DETECTING METHOD

• US 20120196302A1
• Filed: 02/13/2012
• Published: 08/02/2012
• Est. Priority Date: 10/07/2008
• Status: Abandoned Application

• Current Assignee: National Synchrotron Radiation Research Center
• Original Assignee: National Synchrotron Radiation Research Center
• Inventors: LAI, Lee Jene, HSIEH, Yi Heui, LIU, Shih Jen, Chen, Hsin Wei
• Application Number:
  US13/372,387
• Publication Number:
  US 20120196302A1
• Time in Patent Office: Days
• US Class Current: 435/7.24
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53

Abstract

A detecting method is herein disclosed. The method includes steps of providing an eukaryotic cell, having a cell nucleus and a cell membrane, wherein the cell nucleus endogenetically translates a first receptor and a second receptor, and wherein the first receptor and the second receptor pass through the cell nucleus and translocate to the cell membrane; coupling the first receptor to a first bioactive ligand with a quantum dot; washing the cell coupled with the first bioactive ligand and the quantum dot by centrifugation; coupling the second receptor to a second bioactive ligand with a magnetic bead; washing the cell coupled with the first bioactive ligand and the second bioactive ligand by magnetic separation; irradiating a exciting energy for the quantum dot to emit a fluorescence, wherein the quantum dot coupled with the cell is excited in a pH range from pH 9 to pH 14; and detecting the fluorescence.

Claims

1. A detecting method, comprising steps of:

providing an eukaryotic cell, having a cell nucleus and a cell membrane, wherein the cell nucleus endogenetically translates a first receptor and a second receptor, and wherein the first receptor and the second receptor pass through the cell nucleus and translocate to the cell membrane;

coupling the first receptor to a first bioactive ligand with a quantum dot;

washing the cell coupled with the first bioactive ligand and the quantum dot by centrifugation;

coupling the second receptor to a second bioactive ligand with a magnetic bead;

washing the cell coupled with the first bioactive ligand and the second bioactive ligand by magnetic separation;

irradiating an exciting energy for the quantum dot to emit a fluorescence, wherein the quantum dot coupled with the cell is excited in a pH range from pH 9 to pH 14; and

detecting the fluorescence.

2. The detecting method as claimed in claim 1, wherein the first receptor is selected from a sigma receptor, a T cell receptor, a CD3 marker, a CD4 marker, a CD19 marker, a CD40 marker, and a CD8 marker.

3. The detecting method as claimed in claim 1, wherein the second receptor is selected from a sigma receptor, a T cell receptor, a CD3 marker, a CD4 marker, a CD19 marker, a CD40 marker, and a CD8 marker, and the second receptor is different from the first receptor.

4. The detecting method as claimed in claim 1, wherein the first bioactive ligand, in response to the first receptor, is selected from a pentazocine, an anisamide, a haloperidol, an antomer, a major histocompatibility complex (MHC) molecule, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD19 antibody, an anti-CD40 antibody, and an anti-CD8 antibody.

5. The detecting method as claimed in claim 1, wherein the second bioactive ligand, in response to the second receptor, is selected from a pentazocine, an anisamide, a haloperidol, an antomer, a major histocompatibility complex (MHC) molecule, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD19 antibody, an anti-CD40 antibody, and an anti-CD8 antibody.

6. The detecting method as claimed in claim 1, wherein the quantum dot comprises a PbS quantum dot, a II-VI quantum dot or a III-V quantum dot.

7. The detecting method as claimed in claim 6, wherein the II-VI quantum dot comprises a CdSe quantum dot or a CdTe quantum dot.

8. The detecting method as claimed in claim 6, wherein the II-VI quantum dot is encapsulated with a ZnS coating.

9. The detecting method as claimed in claim 6, wherein the III-V quantum dot comprises an InP quantum dot, a GaN quantum dot, or an InAs quantum dot encapsulated with a GaAs coating.

10. The detecting method as claimed in claim 1, wherein the quantum dot and the first bioactive ligand is coupled by a streptavidin and a biotin.

11. The detecting method as claimed in claim 1, wherein the magnetic bead and the second bioactive ligand are coupled through an interaction between a streptavidin and a biotin.

12. The detecting method as claimed in claim 1, wherein a first unit is composed of the cell, the first receptor, the first bioactive ligand, and the quantum dot and a second unit is composed of the first unit, the second receptor, the magnetic bead, and the second bioactive ligand, the second unit is irradiated in a pH value between pH 9 and pH 14.

13. The detecting method as claimed in claim 1, wherein the diameter of the magnetic bead is between 25 nm and 5000 nm.

14. The detecting method as claimed in claim 1, wherein the exciting energy is emitted from an excitation light source, the excitation light source is selected from an ultraviolet light, a light-emitting diode, X-ray, synchrotron radiation light source, infrared ray and a laser light, and the excitation light source excites the quantum dot in a time-period from 7 to 40 minutes.

15. The detecting method as claimed in claim 1, wherein the temperature for detecting the fluorescence by a photomultiplier tube is between −

200 and 25°

C.

16. The detecting method as claimed in claim 15, wherein the photomultiplier tube is cooled in a vacuumed or non-vacuumed way to lower the background current of the photomultiplier tube.

17. The detecting method as claimed in claim 15, wherein the photomultiplier tube comprises a pulse mode used for converting the fluorescence measured by the photomultiplier tube into a pulse signal.

18. The detecting method as claimed in claim 15, wherein the photomultiplier tube comprises a current mode used for converting the fluorescence measured by the photomultiplier tube into a current signal.

19. The detecting method as claimed in claim 1, further comprising a step of transfecting nucleotides including sequences of the first receptor and the second receptor.

20. The detecting method as claimed in claim 1, wherein after the quantum dot is excited from 7 to 40 minutes, the fluorescence is analyzed.