Abstract

Detection of body fluids and fingerprints using a cell-free spray

In the case of detection of bodily fluids and age determination of fingerprints, both were planned as a cell-free system, possibly with a spray-based application. Therefore, there are risks related to how our detectors could be applied at a crime scene.

In terms of general contamination of the environment, there would be no release of GMOs in this case and no contamination with DNA. It should be considered whether the proteins and beads are toxic to other organisms and for how long they would persist in the environment when placed there. Extensive tests for interactions between the used proteins and different materials, surfaces, and household products need to be made in order to determine the specificity.

In addition, potential risks for forensic scientists using the spray, and people nearby such as police officers, need to be assessed. As a comparison of sprayed other proteins in common use, hairsprays containing keratin were considered. The risk assessment for keratin containing hairsprays suggests that most droplets/particles incidentally inhaled from cosmetic sprays would be deposited in the nasopharyngeal and bronchial regions and would not be respirable (i.e., able to enter the lungs) to any appreciable amount. However, toxicological studies are still recommended before marketing a spray containing proteins. Until then, appropriate PPE, including gloves and a mask, will have to be recommended as a safety precaution.

We need to consider how the engineered proteins in the body fluid detection system (Haptoglobin, PotD/Spermine Binding Protein, Lactoferrin Binding Protein, Odorant Binding Protein, Lanosterol Syntase) might interact with substances at the crime scene that are not desired targets. In other words, how do we avoid false positives or false negatives? Where substances that can lead to false positives are commonly found this will also have an impact on where the spray can be used. It is possible that it is only reasonable to use it for specific crimes, as the spray would not be effective enough in a complex environment.

Due to the specific application of this spray, it is also important to consider how efficiently the spray can be applied at a crime scene, and what quality of evidence it would return. It is crucial that the spray highlights traces of bodily fluids and fingerprints, but does not wash them away or smear them across a surface while doing so.

It also needs to be considered how the proteins in the spray might interact with each other, since several different proteins are stored in one container, or whether individual sprays will be required. It is crucial that the different detectors do not form aggregates with each other, or with the attached fluorescent beads, or interact with each other in any other way. This can be tested in a laboratory setting e.g. by biolayer interferometry or another biomolecular interaction technique such as isothermal titration calorimetry.

We also need to consider about storage of spray cans, i.e. what material does the can need to be made of, what temperature does it need to be stored at, what is the expiration date? Taking all the above considerations into account, would it then still be user-friendly and affordable?

For manufacturing the spray it is crucial that a well-developed production-pipeline is assembled that leads to good yields of the product at an acceptable cost. Reagents required for the manufacturing process will need to be considered as well as potential waste products and their disposal. A careful cost-benefit calculation needs to be made in order to see if the price the spray would have is still acceptable for crime scene managers.

A final and key point is to work out how to clean up spray-residues after its use. Leaving it on a crime scene after an investigation is finished could lead to potential risks due to long term exposure.

Cell-based chromium detector

The chromium detector is designed and intended as a laboratory-based device, which can be used in a basic laboratory of Containment Level 1. Safety precautions are hence in place that avoid the release of genetically modified organism into the environment, as well as precautions against infections.

From the user's point of view, chromium is the important constituent in stainless steel alloys, but chromate is the substrate for this sensor. In order to get one from the other, chemical modification (such as acid treatment) of samples would be required. Careful consideration of the risk and the benefit of this chemical transformation is required. In addition, chromate is an environmental toxin, so disposal of samples after testing would have to be carefully regulated.

Potential problems with the device are substances that lead to non-specific induction of the GFP readout, and their prevalence with the bone samples that would be analysed, would need to be investigated. This can be done by exposing the sensor to several different substances that are commonly found in the environment, and rapidly testing the effect in a high-throughput, possibly automated, manner: for example in a 384-well plate reader experiment.

Apart from specificity, sensitivity is also an important consideration of the chromate sensor. If no appreciable amounts of chromate can be found on an incision on bone, the conclusion may be that there is not enough chromate present.

Assessment of the Bacterial Strains used

Strains of E. coli deliberately adapted to have biological limitations such that they are unlikely to infect or colonise the human body and that have a history of safe use in the laboratory environment, e.g. multiply auxotrophic or recombination deficient mutants. This category primarily consists of E coli K12 and B strain derivatives, including BL21.

Note: Genetic modification of such bacteria does not change this assessment providing (1) the GM cell line has been subject to the GM risk assessment process and deemed to be no more hazardous than the unmodified parent and (2) the vector used is not a viral vector capable of infecting human cells, regardless of whether it is replication competent or defective.

Bacterial strains in this category are deemed to be unlikely to cause disease in humans, i.e. equivalent to ACDP Hazard Group 1. No infection is anticipated. Direct dermal inoculation (i.e. injection via sharps) may cause local inflammation.

Risk Assessment of chemicals used:

Beta-mercapto-ethanol was used as a reducing agent for cysteine residues prior to SDS-PAGE experiments. Additionally to standard PPE, this substance was handled in a fume hood.

In order to assay the chromate responsive system cloned into E. coli, different chromium salts and control substances were required. The substances used were: Potassium Chromate, Potassium Dichromate, Potassium Sulphate, and Chromium(III) Chloride. PPE was worm according to recommendation from MSDS: Nitrile Gloves, Lab coat, Dust respirator, Safety glasses.