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Label-free detection of rare cell in human blood using gold nano slit surface plasmon resonance.

Mousavi MZ, Chen HY, Hou HS, Chang CY, Roffler S, Wei PK, Cheng JY - Biosensors (Basel) (2015)

Bottom Line: The suspension containing the captured cells (MNPs-cells) is then introduced into a microfluidic chip integrated with a gold nanoslit film.MNPs-cells bind with the second specific antibody immobilized on the surface of the gold nanoslit and are therefore captured on the sensor active area.The cell binding on the gold nanoslit was monitored by the wavelength shift of the SPR spectrum generated by the gold nanoslits.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Applied Sciences, Academia Sinica, Taipei 11529, Taiwan. m.mousavi07@gmail.com.

ABSTRACT
Label-free detection of rare cells in biological samples is an important and highly demanded task for clinical applications and various fields of research, such as detection of circulating tumor cells for cancer therapy and stem cells studies. Surface Plasmon Resonance (SPR) as a label-free method is a promising technology for detection of rare cells for diagnosis or research applications. Short detection depth of SPR (400 nm) provides a sensitive method with minimum interference of non-targets in the biological samples. In this work, we developed a novel microfluidic chip integrated with gold nanoslit SPR platform for highly efficient immunomagnetic capturing and detection of rare cells in human blood. Our method offers simple yet efficient detection of target cells with high purity. The approach for detection consists of two steps. Target cells are firs captured on functionalized magnetic nanoparticles (MNPs) with specific antibody I. The suspension containing the captured cells (MNPs-cells) is then introduced into a microfluidic chip integrated with a gold nanoslit film. MNPs-cells bind with the second specific antibody immobilized on the surface of the gold nanoslit and are therefore captured on the sensor active area. The cell binding on the gold nanoslit was monitored by the wavelength shift of the SPR spectrum generated by the gold nanoslits.

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The binding potency analysis of anti-EphA2 Mabs on CL1-5 cell by flow cytometry. Mouse anti human EphA2 antibodies (3F7) was used to detect EphA2 on CL1-5 cells. The cells were harvested from the tissue-cultured flask with versene (EDTA-PBS) and washed with ice cold 1% FBS/PBS. The cells were incubated with antibody (0.0625 mg/3 × 105 cells) for 30 min on ice and washed with 1% FBS/PB. The cells were incubated with FITC-conjugated donkey anti-mouse IgG antibody (0.2 mg/3 × 105 cells) for another 30 min on ice and washed with 1% FBS/PBS. The fluorescence intensity was determined by BD FACSCalibur (BD Biosciences). Data were analyzed by software FlowJo 7.51. The result of the flow cytometry analysis shows antibody3F7 has the high affinity to bind with EphA2 on CL1-5 cells.
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biosensors-05-00098-f010: The binding potency analysis of anti-EphA2 Mabs on CL1-5 cell by flow cytometry. Mouse anti human EphA2 antibodies (3F7) was used to detect EphA2 on CL1-5 cells. The cells were harvested from the tissue-cultured flask with versene (EDTA-PBS) and washed with ice cold 1% FBS/PBS. The cells were incubated with antibody (0.0625 mg/3 × 105 cells) for 30 min on ice and washed with 1% FBS/PB. The cells were incubated with FITC-conjugated donkey anti-mouse IgG antibody (0.2 mg/3 × 105 cells) for another 30 min on ice and washed with 1% FBS/PBS. The fluorescence intensity was determined by BD FACSCalibur (BD Biosciences). Data were analyzed by software FlowJo 7.51. The result of the flow cytometry analysis shows antibody3F7 has the high affinity to bind with EphA2 on CL1-5 cells.

Mentions: Two different antibodies were used to increase the specificity of the cell capturing in this study. Antibody I and antibody II were selected based on the specificity and binding affinity to CL1-5 cell surface receptors. To identify such antibodies, three candidate antibodies, anti-EGFR, anti-CD44 and anti-EphA2, were tested. High expression of EGFR [39] and CD44 [40] on CL1-5 cells have been reported. The overexpression of the receptor EphA2 has been reported in non-small cell lung carcinoma cells [41,42]. The binding potency of anti-EphA2 monoclonal antibody to CL1-5 cells was analyzed by flow cytometry and is shown in Supplementary Figure A3.


