An integrated biosensor and mobile workstation for on-site species and sex identification from bloodstains

Bloodstains are the most common biological specimens at a crime scene. Rapid and sensitive species and sex identification from bloodstains can expedite crime investigation. Nevertheless, time-consuming protocols and specialized equipment are often required in forensic practice. Taking advantage of speediness and convenience, loop-mediated isothermal amplification (LAMP) has been used for bloodstain analysis. However, only a single characteristic gene can be targeted per reaction, leading to specimen consumption and limited throughput. Herein, a portable biosensor is first developed for on-site simultaneous species and sex identification from bloodstains without DNA purification. By combining multiplex LAMP and a triplex lateral flow device (LFD), minute amounts of specimen can be identified without any sophisticated apparatus. Using gold magnetic nanoparticle as nanoprobe for amplicon hybridization, the results can be interpreted with naked eye. Meanwhile, the relative intensity of tinctorial signal can be quantitated using a smartphone to eliminate subjective bias. Besides, carryover contamination from uncapping is thoroughly prevented by the uracil-DNA glycosylase-based anti-aerosol strategy while holding constant reaction temperature. Based on the simplicity of the whole "sample to result" workflow, a mobile workstation containing lyophilized LAMP reaction mixtures and a compact heater is customized for on-site detection, rendering this biosensor competent in various scenarios. As a demonstration, species and sex can be identified within 1 h by targeting cytochrome b gene and Y-chromosomal amelogenin gene. Even bloodstains stored for over 6 years can be accurately identified, indicating the significant potential of this biosensor for forensic nucleic acid analysis.