore, the Bradford assay possesses an unparalleled simplicity in comparison to the ninhydrin chemical assay and the L-amino acid oxidase/horseradish peroxidase enzymatic assay – both of which we have been able to successfully identify biological sex from fingerprints – in that it there is little to no assay preparation (i.e., no heating step like ninhydrin and no additional substrates required like the enzyme cascade) aside from extracting the fingerprint content. Of the developed methods, the Bradford assay would unequivocally be the ideal method to be incorporated into an on-site platform.

 

Link to the research team: https://www.halameklab.com/

 

More studies of identifying originator attributes from crime scene evidence:

• Huynh, C.; Brunelle, E.; Agudelo, J.; & Halámek, J. “Bioaffinity-based assay for the sensitive detection and discrimination of sweat aimed at forensic applications.” Talanta. 2017, 170, 210–214.
• Agudelo, J.; Halámková, L.; Brunelle, E.; Rodrigues, R.; Huynh, C.; & Halámek, J. “Ages at a crime scene: Simultaneous estimation of the time since deposition and age of its originator.” Analytical Chemistry. 2016, 88 (12), 6479–6484.
• Brunelle, E.; Huynh, C.; Le, A.-M.; Halámková, L.; Agudelo, J.; & Halámek, J. “New Horizons for Ninhydrin: Colorimetric Determination of Gender from Fingerprints.” Analytical Chemistry. 2016, 88 (4), 2413–2420.
• Brunelle, E; & Halámek, J. “Biocomputing Approach in Forensic Analysis.” International Journal of Parallel, Emergent and Distributed Systems. Published Online, February 2016.
• Huynh, C.; Brunelle, E.; Halámková, L.; Agudelo, J.; & Halámek, J. “Forensic identification of gender from fingerprints.” Analytical Chemistry. 2015, 87 (22), 35–44.