Label-free detection of rare cell in human blood using gold nano slit surface plasmon resonance.

Mousavi MZ, Chen HY, Hou HS, Chang CY, Roffler S, Wei PK, Cheng JY - Biosensors (Basel) (2015)

The binding potency analysis of anti-EphA2 Mabs on CL1-5 cell by flow cytometry. Mouse anti human EphA2 antibodies (3F7) was used to detect EphA2 on CL1-5 cells. The cells were harvested from the tissue-cultured flask with versene (EDTA-PBS) and washed with ice cold 1% FBS/PBS. The cells were incubated with antibody (0.0625 mg/3 × 105 cells) for 30 min on ice and washed with 1% FBS/PB. The cells were incubated with FITC-conjugated donkey anti-mouse IgG antibody (0.2 mg/3 × 105 cells) for another 30 min on ice and washed with 1% FBS/PBS. The fluorescence intensity was determined by BD FACSCalibur (BD Biosciences). Data were analyzed by software FlowJo 7.51. The result of the flow cytometry analysis shows antibody3F7 has the high affinity to bind with EphA2 on CL1-5 cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384085&req=5

biosensors-05-00098-f010: The binding potency analysis of anti-EphA2 Mabs on CL1-5 cell by flow cytometry. Mouse anti human EphA2 antibodies (3F7) was used to detect EphA2 on CL1-5 cells. The cells were harvested from the tissue-cultured flask with versene (EDTA-PBS) and washed with ice cold 1% FBS/PBS. The cells were incubated with antibody (0.0625 mg/3 × 105 cells) for 30 min on ice and washed with 1% FBS/PB. The cells were incubated with FITC-conjugated donkey anti-mouse IgG antibody (0.2 mg/3 × 105 cells) for another 30 min on ice and washed with 1% FBS/PBS. The fluorescence intensity was determined by BD FACSCalibur (BD Biosciences). Data were analyzed by software FlowJo 7.51. The result of the flow cytometry analysis shows antibody3F7 has the high affinity to bind with EphA2 on CL1-5 cells.
Mentions: Two different antibodies were used to increase the specificity of the cell capturing in this study. Antibody I and antibody II were selected based on the specificity and binding affinity to CL1-5 cell surface receptors. To identify such antibodies, three candidate antibodies, anti-EGFR, anti-CD44 and anti-EphA2, were tested. High expression of EGFR [39] and CD44 [40] on CL1-5 cells have been reported. The overexpression of the receptor EphA2 has been reported in non-small cell lung carcinoma cells [41,42]. The binding potency of anti-EphA2 monoclonal antibody to CL1-5 cells was analyzed by flow cytometry and is shown in Supplementary Figure A3.

Bottom Line: The suspension containing the captured cells (MNPs-cells) is then introduced into a microfluidic chip integrated with a gold nanoslit film.MNPs-cells bind with the second specific antibody immobilized on the surface of the gold nanoslit and are therefore captured on the sensor active area.The cell binding on the gold nanoslit was monitored by the wavelength shift of the SPR spectrum generated by the gold nanoslits.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Applied Sciences, Academia Sinica, Taipei 11529, Taiwan. m.mousavi07@gmail.com.

ABSTRACT
Label-free detection of rare cells in biological samples is an important and highly demanded task for clinical applications and various fields of research, such as detection of circulating tumor cells for cancer therapy and stem cells studies. Surface Plasmon Resonance (SPR) as a label-free method is a promising technology for detection of rare cells for diagnosis or research applications. Short detection depth of SPR (400 nm) provides a sensitive method with minimum interference of non-targets in the biological samples. In this work, we developed a novel microfluidic chip integrated with gold nanoslit SPR platform for highly efficient immunomagnetic capturing and detection of rare cells in human blood. Our method offers simple yet efficient detection of target cells with high purity. The approach for detection consists of two steps. Target cells are firs captured on functionalized magnetic nanoparticles (MNPs) with specific antibody I. The suspension containing the captured cells (MNPs-cells) is then introduced into a microfluidic chip integrated with a gold nanoslit film. MNPs-cells bind with the second specific antibody immobilized on the surface of the gold nanoslit and are therefore captured on the sensor active area. The cell binding on the gold nanoslit was monitored by the wavelength shift of the SPR spectrum generated by the gold nanoslits.

Show MeSH
Related in: MedlinePlus