Original Article A Fluorescence Sensing Method with Reduced DNA Typing and Low-Cost Instrumentation for Detection of Sample Tampering Cases in Urinalysis NUNO M. M. PIRES, 1,2 TAO DONG , 2 ZHAOCHU YANG, 1 and SIMA ˜ O M. B. SANTOS 1,2 1 Chongqing Key Laboratory of Micro-Nano Systems and Smart Transduction, Collaborative Innovation Center on Micro- Nano Transduction and Intelligent Eco-Internet of Things, Chongqing Key Laboratory of Colleges and Universities on Micro- Nano Systems Technology and Smart Transducing, National Research Base of Intelligent Manufacturing Service, Chongqing Technology and Business University, Nan’an District, Chongqing 400067, China; and 2 Department of Microsystems – IMS, Faculty of Technology, Natural Sciences and Maritime Sciences, University of South-Eastern Norway-USN, P.O. Box 235, 3603 Kongsberg, Norway (Received 15 May 2019; accepted 4 October 2019) Associate Editor Stefan M. Duma oversaw the review of this article. Abstract—This work presents a method to unequivocally detect urine sample tampering in cases where integrity of the sample needs to be verified prior to urinalysis. The technique involves the detection of distinct patterns of a triplex short tandem repeats system in DNA extracted from human urine. The analysis is realized with single-dye fluorescence detection and using a regular smartphone camera. The experimental results had demonstrated the efficacy of the analytical approach to obtaining distinct profiles of amplicons in urine from different sample providers. Reproducibility tests with fresh and stored urine have revealed a maximum variation in the profiles within an interval of 5 to 9%. Cases of urine sample tampering via mixture were simulated in the study, and the experiments have identified patterns of mixed genotypes from dual mixtures of urine samples. Moreover, sample adulteration by mixing a non-human fluid with urine in a volume ratio over 25% can be detected. The low cost of the approach is accompanied by the compatibility of the technique to use with different DNA sample preparation protocols and PCR instrumentation. Furthermore, the pos- sibility of realizing the method in an integrated microchip system open great perspectives to conducting sample integ- rity tests at the site of urine sample reception and/or at resource-limited settings. Keywords—Sample tampering, Bioanalytical methods, Clin- ical/biomedical analysis, Drug monitoring/drug screening, Optical sensors, On-site analysis. INTRODUCTION Urine collection and analysis is a common proce- dure in drug abuse or sports drug testing. 27,31 Several illicit substances can be found in urine with high sen- sitivity; nevertheless, their presence can be masked by prohibited actions done by athletes and drug abusers. There have been reported cases of sample manipula- tion and/or its substitution before samples are deliv- ered for drug testing. 4,13,17 Therefore, methods of proving sample integrity are essential in drug screening and sports doping control; however, many of current procedures are still limited to empirical analysis, such as use of sealed containers, visual inspection during sampling procedure, or meticulous recording of the whole sample delivery process. 32,33 Some of these procedures are cumbersome, and often embarrassing to the subjects as the cases of visual proving of sample delivery. Moreover, the authorities continuously face challenges in verifying intentional tampering of the delivered samples. Sophisticated methods to identify unequivocal sample manipulation by mixture with non-human liquids (e.g. water or beverages) or other person’s urine are a demand. 10 It is also of high interest that new methods can be realized in point-of-use sys- tems, 11 proving eventual sample manipulation before samples are sent to urinalysis laboratories. DNA typing using short tandem repeats (STRs) is an indispensable tool in modern genetics and particu- larly in forensic analysis. STR loci is characterized by the high polymorphism, which is exploited by forensic personnel to unequivocally identify individuals. Situ- Address correspondence to and Tao Dong, Department of Microsystems – IMS, Faculty of Technology, Natural Sciences and Maritime Sciences, University of South-Eastern Norway-USN, P.O. Box 235, 3603 Kongsberg, Norway. Electronic mail: tao.dong@ usn.no Annals of Biomedical Engineering (Ó 2019) https://doi.org/10.1007/s10439-019-02386-y BIOMEDICAL ENGINEERING SOCIETY Ó 2019 Biomedical Engineering Society