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Paramagnetic nanoparticles as a platform for FRET-based sarcosine picomolar detection.

Heger Z, Cernei N, Krizkova S, Masarik M, Kopel P, Hodek P, Zitka O, Adam V, Kizek R - Sci Rep (2015)

Bottom Line: Successful binding of sarcosine leads to assembly of a sandwich construct composed of anti-sarcosine antibodies keeping the Förster distance (Ro) of FRET pair in required proximity.The detection is based on spectral overlap between gold-functionalized green fluorescent protein and antibodies@quantum dots bioconjugate (λex 400 nm).Assembled biosensor was then successfully employed for sarcosine quantification in prostatic cell lines (PC3, 22Rv1, PNT1A), and urinary samples of prostate adenocarcinoma patients.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00, Czech Republic, European Union [2] Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno, Czech Republic, European Union.

ABSTRACT
Herein, we describe an ultrasensitive specific biosensing system for detection of sarcosine as a potential biomarker of prostate carcinoma based on Förster resonance energy transfer (FRET). The FRET biosensor employs anti-sarcosine antibodies immobilized on paramagnetic nanoparticles surface for specific antigen binding. Successful binding of sarcosine leads to assembly of a sandwich construct composed of anti-sarcosine antibodies keeping the Förster distance (Ro) of FRET pair in required proximity. The detection is based on spectral overlap between gold-functionalized green fluorescent protein and antibodies@quantum dots bioconjugate (λex 400 nm). The saturation curve of sarcosine based on FRET efficiency (F₆₀₄/F₅₁₀ ratio) was tested within linear dynamic range from 5 to 50 nM with detection limit down to 50 pM. Assembled biosensor was then successfully employed for sarcosine quantification in prostatic cell lines (PC3, 22Rv1, PNT1A), and urinary samples of prostate adenocarcinoma patients.

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The characterization of paramagnetic microparticles, composed of nanomaghemite core, modified with polyvinylpyrrolidone and gold expressed as: (A) SEM micrograph (length of scale bar is 200 nm). (B) Particles size distribution, with expression of their zeta potential (determined in PBS, pH 7.4). (C) XRF spectrum showing the most abundant elements in paramagnetic particles. (D) SECM scans expressing the electrochemical current response behavior of immobilized nanoparticles (800 × 800 μm). (E) Scan was further converted to more detailed 3D scan (80 × 80 μm) that shows the decrease of relative current response influenced by nanoparticles presence. (F) Results of recoveries of sarcosine (2 μM) binding to antibodies at nanoparticles, obtained from IEC analyses. Values are means of three independent replicates (n = 3). Vertical bars indicate standard error. SDS-PAGE showing the binding capacity of nanoparticles towards sarcosine antibodies (0.75–1.2 mg.mL−1 of Abs) are illustrated too.
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f1: The characterization of paramagnetic microparticles, composed of nanomaghemite core, modified with polyvinylpyrrolidone and gold expressed as: (A) SEM micrograph (length of scale bar is 200 nm). (B) Particles size distribution, with expression of their zeta potential (determined in PBS, pH 7.4). (C) XRF spectrum showing the most abundant elements in paramagnetic particles. (D) SECM scans expressing the electrochemical current response behavior of immobilized nanoparticles (800 × 800 μm). (E) Scan was further converted to more detailed 3D scan (80 × 80 μm) that shows the decrease of relative current response influenced by nanoparticles presence. (F) Results of recoveries of sarcosine (2 μM) binding to antibodies at nanoparticles, obtained from IEC analyses. Values are means of three independent replicates (n = 3). Vertical bars indicate standard error. SDS-PAGE showing the binding capacity of nanoparticles towards sarcosine antibodies (0.75–1.2 mg.mL−1 of Abs) are illustrated too.

Mentions: The morphology of prepared nanoparticles was determined by scanning electron microscopy. As it can be seen in Fig. 1A (scale bar 200 nm), nanoparticles form aggregates, due to their low zeta potential, determined as - 7.09 ± 0.13 mV (upper inset in Fig. 1B). Generally, iron oxide nanoparticles exhibit their absolute zeta potential within the pH range 4–1013, and thus using PBS (pH = 7.4) leads to aggregation of nanometric particles that was partially reduced using ultrasonic homogenizer (Bandelin electronic, Berlin, Germany) to form suspension containing particles with relatively uniform size (d = 23 ± 5 nm, Fig. 1B).


