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

Subscriber access provided by UNIV OF CAMBRIDGE

Article

Coomassie Brilliant Blue G-250 Dye: New Application for Forensic Fingerprint Analysis Erica Brunelle, Anh Minh Le, Crystal Huynh, Kelly Wingfield, Lenka Halámková, Juliana Agudelo, and Jan Halámek Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00510 • Publication Date (Web): 15 Mar 2017 Downloaded from http://pubs.acs.org on March 16, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Coomassie Brilliant Blue G-250 Dye: New Application for Forensic Fingerprint Analysis Authors: Erica Brunelle,1 Anh Minh Le,1 Crystal Huynh1, Kelly Wingfield,1 Lenka Halámková1, Juliana Agudelo,1 and Jan Halámek1* Affiliations: 1 Dept. of Chemistry, University at Albany, State University of New York, 1400 Washington Ave., 12222, NY *

Correspondence to: [email protected]

Abstract The Bradford reagent, comprised of the Coomassie Brilliant Blue G-250 dye, methanol and phosphoric acid, has been traditionally used for quantifying proteins. Use of this reagent in the Bradford assay relies on the binding of the Coomassie Blue G-250 dye to proteins. However, the ability of the dye to react with a small group of amino acids – arginine, histidine, lysine, phenylalanine, tyrosine, and tryptophan – makes it a viable chemical assay for fingerprint analysis in order to identify the biological sex of the fingerprint originator. It is recognized that the identification of biological sex has been readily accomplished using two other methods, however, both of those systems are reliant upon a large group of amino acids – 23 to be precise. The Bradford assay, described here, was developed specifically to aid in the transition from targeting large groups of amino acids, as demonstrated in the previous studies, to targeting only a single amino acid without compromising the intensity of the response and or the ability to differentiate between two attributes – in this case, female fingerprints from male fingerprints.

Introduction Forensic investigations have become increasingly modern with the use of biological samples, such as fingerprints, as evidence since they elucidate important facts about the crime scene and the crime itself. Biological traces found at crime scenes represent important leads in the identification of possible suspects. Chemical and biochemical techniques utilized for the analysis of biological traces at crime scenes are the main scientific support of criminal investigations and subsequent prosecutions.1 The history of chemical assays specifically resulting in colorimetric determinations is extensive but, at the same time, is rather limited. Of the chemical methods that are used to develop latent fingerprints such as 1,2-diazafluoren-9-one (DFO) which fluoresces when irradiated with blue-green light,2,3 the ninhydrin method is the most well-known and widely used. Federal, state, and city crime laboratories have been implementing this technique for almost 50 years4,5-7 and it has been used to detect and develop fingerprints that are more than 30 years old.8 This method has proven to be simple and convenient in comparison to instrumental methods such as HPLC,9,10 GC,11 or GC-MS12-15 in that no complex instrumentation is required and it can be used for the routine analysis of large sample populations.16 However, one significant downfall to this reaction is that it is known to yield the same end-product – diketohydrindylidene-

