Quantitative determination of testosterone levels with biolayer interferometry
Graphical abstract
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.
References (32)
- et al.
Urinary excretion rates of natural estrogens and androgens from humans, and their occurrence and fate in the environment: A review
Sci. Total Environ.
(2009) - et al.
Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences
Aquaculture
(2002) - et al.
The Comamonas testosteroni, steroid biosensor system (COSS) – reflection on other methods
J. Steroid Biochem. Mol. Biol.
(2010) - et al.
Comparative study of an estradiol enzyme-linked immunosorbent assay kit, liquid chromatography-tandem mass spectrometry, and ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry for part-per-trillion analysis of estrogens in water samples
J. Chromatogr. A
(2007) - et al.
Analysis of anabolic steroids in the horse: development of a generic ELISA for the screening of 17α-alkyl anabolic steroid metabolites
J. Steroid Biochem. Mol. Biol.
(2005) - et al.
Sensitive determination of estriol-16-glucuronide using surface plasmon resonance sensing
Steroids
(2009) - et al.
Picogram-detection of estradiol at an electrochemical immunosensor with a gold nanoparticle/Protein G-(LC-SPDP)-scaffold
Talanta
(2009) - et al.
A model on the regulation of 3α-hydroxysteroid dehydrogenase/carbonyl reductase expression in Comamonas testosteroni
J. Biol. Chem.
(2001) - et al.
Cis-and trans-regulatory elements of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase as biosensor system for steroid determination in the environment
Chem. Biol. Interact.
(2009) - et al.
Higher-throughput, label-free, real-time molecular interaction analysis
Anal. Biochem.
(2007)
A model on the regulation of 3α-hydroxysteroid dehydrogenase/carbonyl reductase expression in Comamonas testosteroni
J. Biol. Chem.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
Application of Bio-Layer Interferometry for the analysis of protein/liposome interactions
J. Pharm. Biomed. Anal.
Manure-borne estrogens as potential environmental contaminants: a review
Environ. Sci. Technol.
Dairy wastewater, aquaculture, and spawning fish as sources of steroid hormones in the aquatic environment
Environ. Sci. Technol.
Occurrence and fate of hormone steroids in the environment
Environ. Int.
Cited by (6)
Optical fiber biosensors toward in vivo detection.
2024, Biosensors and BioelectronicsOne-step high-throughput detection of low-abundance biomarker BDNF using a biolayer interferometry-based 3D aptasensor
2022, Biosensors and BioelectronicsPhosphorescent palladium-tetrabenzoporphyrin indicators for immunosensing of small molecules with a novel optical device
2021, TalantaCitation Excerpt :Surface plasmon resonance (SPR) is an optical signal-transducer allowing real-time monitoring of label-free biomolecular interactions [8,9]. Other label-free detection technologies like antibody based SPR enhanced or not by nanoparticles [10–15], enzyme based SPR [16,17], surface-enhanced Raman spectroscopy (SERS) including antibody based SERS [18], aptamer-based SERS [6,19], molecular imprinted polymer (MIP)-based SERS [5,20,21], and interferometry including interferometric sensors based on aptamer [22,23], antibodies [24–28], polymers [29,30] and proteins [31] as well as electrochemical sensors (ES) [32–34] such as MIP based ES [35], aptamer based ES [36], and protein based ES [37]. Immunosensors rely on recognition of an antigen by an antibody which serves as a biorecognition element [10,38].
Interpol review of controlled substances 2016–2019
2020, Forensic Science International: SynergyCitation Excerpt :Tapentadol: 2016 Diversion and illicit sale of extended release tapentadol in the United States [456]; literature review on tramadol related scientific studies [457] 2017 Systematic review and meta-analysis of the efficacy and safety of tapentadol [458]; synthetic routes towards homochiral tapentadol [459]; 2018 efficacy and safety of tapentadol prolonged release formulation [460]; assessment of tapentadol API Abuse Liability [461]; review of tapentadol [462]; incorporation of tapentadol into validated screening and quantitative methods [463]; HPLC method (LC-MS compatible) developed and validated for identification and characterization of tapentadol and degradation products [464]; 2019 electrochemical sensor for determination of tapentadol in the presence of paracetamol in pharmaceutical samples [465]; optical, thermal, spectroscopic and structural analyses of the phase transformation occurring in tapentadol hydrochloride was studied using single-crystal X-ray diffraction, differential scanning calorimetry and Raman scattering measurements [466]. Testosterone: 2016 high-resolution C-13 NMR spectroscopy compared to H-1 NMR for the detection of cation chelation and cation-induced signal shift effects for testosterone [467]; HPLC-DAD method for quantification of testosterone esters in an oil-based injectable dosage form [468]; availability and acquisition of illicit anabolic androgenic steroids and testosterone preparations on the internet [469]; 2017 UHPLC-ESI analysis of testosterone and other steroids in drinking water [470]; AB-ELISA method for the detection of testosterone and other anabolic androgenic steroids in dietary supplements [471]; electrochemical biosensor for determination of testosterone via electrochemical impedance spectroscopy measurements [472]; TLC method for quantification of synthetic testosterone derivative, methyltestosterone, in pharmaceutical formulations [473]; SPE-LC-MS/MS method for determination of testosterone and other endocrine disrupting compounds in tropical estuarine sediments [474]; biosensor system based on biolayer interferometry for quantitative determination of testosterone in the environment [475]; 2018 SPE/LC-(ESI) MS-MS method for simultaneous quantitative monitoring of testosterone and related pharmaceuticals and hormones in environmental water samples [476]; molecularly imprinted polymer photonic film for the detection of testosterone in water [477,478]; 2019 certification of a testosterone calibration standard and detection and quantification of impurities using GC-FID and NMR [479]; SPE-UHPLC-MS/MS method for detection of 13 hormones including testosterone in diverse water matrices [480]; Tianeptine: 2017 Case report of Tianeptine use purchased on the internet in the United States [481]; gold and silver nanoparticle electrodes combined with amperometric monoaminoxidase biosensors for the determination of tianeptine and other antidepressant drugs (moclobemide and amitriptyline) [482]; 2018 case reports of two known tianeptine fatalities in the United States [483]; characteristics of Tianeptine exposures reported to the National Poison Data System - United States, 2000–2017 [484,485]; New York State Poison Control Centers experience with calls related to tianeptine [486].
Aptamer-Protein Interactions: From Regulation to Biomolecular Detection
2023, Chemical ReviewsLabel-free optical resonator-based biosensors
2020, Sensors (Switzerland)