Elsevier

Chemico-Biological Interactions

Volume 276, 1 October 2017, Pages 141-148
Chemico-Biological Interactions

Quantitative determination of testosterone levels with biolayer interferometry

https://doi.org/10.1016/j.cbi.2017.05.013Get rights and content

Highlights

  • We developed a biolayer interferometry method to determine environmental steroids.

  • In the method, DNA and protein interaction was detected easily.

  • Changes in the interference of white light from the fiber surface enable detection.

  • Monitoring binding does not require radioactive, enzymatic, or fluorescent labels.

Abstract

Natural and synthetic steroid hormones are widely spread in the environment and are considered as pollutants due to their endocrine activities, even at low concentrations, which are harmful to human health. To detect steroid hormones in the environment, a novel biosensor system was developed based on the principle of biolayer interferometry. Detection is based on changes in the interference pattern of white light reflected from the surface of an optical fiber with bound biomolecules. Monitoring interactions between molecules does not require radioactive, enzymatic, or fluorescent labels. Here, 2 double-stranded DNA fragments of operator 1 (OP1) and OP2 containing 10-bp palindromic sequences in chromosomal Comamonas testosteroni DNA (ATCC11996) were surface-immobilized to streptavidin sensors. Interference changes were detected when repressor protein RepA bound the DNA sequences. DNA–protein interactions were characterized and kinetic parameters were obtained. The dissociation constants between the OP1 and OP2 DNA sequences and RepA were 9.865 × 10−9 M and 2.750 × 10−8 M, respectively. The reactions showed high specifically and affinity. Because binding of the 10-bp palindromic sequence and RepA was affected by RepA–testosterone binding, the steroid could be quantitatively determined rapidly using the biosensor system. The mechanism of the binding assay was as follows. RepA could bind both OP1 and testosterone. RepA binding to testosterone changed the protein conformation, which influenced the binding between RepA and OP1. The percentage of the signal detected negative correlation with the testosterone concentration. A standard curve was obtained, and the correlation coefficient value was approximately 0.97. We could quantitatively determine testosterone levels between 2.13 and 136.63 ng/ml. Each sample could be quantitatively detected in 17 min. These results suggested that the specific interaction between double-stranded OP1 DNA and the RepA protein could be used to rapidly and quantitatively determine environmental testosterone levels by the biolayer interferometry technique.

Introduction

Natural and synthetic steroid hormones are continuously released into the environment from humans, livestock, and aquaculture sources [1], [2], [3], [4]. Synthetic steroids, such as the androgens testosterone (T) and trenbolone acetate (TbA) and the estrogens 17β-estradiol (E2) and zeranol, are primary growth promoters used in the United States livestock industry to increase animal growth [5]. These steroids have been widely detected in sewage treatment plants [6], [7], rivers [8], and drinking water [9].

Steroids can interfere with the hormone systems of humans and other organisms by mimicking physiological hormones, inhibiting signaling pathways as endocrine disruptors, interfering with normal biological responses [10].

In most fish, environmental steroids can alter gonad development even after sex differentiation has occurred, and these exogenous steroids can cause sex reversal [11]. For example, the expression of vitellogenin in fish can be induced by 17α-ethinylestradiol (EE2), even at a concentration as low as 0.1 ng/L, which can affect sex differentiation of the fish [12], [13].

To date, several reviews have been published on steroid-analysis methods [14], [15]. A variety of classical analytical techniques for specifically estimating steroids has already been reported, such as high-performance liquid chromatography (HPLC), liquid chromatography coupled with fluorescence measurement, or mass spectrometry [16], [17]. For screening purposes, enzyme-linked immunosorbent assays are widely used [18]. Novel developments for quantitating low levels of estrogens include highly sensitive immunoassays with surface plasmon resonance systems [19] or electrochemical immune sensors [20]. Although the problem of pollution with steroids has received substantial attention, data regarding their concentrations in the environment are lacking. The methods currently used to determine environmental steroid levels are expensive and complex. Therefore, new methods for low-cost, rapid, easy steroid detection and quantification are urgently needed. The method used should be sensitive enough to detect low concentrations of steroids in the environment.