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

diketohydrindamine (DYDA) or Ruhemann's purple5,17 – from other α-NH2 compounds such as ammonium salts.18 Fingerprint analysis, regardless of the method, has mainly stalled at simple pictorial comparison and matching for the past 110 years.19 Currently, only the shape, size, and unique patterns associated with fingerprints are compared visually as well as via computational and biometric identification software – the ninhydrin method for the development of latent fingerprints is no exception, despite some improvements to light sources, image capturing devices, and chemical imaging software.20 Other improvements that have surfaced include the introduction of proteolytic enzymes such as trypsin and chymotrypsin as well as the formation of metal ion complexes.21,22 As mentioned in previous publications, 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. 23 The same is true for DNA.24 It has been established that the contents of fingerprints are produced by multiple hormonebased control mechanisms25 and are thus, a function of physical properties such as gender, age, ethnicity, or health status.26-33 In our recently published work,34,35 we have demonstrated that fingerprints have the potential to generate much more information because they are samples of biological origin analogous to other body fluid such as blood, using new methods for the analysis of fingerprints in order to determine the biologicals sex of the originator. This involved both enzymatic and chemical assays which focused on the detection of 23 amino acids – 20 natural and 3 unnatural – where the overall concentration of amino acids correlates to the biological sex of the fingerprint originator with females having the higher concentration. Despite these successes, our group’s intentions are to establish a method where, ideally, only one amino acid corresponds to only one originator attribute. Multi-analyte assays that target a larger number of amino acids, such as the ones previously developed, 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. For example, if attribute A causes amino acid 1 to increase and but attribute B causes the same amino acid to decrease the change is negated and neither attribute can be identified. Ultimately, the ability to make such correlations would allow for multiple attributes to be identified from a single sample. For the reasons mentioned above, ways to minimize the number of amino acid targets, while not compromising the sensitivity or selectivity of the concept that has already been established were investigated. This investigation and the principle that proteins are composed of amino acid chains led to the Bradford assay which has been traditionally used for quantifying proteins. This method has become the preferred method for many laboratories as it is simpler, faster, and more sensitive than other quantification methods, such as the Lowry method. When compared with the Lowry method, the Bradford assay is less effected by common biochemical reagents and non-protein components of biological samples.36 Upon identifying the Bradford assay as a potential chemical assay for the identification of originator attributes from fingerprint content, it was essential to determine if the assay could target the components of proteins, rather than just proteins as a whole. To this effect, studies have identified that the Coomassie dye specifically targets amino acids with basic side chains – arginine, histidine, and lysine – as well as those with aromatic groups – tyrosine, tryptophan, and phenylalanine.37,38 Since there are specific amino acids that the Bradford assay can target, it became a viable option for the concept of identifying originator attributes from fingerprints. In


Page 2 of 10

Page 3 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


addition to having appropriate targets for our studies, the Bradford assay is time-efficient, simple, and cost-effective, qualities that are especially intriguing to the forensic science and law enforcement community. The assay itself relies solely on the Bradford Reagent which contains Coomassie Brilliant Blue G-250 dye, methanol and phosphoric acid. The Coomassie Brilliant Blue G-250 dye is the key component to this assay and is known to exist in three forms: cationic, neutral, and anionic.37,39 Due to this nature, the dye also takes on three colors: red, green, and blue, respectively. The most common form of the dye is the acidified form (cationic) and is thus a red color, which is spectrophotometrically measured at 470 nm.38,40 The anionic blue form of dye allows for binding to the proteins. This blue color that can be measured spectrophotometrically at 595 nm37-40 and can then be used to quantify the amount of protein in the sample by identifying the amount of blue dye present. While this wavelength is well-reported,36-40 a full spectrum of the blue-colored complex was measured and reported for confirmation. In addition, the use of biological sex was again used as the target originator attribute as it has been well established and served as a reliable control.

Experimental Ethics Statement The Institutional Review Board, Office of Pre-Award and Compliance at the University at Albany has fully approved the experimental protocols demonstrated in this manuscript. These protocols were carried out in accordance to the office’s requirement of obtaining informed consent, in the form of a signature from each volunteer, acknowledging that they are aware of the procedure that will take place, any risks or benefits that may accompany the study, as well as acknowledging that they will not receive any payment for their participation. Informed consent from all volunteers who participated in this research study was obtained. Chemical Assay Components and Extraction Coomassie Brilliant Blue G-250 dye, methanol, and phosphoric acid (Bradford Reagent) was purchased from Sigma-Aldrich. Hydrochloric acid, purchased from EMD Millipore, was used for the amino acid extraction procedure. Water used in all of the experiments was ultrapure (18.2 MΩ cm) water from PURELAB flex, an ELGA water purification system. The amino acid content for this experiment was obtained using an extraction protocol that was developed34 and optimized35 in our previous work. The protocol used here can be found in the publication involving the use of ninhydrin.35 This protocol was also used for extracting amino acids from fingerprints taken from 5 different surfaces. Amino Acid Detection Using Authentic Samples The chemical assay depicted in Scheme 1 was designed and optimized in this study for use in identifying originator attributes from authentic fingerprint content. The assay was developed using 300 µL-volume polystyrene (96 well) microtiter plates, the Bradford reagent, ultrapure (18.2 MΩ cm) water and the extracted fingerprint sample. The reaction is initiated when the Bradford reagent interacts with the amino acids present in the sample and ultimately generates the blue color that can be observed visually by the naked eye or measured spectrophotometrically at 595 nm. The rate of production and the intensity of this color is subsequently proportional to the overall amino



