A Fluorescence Polarization Assay To Detect Steroid Hormone Traces in Milk

Varriale, Antonio; Pinto, Gabriella; Oliviero, Giorgia; D’Errico, Stefano; Majoli, Adelia; Scala, Andrea; Capo, Alessandro; Pennacchio, Anna; Giovanni, Stefano Di; Staiano, Maria; D’Auria, Sabato

doi:10.1021/acs.jafc.5b03689

Cited by 26 publications

(20 citation statements)

References 25 publications

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“…An estradiol–coumarin conjugate (prepared as described in ref ( 21 )) was added to a final concentration of 10 μM to either freshly harvested or lyophilized E. coli at an OD 600 of 0.01 in M9 medium. Following a 20 min incubation, cells were purified from unbound E2–coumarin by spin filtration (10K; 5 min, 5000 rcf).…”

Section: Methodsmentioning

confidence: 99%

Quantifying Hormone Disruptors with an Engineered Bacterial Biosensor

2017

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Endocrine disrupting compounds are found in increasing amounts in our environment, originating from pesticides, plasticizers, and pharmaceuticals, among other sources. Although the full impact of these compounds is still under study, they have already been implicated in diseases such as obesity, diabetes, and cancer. The list of chemicals that disrupt normal hormone function is growing at an alarming rate, making it crucially important to find sources of contamination and identify new compounds that display this ability. However, there is currently no broad-spectrum, rapid test for these compounds, as they are difficult to monitor because of their high potency and chemical dissimilarity. To address this, we have developed a new detection strategy for endocrine disrupting compounds that is both fast and portable, and it requires no specialized skills to perform. This system is based on a native estrogen receptor construct expressed on the surface of Escherichia coli, which enables both the detection of many detrimental compounds and signal amplification from impedance measurements due to the binding of bacteria to a modified electrode. With this approach, sub-ppb levels of estradiol and ppm levels of bisphenol A are detected in complex solutions. Rather than responding to individual components, this system reports the total estrogenic activity of a sample using the most relevant biological receptor. As an applied example, estrogenic chemicals released from a plastic baby bottle following microwave heating were detectable with this technique. This approach should be broadly applicable to the detection of chemically diverse classes of compounds that bind to a single receptor.

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Section: Methodsmentioning

confidence: 99%

Quantifying Hormone Disruptors with an Engineered Bacterial Biosensor

2017

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“…Western blot experiments were performed according to Varriale et al 44 . In brief, aliquots of 15 mL of L. monocytogenes bacterial cultures growth with two different medium (TSB and BHI) and at different time of growth (1 h to 8 h, and 24 h), were heated at 95 °C in the presence of Laemmli buffer 45 and were separated on a sodium dodecyl sulphate polyacrylamide gel electrophoresis (12% SDS-PAGE).…”

Section: Methodsmentioning

confidence: 99%

A fluorescence immunoassay for a rapid detection of Listeria monocytogenes on working surfaces

Capo

D’Auria

Lacroix³

2020

Sci Rep

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Listeria monocytogenes is a foodborne pathogen responsible for human listeriosis. The increasing incidence of listeriosis induced governments and food manufacturing enterprises to act to diminish the problem. Several methods for the detection of Listeria monocytogenes in food industries were developed. However, they are time-consuming and require the use of specialized equipment. To reduce the detection time of Listeria monocytogenes in food, in this work we developed a fluorescence sandwich immunoassay based on the use of an innovative chitosan-cellulose nanocrystal (CNC) membrane that improves the antigen capture during bacterial growth. The combined use of CNC film for the capture of p60 protein-specific antigen together with the use of fluorescence detection reduced the time of analysis from 24 to 12 h with a limit of detection (LOD) of the assay of 102 CFU/mL (2 Log). In addition, the use of monoclonal anti-PepD covalently immobilized to a CNC membrane assured a high specificity of the assay. Interestingly, the obtained results show no cross-reactivity with the five most diffused pathogen bacteria strains tested.

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“…Fluorescence polarization (FP) assay is founded on the exceptional specificity of antibodies for their target antigens and has been extensively employed in clinical diagnostics . Nowadays, the receptor‐based FP assay has already been successfully applied to the detection of ochratoxin A , 17β‐estradiol , ractopamine , and phthalate esters . As a simple and rapid method, broad‐specific receptor‐based FP assay has several advantages, such as short assay time and absence of washing step in comparison with the enzyme‐linked immunosorbent assay.…”

Section: Introductionmentioning

confidence: 99%

Estrogen receptor‐based multi‐residue screening of bisphenol compounds in urine

Guan

Sun

et al. 2018

Biotech and App Biochem

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Human exposure to bisphenol compounds (BPs) has been implicated in the development of several chronic diseases. Instead of exploiting the traditional methods for determination of BPs, this work confirms that the human estrogen receptor α ligand binding domain (hERα-LBD) is a powerful recognition element that can be used to monitor multi-residue of BPs in urine samples by fluorescence polarization (FP) assay. Test parameters were optimized for the best performance. Under the optimal conditions, the IC 50 values of BPs are in the range of 0.04-1.61 μg mL −1 . Recovery experiments were then performed to assess the accuracy and precision of the established method. The results detected by FP assay show good agreements with that of liquid chromatography-tandem mass spectrometry method with a fit of R 2 = 0.9372 and 0.9640 for BPE and BPAP, respectively. A computational methodology, ligand-based pharmacophore model, was also employed to further explore the broad-specific of tested compounds. It was found that the two hydrogen bond acceptor features and one hydrophobic aliphatic feature were essential for the corresponding cross-reactivity results from the FP assay. All these results suggest that the established method can be successfully applied to monitor the occurrence

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A Fluorescence Polarization Assay To Detect Steroid Hormone Traces in Milk

Cited by 26 publications

References 25 publications

Quantifying Hormone Disruptors with an Engineered Bacterial Biosensor

Quantifying Hormone Disruptors with an Engineered Bacterial Biosensor

A fluorescence immunoassay for a rapid detection of Listeria monocytogenes on working surfaces

Estrogen receptor‐based multi‐residue screening of bisphenol compounds in urine

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