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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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An utilization of antibodies for sarcosine isolation from the real samples of urine.(A) Sarcosine amount isolated from prostatic cell lines (PC3, 22Rv1, PNT1A). (B) Urinary samples were spiked with 40 nM sarcosine and various dilution were tested (0×; 10×; 100×; and 1000×) and compared to control (C) without sarcosine spike. Analyses were performed using FRET at λexc 400 nm and concentrations were calculated according to calibration curve. (C) Sarcosine concentration determined in real samples of patients suffering from acinar adenocarcinoma of prostate. The urinary samples were diluted 100× prior to isolation. Values are means of three independent replicates (n = 3) analyzed with different batches of system. Vertical bars indicate standard error.
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f5: An utilization of antibodies for sarcosine isolation from the real samples of urine.(A) Sarcosine amount isolated from prostatic cell lines (PC3, 22Rv1, PNT1A). (B) Urinary samples were spiked with 40 nM sarcosine and various dilution were tested (0×; 10×; 100×; and 1000×) and compared to control (C) without sarcosine spike. Analyses were performed using FRET at λexc 400 nm and concentrations were calculated according to calibration curve. (C) Sarcosine concentration determined in real samples of patients suffering from acinar adenocarcinoma of prostate. The urinary samples were diluted 100× prior to isolation. Values are means of three independent replicates (n = 3) analyzed with different batches of system. Vertical bars indicate standard error.

Mentions: Based on obtained results we hypothesized that our approach can be applicable also for evaluation of sarcosine levels in prostatic cell lines and clinical urinary samples. Firstly, Abs@PMPs conjugate was employed to quantify sarcosine in disrupted prostatic cells. It was shown that sarcosine is present in all types of prostatic cells, including normal prostatic epithelial cells (PNT1A) - 0.03 nmol per 106 cells, human carcinoma epithelial cells derived from a xenograft propagated in mice (22Rv1) - 0.07 nmol per 106 cells, and human cell line from androgen independent prostatic adenocarcinoma derived from metastatic site in bone (PC3) - 0.11 nmol per 106 cells (Fig. 5A). These results are in agreement with results published by Khan and colleagues used glycine-N-methyl transferase (GNMT) knockdown cell lines for sarcosine assessment21.


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)

An utilization of antibodies for sarcosine isolation from the real samples of urine.(A) Sarcosine amount isolated from prostatic cell lines (PC3, 22Rv1, PNT1A). (B) Urinary samples were spiked with 40 nM sarcosine and various dilution were tested (0×; 10×; 100×; and 1000×) and compared to control (C) without sarcosine spike. Analyses were performed using FRET at λexc 400 nm and concentrations were calculated according to calibration curve. (C) Sarcosine concentration determined in real samples of patients suffering from acinar adenocarcinoma of prostate. The urinary samples were diluted 100× prior to isolation. Values are means of three independent replicates (n = 3) analyzed with different batches of system. Vertical bars indicate standard error.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: An utilization of antibodies for sarcosine isolation from the real samples of urine.(A) Sarcosine amount isolated from prostatic cell lines (PC3, 22Rv1, PNT1A). (B) Urinary samples were spiked with 40 nM sarcosine and various dilution were tested (0×; 10×; 100×; and 1000×) and compared to control (C) without sarcosine spike. Analyses were performed using FRET at λexc 400 nm and concentrations were calculated according to calibration curve. (C) Sarcosine concentration determined in real samples of patients suffering from acinar adenocarcinoma of prostate. The urinary samples were diluted 100× prior to isolation. Values are means of three independent replicates (n = 3) analyzed with different batches of system. Vertical bars indicate standard error.
Mentions: Based on obtained results we hypothesized that our approach can be applicable also for evaluation of sarcosine levels in prostatic cell lines and clinical urinary samples. Firstly, Abs@PMPs conjugate was employed to quantify sarcosine in disrupted prostatic cells. It was shown that sarcosine is present in all types of prostatic cells, including normal prostatic epithelial cells (PNT1A) - 0.03 nmol per 106 cells, human carcinoma epithelial cells derived from a xenograft propagated in mice (22Rv1) - 0.07 nmol per 106 cells, and human cell line from androgen independent prostatic adenocarcinoma derived from metastatic site in bone (PC3) - 0.11 nmol per 106 cells (Fig. 5A). These results are in agreement with results published by Khan and colleagues used glycine-N-methyl transferase (GNMT) knockdown cell lines for sarcosine assessment21.

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