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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.

acid concentration of all 6 amino acids present in the sample that known to react with the Bradford reagent. The Bradford assay was first optimized by varying the amount of the Bradford reagent, while keeping the fingerprint at a constant volume of 100 µL. The total volume was subsequently brought to 300 µL with ultrapure (18.2 MΩ cm) water. Ultimately, it was determined that the best volumes were 150 µL Bradford reagent, 100 µL of the fingerprint sample, and 50 µL water. Five fingerprints samples were collected from two groups of volunteers, 5 males and 5 females, totaling 50 authentic fingerprint samples. It is important to note that the fingerprint samples were not collected at the same time or on the same day. This is important because it allows us to confirm that the amino acid content in fingerprints only varies slightly from day to day but do differ significantly between two demographic groups (males and females).26,27 The fingerprints were collected on polyethylene film according to an established procedure described by Croxton et al.26,29 In order to keep this project as closely related to authentic crime scene scenarios as possible, it was unknown whether or not volunteers had washed their hands prior to fingerprint deposition. Evidence supporting the lack of statistical difference between washed and unwashed hands is demonstrated by Alessandrini et al.24 Following deposition, the extraction protocol35 was applied. Once the three components of the assay were placed in the microplate wells, spectral readings of the samples were performed from 530 to 630 nm with a 5 nm step. All optical measurements were performed at 37 °C using a SpectraMax Plus384 (Molecular Devices, CA) microplate reader with polystyrene (96 well) microtiter plates. Statistical Analysis of Authentic Fingerprint Samples Given the success of the ninhydrin chemical assay,35 the Bradford chemical assay was used to analyze 25 authentic male fingerprint samples and 25 authentic female fingerprint samples, totaling 50 authentic fingerprint samples. Statistical analysis of these results was performed using R-project software27-29,34,35,41-45 in order to determine the diagnostic potential, the ability to correctly differentiate between male and female fingerprints, of the Bradford chemical assay. Specifically, receiver operating characteristic (ROC) analysis was utilized. ROC curves and Areas Under the Curve (AUCs) were calculated to estimate the discriminatory power of the Bradford assay.34,35 The ROC analysis captures the trade-off between sensitivity and specificity while changing a discrimination threshold, but it can be summarized as a single measurement (AUC). The sensitivity (true positive rate) was plotted against the specificity (true negative rate) in the ROC curve as a function of a variety of thresholds of class prediction probabilities. The overall assay accuracy depends on the overlap of the output signal distributions for the two classes


Page 4 of 10

Page 5 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


– in this case, male and female. Values range between 0.5 and 1.0, where a value of 0.5 indicates that the two distributions are identical and a value of 1.0 indicates that there is no overlap in the distributions of output signals for the two classes. The AUC is used as a lone measure of evaluating the efficiency of the model ranked subjects according to the probability assigned to the positive class. The AUC of the ROC curve was calculated by the trapezoidal method of integration with the corresponding 95% confidence intervals (CI) as described by De Long et al.46 Using this method, the best threshold (the point at which the signal changes correspond to only one of the groups) that yielded the maximum accuracy was determined. The goal of using ROC analysis was to evaluate the performance of our assay when it came to discriminating between female and male groups based on the output absorbance values. We chose ROC analysis for performance evaluation above other methods because the AUC, as an overall accuracy index, is considered a robust index for meaningful interpretation and understanding in diagnostic and forensic accuracy situations. We accommodated the distribution shape of the markers’ concentrations for the experiments based on the approximate known concentration distribution in the general population. Depending on the available data, it is possible to end up with a very wide confidence interval for the AUC, especially with rather small sample sizes. In our study we have achieved a very high AUC for female/male group discrimination with very low variance. Amaral et al47 has achieved similarly high AUCs using forced oscillation (FO) measurements for chronic obstructive pulmonary disease (COPD) diagnostics. They have used several classifiers based on machine learning algorithms and were getting very high and consistent AUCs when using patients and control groups, both of which included 25 subjects, as was the case for this study.

