High affinity truncated DNA aptamers for the development of fluorescence based progesterone biosensors Hani A. Alhadrami a, 1 , Raja Chinnappan b, 1 , Shimaa Eissa b , Anas Abdel Rahamn c , Mohammed Zourob b, c, * a Faculty of Applied Medical Sciences, King Abdulaziz University, Post Office Box 80402, Jeddah 21589, Saudi Arabia b Department of Chemistry, Alfaisal University, Al Zahrawi Street, Al Maather, AlTakhassusi Rd, Riyadh 11533, Saudi Arabia c King Faisal Specialist Hospital and Research Center, Zahrawi Street, Al Maather, Riyadh 12713, Saudi Arabia article info Article history: Received 1 January 2017 Received in revised form 12 February 2017 Accepted 20 February 2017 Available online 24 February 2017 Keywords: Aptamers Progesterone Hormones in water abstract Aptamers have shown a number of potential applications in sensing and therapeutic due to the high affinity and specificity towards their target molecules. Not all the nucleotides in the full length aptamers are involved in the binding with their targets. The non-binding domain of the aptamer may affect the binding affinity of the aptamer-target complex. Mapping the aptamer binding region could increase the affinity and the specificity. In this paper, we designed aptamer-based fluorescence sensors from a truncated progesterone (P4) aptamer. Then, fluorescein and quencher labelled aptamer complementary oligonucleotide sequences were hybridized to the truncated aptamer at different sites to form duplex structures. We used fluorescence-quencher pair displacement assay upon progesterone binding for the determination of P4. One of the truncated sequences has shown high binding affinity with 16 fold in- crease in the dissociation constant, K d (2.1 nM) compared to the original aptamer. The aptasensor was highly selective for P4 against similar compounds such as 17-b estradiol, bisphenol-A and vitamin D. The sensor has been applied for the detection of P4 in spiked tap water and in urine samples showing good recovery. This new developed aptamer-based fluorescence biosensor can be applied in food, pharma- ceutical, and clinical industries. © 2017 Elsevier Inc. All rights reserved. 1. Introduction Food products and drinking water contamination with endocrine-disrupting chemicals (EDCs) may have a serious effect on hormonal functions such as metabolism, biosynthesis, etc [1]. Progesterone (P4) is a small hydrophobic steroid hormone secreted from corpusluteum, which plays a major role in mammalian pregnancy, animal growth and development. Progesterone level is used as an indicator for early pregnancy. Many clinical assays are currently used to measure hormonal levels in a variety of body fluids. The progesterone concentration is 1 ng/ml in serum during the pre-ovulation period, 20 ng/ml in the mid cycle and more than 300 ng/ml at the pregnancy period. Elevated levels of P4 leads to headache, breast tenderness, stomach upset, constipation, diarrhea, body pain, tiredness, virginal discharge and urinal in- fections [2]. Consumption of high levels of progesterone in cow milk may case breast and lung cancers [3,4] and it affect the gonadotropin (GnRH) releasing hormone secretion in males [5]. When high amount of P4 is consumed, the body retains certain amount and the rest is released to the environment as waste. Therefore, it is highly important to monitor P4 levels in environ- mental and clinical samples. Several methods are applied for the detection of P4. Instru- mental analysis methods such as high performance liquid chro- matography (HPLC), gas chromatography-mass spectrometry (GC/ MS) and liquid chromatography-mass spectrometry (LC-MS) are highly sensitive. However, they need well-trained operators, high cost and not suitable for field applications. Immunoassays like enzyme linked immunosorbent assay (ELISA), radioimmune assays, electrophoresis-chemiluminescence and non-competitive audio- metric assays are used for the detection of P4 in different samples [6e8]. However, the limited stability and high cost of the immu- noassays are still major challenges. * Corresponding author. Department of Chemistry, Alfaisal University, Al Zahrawi Street, Al Maather, AlTakhassusi Rd, Riyadh 11533, Saudi Arabia. E-mail address: mzourob@alfaisal.edu (M. Zourob). 1 equal Contribution. Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio http://dx.doi.org/10.1016/j.ab.2017.02.014 0003-2697/© 2017 Elsevier Inc. All rights reserved. Analytical Biochemistry 525 (2017) 78e84