• Sabina Bunescu IML Iasi
  • Bogdan Alexandru Stoica IML Iasi
  • Dragos Peptanariu ICMPP Iasi
  • Liliana Foia UMF Iasi
Keywords: Forensic Science, salivary amylase, Amplex Red, saliva detection, fluorimetry, body fluids detection, serology


A new sensitive method for saliva detection was developed, based on salivary amylase detection but with a final fluorescent product, which increases its sensitivity. After the starch is degraded due to the presence of salivary amylase, glucose is oxidised and generates hydrogen peroxide which is able to transform Amplex Red in resorufin - a highly fluorescent product. The final product is visible both under normal and UV light. The method is fast, accurate, can detect trace amounts of saliva and shows little to no interference with other body fluids. A further increase in sensitivity could be obtained by using horseradish peroxidase in the final step, but this would also lead to an increased background signal and stronger interference with urine.


Aps, J. K., Martens, L. C. (2005): Review: The physiology of saliva and transfer of drugs into saliva. Forensic Sci Int, 150(2-3):119-31.
Carboni, I., Rapi, S., Ricci, U. (2014): Stability of human alpha-salivary amylase in aged forensic samples. Leg Med (Tokyo), 16:214-17.
Ceska, M., Birath, K., Brown, B. (1969): A new and rapid method for the clinical determination of α-amylase activities in human serum and urine. Optimal conditions. Clin Chim Acta, 26(3):437-44.
Groschl, M. (2017): Saliva: a reliable sample matrix in bioanalytics. Bioanalysis, 9(8):655-68.
Han, J. A., Lim, S. T. (2004): Structural changes of corn starches by heating and stirring in DMSO measured by SEC-MALLS-RI system. Carbohydr Polym, 55:265-72.
Kuwayama, K., Miyaguchi, H., Yamamuro, T., Tsujikawa, K., et al. (2016): Effectiveness of saliva and fingerprints as alternative specimens to urine and blood in forensic drug testing. Drug Test Anal, 8(7):644-51.
Mueller, B. (1928): Über den Nachweis eingetrockneten Speichels in Tüchern. Dtsh Z Gerichtl Med, 11:211-24.
Myers, J. R., Adkins, W. K. (2008): Comparison of modern techniques for saliva screening. J Forensic Sci, 53(4):862-7.
Nakanishi, H., Kido, A., Ohmori, T., Takada, A., et al. (2009): A novel method for the identification of saliva by detecting oral streptococci using PCR. Forensic Sci Int, 183(1-3):20-3.
Old, J. B., Schweers, B. A., Boonlayangoor, P. W., Reich, K. A. (2009): Developmental validation of RSID-saliva: a lateral flow immunochromatographic strip test for the forensic detection of saliva. J Forensic Sci, 54(4):866-73.
Park, H. Y., Son, B.N., Seo, Y. I., Lim, S. K. (2015): Comparison of Four Saliva Detection Methods to Identify Expectorated Blood Spatter. J Forensic Sci, 60(6):1571-6.
Quarino, L., Hess, J., Shenouda, M., Ristenbatt, R. R., et al. (1993): Differentiation of alpha-amylase from various sources: an approach using selective inhibitors. J Forensic Sci Soc, 33(2):87-94.
Saxena, S., Kumar, S. (2017): Saliva in forensic odontology: A comprehensive update. J Oral Maxillofac Pathol: JOMFP, 19(2):263-65.
Silva, F. S., Starostina, I. G., Ivanova, V. V., Rizvanov A. A., et al. (2016): Determination of Metabolic Viability and Cell Mass Using a Tandem Resazurin/Sulforhodamine B Assay. Curr Protoc Toxicol, 68(4):2.24.1-15.
Soyama, K., Ono, E. (1983): Pancreatic and salivary amylase determination using a short-chain chromogenic substrate (alpha-4-nitrophenyl-maltoheptaoside) and an amylase inhibitor. Clin Chim Acta, 131(1-2):149-54.
Tsutsumi, H., Higashide, K., Mizuno, Y., Tamaki, K., Katsumata, Y. (1991): Identification of saliva stains by determination of the specific activity of amylase. Forensic Sci Int, 50(1):37-42.
Virkler, K., Lednev, I. K. (2009): Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene. Forensic Sci Int, 188(1-3):1-17.
Wornes, D. J., Speers, S. J., Murakami, J.A. (2018): The evaluation and validation of Phadebas® paper as a presumptive screening tool for saliva on forensic exhibits. Forensic Sci Int, 288: 81-8.
Zakowski, J. J., Bruns, D. E. (1985): Biochemistry of human alpha amylase isoenzymes. Crit Rev Clin Lab Sci, 21(4):283-322.
How to Cite
Bunescu, S., Stoica, B., Peptanariu, D., & Foia, L. (2021). A SENSITIVE METHOD FOR SALIVA DETECTION IN FORENSICS USING SALIVARY AMYLASE COUPLED WITH AMPLEX RED OXIDATION. Journal of Experimental and Molecular Biology, 21(2), 41-46. Retrieved from http://www.jemb.bio.uaic.ro/index.php/jemb/article/view/48