Results Evaluation of Authentic Fingerprint Samples As expected based on previously published results,34,35 the aqueous acidic solution collected from the fingerprint extraction consisted of the amino acids needed for analysis – specifically arginine, histidine, lysine, tyrosine, tryptophan, and phenylalanine – while the nonpolar material stayed on the polyethylene film. The signal corresponding to the concentration of the blue-colored complex with the authentic fingerprint samples was measured spectrophotometrically; the maximum wavelength for this reaction was determined to be 595 nm, which was consistent with the literature value. The success of the extraction of amino acids as well as the chemical assay’s performance is depicted in Figure 1A and 1B. The spectral measurements depicted in Figure 1A allowed for rapid identification of the biological sex – 25 female samples are displayed in red and 25 male samples are displayed in blue – of the fingerprint originator given the short amount of time needed for the measurement. In addition, five blank samples (depicted in black) were analyzed to demonstrate the initial absorbance of the assay without the fingerprint sample as well as to justify the absorbance being higher than zero. For better illustration of the results from the authentic samples, a box and whisker plot was generated, Figure 1B, based on the absorbance (Abs.) at 595 nm. This representation shows the variation in data to allow for visual assessment of the female and male sample distributions, as well as their respective clustering. The red box on the left represents the range of values for the female samples and the blue box represents the male samples. The values for the 25th to 75th percentiles are denoted by the box outline. The whiskers indicate



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1A. 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.

Page 6 of 10

Figure 1B. 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.

the range of values for the 5th and 95th percentiles. In addition, the median values for each groups are represented by the solid lines within the boxes. The maximum, mean, and minimum values are denoted by the three dots, respectively from top to bottom. In order to validate the Bradford assay as a legitimate method for correctly identifying the biological sex of the fingerprint originator, the statistical analysis described above was employed. This was used to evaluate the true diagnostic potential of the chemical assay when using authentic fingerprint samples. An ROC curve, shown in Figure 2, was created using the absorbance changes and was utilized to determine the best absorbance threshold of 0.402. The AUC generated for the authentic fingerprint samples was estimated to be 0.99 (95% CI, 0.9635-1.0000) from the ROC curve (Fig. 2). This means that the Bradford chemical assay has a 99% chance of correctly discriminating between male and female fingerprints. The results from the ROC statistical analysis were also consistent with previously published reports34,35 that amino acid content from fingerprints can be used to identify the biological sex of the fingerprint originator with a high level of sensitivity and accuracy.

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.


Page 7 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Evaluation of Surfaces Just as in the previous work by our group, it was important to ensure the validity of the assay in real crime scene scenarios where samples would not necessarily be found on an ideal surface. To test this, the extraction protocol and the Bradford chemical assay were further tested on various surfaces that could be found at a crime scene. Five female fingerprints and five male fingerprints were deposited onto five surfaces including a door knob, a laminate desktop, a composite benchtop, and a computer screen. Again, this totaled 25 female fingerprints and 25 male fingerprints. The polyethylene film used in the initial experiment was used to remove the fingerprints from the respective surfaces. This film was also used as the fifth surface and thus was the control. Figure 3 demonstrates that the extraction protocol and the chemical assay are capable of identifying a female fingerprint versus a male fingerprint, regardless of the surface from which it was taken. Figure 3A, is representative of the average (n=5) response of the five sample for each

Figure 3A. 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.

Figure 3B. 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.

surface and each sex from 530 nm to 630 nm with a 5 nm step. In addition, to better represent the results of the assay from each surface according to the sex or the originator, a bar graph (Fig. 3B) was generated using the average absorbance values at the maximum wavelength, 595 nm. Error bars were added to the bar graph to demonstrate the success of the fingerprint transfer and extraction of the amino acid content. The results obtained from the analysis of authentic fingerprint samples using the Bradford chemical assay demonstrated a similar trend to that shown in previous reports34,35 for identifying the fingerprint originator via amino acid content. This also agrees with previous reports of females having higher amino acid concentrations than males.48-51



