A one-step fluorescent detection method for lipid fingerprints; Eu(TTA)₃·2TOPO

Abstract

This paper describes the development of a one-step fluorescent lipid reagent that involves europium chelates whose generic formula can be represented as europium(organic ligand)₃·2(synergic reagent). An optimized formulation is presented and a comparison with physical developer was conducted which showed the new technique to be very good when fresh fingerprints were recovered but inferior to physical developer on aged prints (over a week). In addition, the compatibility of the technique with sequential methods such as ninhydrin and DFO is reported. The experimentation, results and chemical formulation of this study were presented to delegates of the International Symposium on Fingerprint Detection and Identification held in Israel in June 1995 [1]. Prior to that a patent application had been made [2].

Introduction

This research evolved from my earlier success on visualizing cyanoacrylate-treated latents with the europium chelates represented by the general formula Eu(organic ligand)₃·2H₂O 3, 4. The most promising was a fluorescent europium chelate, referred to as TEC, produced by the reaction of europium trivalent cations with the aryl-β-diketone, thenoyltrifluoroacetone, as shown in Fig. 1. TEC dissolves in a solution of methyl ethyl ketone (20%) in water. The methyl ethyl ketone transfers into the cyanoacrylate (CA) and carries the TEC with it, while the water helps to quench fluorescence of the TEC left in the background. The spectral characteristics of europium chelates are ideal for visualizing fingerprints, since excitation of the organic ligand occurs in the UV and europium narrow band emission (10 nm) is observed at 614 nm as a result of intramolecular energy transfer. The large Stokes shift of about 260 nm and the narrow emission band allow for flexible filtering of unwanted backgrounds. Others have reported the visualization of CA-treated fingerprints using Eu(TTA)₃Phen, in which the two coordinating water molecules in the TEC structure have been replaced with the bidentate ortho-phenthroline ligand [5].

The generic formula for this type of chelate is Eu(organic ligand)₃·2(synergic reagent) and is similar in structure to the fluorescent tags used by biochemists when performing a time-resolved immunoassay [6]. The purpose of a time-resolved immunoassay is to obtain an accurate measurement of the amount of protein contained within a biological sample. Biologists often use fluorescence as a method of measuring the protein content of a sample by “tagging” the protein with a fluorescent label, called a fluorophore, and then “flushing” the excess fluorophore out of the system. By using the appropriate excitation light they measure the amount of fluorescent light emitted from the “tagged” protein. To do this they use time-resolved fluorometry because often other biological components of the system will fluoresce giving a false reading. The rare-earth metals, europium and terbium, are especially good for this time-resolved fluorometry because when chelated to organic ligands they fluoresce for relatively long periods of time compared to the other biological materials. The biologists delay recording their fluorescent measurement until the other components have faded and this allows them to record only the fluorescence coming from the europium tag. In such systems, the europium is chelated to three molecules of an organic ligand such as thenoyltrifluoroacetone and a further two molecules of a synergic reagent such as trioctylphosphine oxide (see Fig. 2). The fluorophore has an extremely bulky structure which protects the europium from the aqueous environment of the biological medium. To further isolate the europium ion from the water molecules a detergent is added to the system. This combination, of bulky structure and detergent, allows for maximum fluorescence from the europium which in turn allows for sensitive measurement of extremely small amounts of protein.

Previous application of europium for the visualization of fingerprints on paper involved the amino acids present in a fingerprint [7]. First the fingerprint was treated with ninhydrin to produce Ruhemann's Purple. Further reaction of this compound occurs with a solution containing europium ions, reportedly, forming a weakly fluorescent material as a result of energy transfer from the Ruhemann's Purple into the europium ion. Unfortunately the fluorescence is very weak since the emission of the Ruhemann's Purple molecule is not correctly matched to the absorption of the europium ion which inhibits successful energy transfer from the absorbing ligand to the emitting metal ion. Expensive time-resolved imaging equipment is required to obtain reasonable print detail [8].

My previous research into the development of TEC for CA-treated fingerprints encouraged us to extend the use of europium chelates into fingerprints on porous surfaces such as paper products that would not be treated with CA. In contrast to Menzel and Mitchell's previous work on paper which attempted to detect the amino acids (present in sweat) [7], I was interested in visualising the lipids which are present in sebum. I felt that developing a fluorescent lipid reagent was important for several reasons. Firstly, amino acid reagents develop latents which tend to have very spotty, discontinuous ridge detail whereas ridges developed by lipid reagents are even and continuous. Secondly, the only available treatment for lipid prints on paper is physical developer which is non-fluorescent. I hoped that a fluorescent technique would help remove unwanted background patterns which is often a problem with paper products. One potential problem of using europium chelates that are excited by ultraviolet light is that of the inherent background which occurs as a result of the optical brighteners and printing inks within the paper fluorescing under the UV radiation. However, the narrow emission band exhibited by europium would greatly assist in the elimination of this type of background.

It was necessary to establish whether sufficient TEC could be dissolved in the lipids to enable the latent fingerprint to be easily observed. Once this was confirmed I then had to find a suitably polar solvent that would dissolve the hydrophobic chelate without dissolving the lipid fingerprint. My intention was to achieve fingerprint visualization by a physical transfer of the hydrophobic chelate from the polar solution to the non-polar lipid.