Paramagnetic nanoparticles as a platform for FRET-based sarcosine picomolar detection.

Heger Z, Cernei N, Krizkova S, Masarik M, Kopel P, Hodek P, Zitka O, Adam V, Kizek R - Sci Rep (2015)

The characterization of paramagnetic microparticles, composed of nanomaghemite core, modified with polyvinylpyrrolidone and gold expressed as: (A) SEM micrograph (length of scale bar is 200 nm). (B) Particles size distribution, with expression of their zeta potential (determined in PBS, pH 7.4). (C) XRF spectrum showing the most abundant elements in paramagnetic particles. (D) SECM scans expressing the electrochemical current response behavior of immobilized nanoparticles (800 × 800 μm). (E) Scan was further converted to more detailed 3D scan (80 × 80 μm) that shows the decrease of relative current response influenced by nanoparticles presence. (F) Results of recoveries of sarcosine (2 μM) binding to antibodies at nanoparticles, obtained from IEC analyses. Values are means of three independent replicates (n = 3). Vertical bars indicate standard error. SDS-PAGE showing the binding capacity of nanoparticles towards sarcosine antibodies (0.75–1.2 mg.mL−1 of Abs) are illustrated too.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352859&req=5

f1: The characterization of paramagnetic microparticles, composed of nanomaghemite core, modified with polyvinylpyrrolidone and gold expressed as: (A) SEM micrograph (length of scale bar is 200 nm). (B) Particles size distribution, with expression of their zeta potential (determined in PBS, pH 7.4). (C) XRF spectrum showing the most abundant elements in paramagnetic particles. (D) SECM scans expressing the electrochemical current response behavior of immobilized nanoparticles (800 × 800 μm). (E) Scan was further converted to more detailed 3D scan (80 × 80 μm) that shows the decrease of relative current response influenced by nanoparticles presence. (F) Results of recoveries of sarcosine (2 μM) binding to antibodies at nanoparticles, obtained from IEC analyses. Values are means of three independent replicates (n = 3). Vertical bars indicate standard error. SDS-PAGE showing the binding capacity of nanoparticles towards sarcosine antibodies (0.75–1.2 mg.mL−1 of Abs) are illustrated too.
Mentions: The morphology of prepared nanoparticles was determined by scanning electron microscopy. As it can be seen in Fig. 1A (scale bar 200 nm), nanoparticles form aggregates, due to their low zeta potential, determined as - 7.09 ± 0.13 mV (upper inset in Fig. 1B). Generally, iron oxide nanoparticles exhibit their absolute zeta potential within the pH range 4–1013, and thus using PBS (pH = 7.4) leads to aggregation of nanometric particles that was partially reduced using ultrasonic homogenizer (Bandelin electronic, Berlin, Germany) to form suspension containing particles with relatively uniform size (d = 23 ± 5 nm, Fig. 1B).

Bottom Line: Successful binding of sarcosine leads to assembly of a sandwich construct composed of anti-sarcosine antibodies keeping the Förster distance (Ro) of FRET pair in required proximity.The detection is based on spectral overlap between gold-functionalized green fluorescent protein and antibodies@quantum dots bioconjugate (λex 400 nm).Assembled biosensor was then successfully employed for sarcosine quantification in prostatic cell lines (PC3, 22Rv1, PNT1A), and urinary samples of prostate adenocarcinoma patients.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00, Czech Republic, European Union [2] Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno, Czech Republic, European Union.

ABSTRACT
Herein, we describe an ultrasensitive specific biosensing system for detection of sarcosine as a potential biomarker of prostate carcinoma based on Förster resonance energy transfer (FRET). The FRET biosensor employs anti-sarcosine antibodies immobilized on paramagnetic nanoparticles surface for specific antigen binding. Successful binding of sarcosine leads to assembly of a sandwich construct composed of anti-sarcosine antibodies keeping the Förster distance (Ro) of FRET pair in required proximity. The detection is based on spectral overlap between gold-functionalized green fluorescent protein and antibodies@quantum dots bioconjugate (λex 400 nm). The saturation curve of sarcosine based on FRET efficiency (F₆₀₄/F₅₁₀ ratio) was tested within linear dynamic range from 5 to 50 nM with detection limit down to 50 pM. Assembled biosensor was then successfully employed for sarcosine quantification in prostatic cell lines (PC3, 22Rv1, PNT1A), and urinary samples of prostate adenocarcinoma patients.

Show MeSH
Related in: MedlinePlus