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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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To provide the binding sites for oligonucleotide linker, GFP was modified with gold nanoparticles (AuNPs).During the whole workflow, (a) process of GFP conjugation and (b) subsequent binding to a surface on nanomaghemite through oligonucleotide linker, (A) fluorescence recovery was evaluated (λexc 400 nm and λem 510 nm). As the acceptors for fluorescent resonance transfer there were employed quantum dots with λexc 520 nm and λem 604 nm. (B) Their fluorescent properties were tested both in their optimal excitation and in the excitation of GFP (λexc 400 nm). (a) Fluorescence of bare QDs and fluorescence after their conjugation with HWR heptapeptide, carrying sarcosine antibodies was determined at λem 604 nm. Values are means of three independent replicates (n = 3). Vertical bars indicate standard error.
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f3: To provide the binding sites for oligonucleotide linker, GFP was modified with gold nanoparticles (AuNPs).During the whole workflow, (a) process of GFP conjugation and (b) subsequent binding to a surface on nanomaghemite through oligonucleotide linker, (A) fluorescence recovery was evaluated (λexc 400 nm and λem 510 nm). As the acceptors for fluorescent resonance transfer there were employed quantum dots with λexc 520 nm and λem 604 nm. (B) Their fluorescent properties were tested both in their optimal excitation and in the excitation of GFP (λexc 400 nm). (a) Fluorescence of bare QDs and fluorescence after their conjugation with HWR heptapeptide, carrying sarcosine antibodies was determined at λem 604 nm. Values are means of three independent replicates (n = 3). Vertical bars indicate standard error.

Mentions: As it is shown in Fig. 3A, the modified GFP retained 78.9% of its fluorescence, indicating favorable retention of protein structure after functionalization. As gold binds thiols with high affinity and it does not undergo any unusual reactions with them17, we were able to use dsDNA with thiol moieties on both ends to form a linker between modified GFP and conjugate of Abs@PMPs. The resulting structure retained 12.3% of initial fluorescence of unmodified GFP exhibited paramagnetic properties and contained specific binding sites for sarcosine (Fig. 3A). Using atomic absorption spectrometry, the content of gold, originating from both protein functionalization and modification of nanoparticles, was determined as 0.0037 μg.mL−1.


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)

To provide the binding sites for oligonucleotide linker, GFP was modified with gold nanoparticles (AuNPs).During the whole workflow, (a) process of GFP conjugation and (b) subsequent binding to a surface on nanomaghemite through oligonucleotide linker, (A) fluorescence recovery was evaluated (λexc 400 nm and λem 510 nm). As the acceptors for fluorescent resonance transfer there were employed quantum dots with λexc 520 nm and λem 604 nm. (B) Their fluorescent properties were tested both in their optimal excitation and in the excitation of GFP (λexc 400 nm). (a) Fluorescence of bare QDs and fluorescence after their conjugation with HWR heptapeptide, carrying sarcosine antibodies was determined at λem 604 nm. Values are means of three independent replicates (n = 3). Vertical bars indicate standard error.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: To provide the binding sites for oligonucleotide linker, GFP was modified with gold nanoparticles (AuNPs).During the whole workflow, (a) process of GFP conjugation and (b) subsequent binding to a surface on nanomaghemite through oligonucleotide linker, (A) fluorescence recovery was evaluated (λexc 400 nm and λem 510 nm). As the acceptors for fluorescent resonance transfer there were employed quantum dots with λexc 520 nm and λem 604 nm. (B) Their fluorescent properties were tested both in their optimal excitation and in the excitation of GFP (λexc 400 nm). (a) Fluorescence of bare QDs and fluorescence after their conjugation with HWR heptapeptide, carrying sarcosine antibodies was determined at λem 604 nm. Values are means of three independent replicates (n = 3). Vertical bars indicate standard error.
Mentions: As it is shown in Fig. 3A, the modified GFP retained 78.9% of its fluorescence, indicating favorable retention of protein structure after functionalization. As gold binds thiols with high affinity and it does not undergo any unusual reactions with them17, we were able to use dsDNA with thiol moieties on both ends to form a linker between modified GFP and conjugate of Abs@PMPs. The resulting structure retained 12.3% of initial fluorescence of unmodified GFP exhibited paramagnetic properties and contained specific binding sites for sarcosine (Fig. 3A). Using atomic absorption spectrometry, the content of gold, originating from both protein functionalization and modification of nanoparticles, was determined as 0.0037 μg.mL−1.

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