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Conclusions 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 – arginine, histidine, lysine, tyrosine, tryptophan, and phenylalanine – from real fingerprint samples collected from five male and five female volunteers; 5 thumbprints from each of the volunteer. The results from the analysis of authentic fingerprint samples further demonstrated the ability of the chemical assay to differentiate between male and female fingerprint samples based on the significant difference in absorbance intensities. In addition, 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. Furthermore, the extraction protocol was successfully utilized, along with a Bradford assay, to remove fingerprints from multiple surfaces and was still able to identify the biological sex of the originator regardless of the surface the fingerprint was removed from. In addition to providing another chemical assay that can be used for the analysis of authentic fingerprints, the most important observation that is obtained 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 sufficient 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. Ideally, we anticipate to be able to identify one individual amino acid that can be correlated to a single originator attribute with the intentions of someday being able to identify multiple originator attributes from a single fingerprint sample. To conclude, the Bradford assay possesses an unparalleled simplicity in comparison to ninhydrin chemical assay and the L-amino acid oxidase/horseradish peroxidase enzymatic assay 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. Acknowledgements This work was supported by the National Institute of Justice (Award No. 2016-DN-BX-0188).


Page 8 of 10

Page 9 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


References 1

Elkins, K. M. Forensic DNA Biology: A Laboratory Manual, Academic Press, Oxford, UK, 2012. Lee, H.; Ramotowski, R.; Gaensslen, R.E. Advances in Fingerprint Technology, 2nd ed., CRC Press: Boca Raton, FL, 2001. 3 A Simplified Guide to Fingerprint Analysis; National Forensic Science Technology Center: Largo, FL. 4 Crown, D. J. Crim. Law Criminol. Police Sci. 1969, 60, 258−264. 5 Moore, S.; Stein, W. H. J. Biol. Chem. 1948, 176, 367−388. 6 Meyer, H. J. Biochem. 1957, 67, 333−340. 7 Naftalin, L. Nature 1948, 161, 763. 8 Exline, D. L.; Wallace, C.; Roux, C.; Lennard, C.; Nelson, M. P.; Treado, P. J. J. Forensic Sci. 2003, 48, 1047−1053. 9 Aitken, A.; Geisow, M. J.; Findlay, J. B. C.; Holmes, C.; Yarwood, A. In: Protein sequencing: a practical approach; IRL Press, Oxford, UK, 1989, 43−68. 10 Wittmann-Liebold, B.; Salnikow, J. Advanced Methods in Protein Microsequence Analysis; SpringerVerlag Berlin Heidelberg: Germany, 1986, 415. 11 Kaiser, F. E.; Gehrke, C. W.; Zumwalt, R. W.; Kuo, K. C. J. Chrom. 1974, 94, 133. 12 Hamann, C. S.; Myers, D. P.; Rittle, K. J.; Wirth, E. F.; Moe, O. A. J. Chem. Educ. 1991, 68, 438. 13 Rafter, J. J.; Ingleman-Sandberg, M.; Gustafmon, J. A. Acta Biol. Med. Germ. 1979, 38, 321−381. 14 Epstein, P.; Kaine, C. BioChromatogrophy 1990, 5, 97−100. 15 Leimer, K. R. Rice, R. H.; Gehrke,C. W. J. Chrom. 1977, 141, 121−144. 16 Sun, S.-W.; Lin, Y.-C.; Weng, Y.-M.; Chen, M.-J. J. Food Comp. Anal. 2006, 19, 112−117. 17 Ruhemann, S. J. Chem. Soc. 1911, 99, 792, 1306, 1486. 18 Harding, V. J.; Warneford, F. H. S. J. Biol. Chem. 1916, 25, 319–335. 19 Yamashita, B.; French, M. Fingerprint Sourcebook: Rockville, MD, 2010. 20 Menzel, E. R. Anal. Chem. 1989, 61, 557−561. 21 Lorand, L. Proteolytic Enzymes, Part 2; Academic Press, Inc.: San Diego, CA, 1976. 22 Friedman, M. J. Agr. Food Chem. 2004, 52, 385−406. 23 Francese, S.; Bradshaw, R.; Ferguson, L. S.; Wolstenholme, R.; Clench, M. R.; Bleay, S. Analyst 2013, 138, 4215−4228. 24 Alessandrini, F.; Cecati, M.; Pesaresi, M.; Turchi, C.; Carle, F.; Tagliabracci, A. J. Forensic Sci. 2003, 48, 1−7. 25 Thody, A. J.; Shuster, S. Physiol. Rev. 1989, 69, 383−416. 26 Kutyshenko, V. P.; Molchanov, M.; Beskaravayny, P.; Uversky, V. N.; Timchenko, M. A. PLoS ONE 2011, 6, 1−9. 27 Hier, S. W.; Cornbleet, T.; Bergeim, O. J. Biol. Chem. 1946, 166, 327−333. 28 Coltman, C. A. J.; Rowe, N. J.; Atwell, R. J. Am. J. Clin. Nutr. 1966, 18, 373−378. 29 Croxton, R. S.; Baron, M. G.; Butler, D.; Kent, T.; Sears, V. G. Forensic Sci. Int. 2010, 199, 93−102. 30 Khan, M. S.; Thouas, G.; Shen, W.; Whyte, G.; Garnier, G. Anal. Chem. 2010, 82, 4158–4164. 31 Corstjens, A. M.; Ligtenberg, J. J. M.; Horst, I. C. v. d.; Spanjersberg, R.; Lind, J. S. W.; Tulleken, J. E.; Meertens, J. H. J. M.; Zijlstra, J. G. Crit. Care 2006, 10, R135. 32 Haese, A.; Taille, A. d. l.; Poppel, H. v.; Marberger, M.; Stenzl, A.; Mulders, P. F. A.; Huland, H.; Abbou, C. m.-C.; Remzi, M.; Tinzl, M.; Feyerabend, S.; Stillebroer, A. B.; Gils, M. P. M. Q. V.; Schalken, J. A. Eur. Urol. 2008, 54, 1081–1088. 33 Streckfus, C. F.; Bigler, L. R. Oral Dis. 2002, 8, 69−76. 34 Huynh, C.; Brunelle, E.; Halámková, L.; Agudelo, J.; Halámek, J. Anal. Chem. 2015, 87, 11531–11536. 35 Brunelle, E.; Huynh, C.; Le, A.–M.; Halámková, L.; Agudelo, J.; Halamek. J. Anal. Chem. 2016, 88, 2413–2420. 36 Kruger, N.; Walker, J. (Ed.) In: The Protein Protocols Handbook, 2nd ed., Humana Press Inc.: Totowa, NJ, 2002. 37 Compton, S. J.; Jones, C. G. Anal. Biochem. 1985, 151, 369–374. 2