In early investigations, 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) was found to act as a key enzyme in degrading Comamonas testosteroni steroids. In the bacterium, the RepA protein functions as a repressor that inhibits 3α-HSD/CR transcription. RepA can bind to 2 palindromic operator sequences (OP1 and OP2), thereby blocking 3α-HSD/CR transcription. When testosterone binds to RepA, the conformation of the RepA protein changes, which leads to decreased affinities between RepA and the operator sequences, and increased 3α-HSD/CR expression in C. testosteroni [21]. Based on these findings, a novel, rapid and fluorescence-based screening method was generated for steroid determination using a cell-free biosensor system, and the limit of detection (LOD) was as low as 28 pg/ml for testosterone and 0.029 fg/ml for estradiol [22].

The Biolayer interferometry (BLI) Octet RED96 system (Pall ForteBio, USA) includes a white light source, a transmission optical fiber, an optical fiber sensor, and optics spectrometers. White light is launched into the optical fiber sensor with a polymer-coated tip. After the light reaches the sensor's fiber top, some of the light is reflected into the fiber. Some light continues through the fiber and is reflected when it encounters molecules immobilized on the top of the sensor's fiber, while the rest of the light continues into the biomolecular solution. Both beams of reflected light can interfere with each other, leading to a shift in the wavelength of the detected light. The shift is related to the thickness of the layer immobilized on the top of the sensor fiber. As a result, the system essentially measures the increase in thickness on the top of the fiber as molecules bind to it [23]. The binding process can be monitored in real time, and biomolecular interaction analysis (BIA) can be achieved.

In this work, based on BLI technique, BIA of the RepA protein with the dsDNA OP1 and OP2 templates was rapidly performed in vitro. Subsequently, the quantitative determination of testosterone based on RepA and dsDNA interactions was established with the BLI technique. The new method does not require label radioactive, enzymatic, fluorescence materials, or washing steps. Microfluidic flow-through systems are not used; therefore, the potential problem of clogging the micro-channels is avoided [24].

Section snippets

Bacterial strain and plasmid

The host strain Escherichia coli BL21 (DE3) pLysS (TransGen Biotech, Beijing, China) and the recombinant plasmid pET-RepA were used to overexpress the RepA protein. The recombinant plasmid pET-RepA containing the ampicillin-resistance gene was a gift from the Institute of Toxicology and Pharmacology, University of Kiel (Kiel, Schleswig-Holstein, Germany) [25].

Growth conditions

Bacterial cells were grown in Luria-Bertani medium (OXOID, Basingstoke, UK) at 37 °C in a 180-rpm shaker. The growth medium contained

Preparation of dsDNA OP1, OP2, and CO templates

Three 38-bp, biotin-labeled dsDNA fragments (OP1, OP2, and CO) were prepared by annealing hybridization with the synthetic oligonucleotides. The G + C percentage of the dsDNA OP1, OP2, and CO fragments were calculated to be 76.3%, 52.6% and 50%, respectively (Fig. 1), and their melting temperatures were 86 °C, 75 °C, and 72.4 °C, respectively. The three dsDNA fragments were denatured at 90 °C for 10 min and allowed to cool down slowly to room temperature for 1.5 h. The wavelength-scanning

Discussion

The BLI technique is a novel optical fiber approach for reflectometry interference spectroscopy of BIA. The advantage of this new method is that optical fiber can be used as a biosensor. The method is simple to perform, involves lower cost than other methods, and provides high sensitivity and reliability. A single bioprobe can be easily prepared as a BIA biosensor for direct real-time monitoring of association and dissociation processes in various kinds of molecular interactions, such as in

Acknowledgements

This work was supported by the Science and Technology Department of Jilin Province (project number: 20150311096YY) of P. R. China.

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