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

38

http://www.bio-rad.com/featured/en/bradford-assay.html http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110065A.pdf 40 https://www.thermofisher.com/order/catalog/product/23236 41 Acree, M. A. Forensic Sci. Int. 1999, 102, 35−44. 42 Gungadin, S. Internet J. Med. Update 2007, 2, 1−4. 43 Lee, Y. P.; Takahashi, T. Anal. Biochem. 1966, 14, 71−77. 44 R Development Core Team R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2011. 45 Gentleman, R.; Ihaka, R. R-project, https://www.r-project.org. 46 DeLong, E. R.; DeLong, D. M.; Clarke–Pearson, D.L. Biometrics, 1944, 44, 837–845. 47 Amaral, J. L. M.; Lopes, A. J.; Jansen, J. M.; Faria, A. C. D.; Melo, P. L. Comp. Meth. Prog. Bio., 2012, 105, 183–193. 48 Mark, H.; Harding, C. R. Int. J. Cosmet. Sci. 2013, 35, 163−168. 49 Hadorn, B.; Hanimann, F.; Anders, P.; Curtius, H. C.; Halverson, R. Nature 1967, 215, 416−417. 50 Liappis, N.; Jkel, A. Arch. Dermatol. Res. 1975, 254, 185−203. 51 Pierce, C. M. The Detailed Composition of Feces, Sweat, and Urine: An Annotated Bibliography; Lockheed Missiles and Space Company: Sunnyvale, CA, 1962. 39


Page 10 of 10

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

Recommend Documents