METHOD AND APPARATUS FOR THE DETECTION OF LABELING ELEMENTS IN A SAMPLE

• US 20070254376A1
• Filed: 09/26/2005
• Published: 11/01/2007
• Est. Priority Date: 10/01/2004
• Status: Abandoned Application

• Current Assignee: Koninklijke Philips N.V.
• Original Assignee: Koninklijke Philips Electronics N.V. (Koninklijke Philips N.V.)
• Inventors: STAPERT, Henk, BALISTRERI, Marcello, WIMBERGER-FRIEDL, Reinhold
• Application Number:
  US11/576,279
• Publication Number:
  US 20070254376A1
• Time in Patent Office: Days
• US Class Current: 436/164
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• 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
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/76 Chemiluminescence; Bioluminescence

Abstract

The invention relates to a method and an apparatus for the detection of occupied binding sites (15) in a sample (10), preferably for the detection of a fluorescent labeling element bound to probes on a solid surface. The method comprises the scanning of said sample (10) with a spot (26) of laser light (22) and the detection of target specific responses, e.g. fluorescent light (32), with a detection unit (30). The size of the scanning spot (26) is chosen small enough such that only one occupied binding site (15) is irradiated at the same time. A location (11) is classified as an occupied binding site (15) if a target specific response is observed in repeated scans of said location. Such repeated scans particularly allow to discriminate between specific and nonspecific binding.

Claims

1. A method for the detection of occupied binding sites (15), which contain at least one labeling element, in a sample (10), the method comprising the steps of

a) scanning the sample (10) with a spot (26) of radiation, wherein the extension (d) and the scanning speed of the spot (26) are such that approximately always maximally one occupied binding site (15) is present within the currently examined spot area during the corresponding examination duration;

b) memorizing each location (11) examined by the spot (26) of radiation as a candidate for an occupied binding site (15) if a target specific response is observed from that location;

c) scanning the locations (11) of said candidates at least one more times;

d) classifying a candidate as detected occupied binding site (15) if it shows a predetermined response behavior in the repeated scanning.

2. The method according to claim 1, characterized in that the sample (10) comprises a solid surface with distributed probes on it that are able to bind labeling elements directly or indirectly, preferably via a prior binding of a target substance.

3. The method according to claim 2, characterized in that said surface of the sample (10) is exposed before or during the scanning procedure to a solution containing labeling elements and/or target substance.

4. The method according to claim 3, characterized in that the concentration of the labeling elements and/or target substance in said solution is determined from the distribution of detected occupied binding sites (15) on said surface of the sample (10).

5. The method according to claim 1, characterized in that at least one of the labeling elements is a single molecule, a collection of a plurality of molecules, preferably a plurality of between 10¹ and 10⁸ molecules, and/or a semi-conducting particle.

6. The method according to claim 1, characterized in that the target specific response comprises the emission of light due to fluorescence or chemiluminescence.

7. The method according to claim 6, characterized in that the intensity of the radiation of the scanning spot (26) corresponds to about 10% to 90% of the saturation level of the fluorescence label, preferably to about 30% to 80%.

8. The method according to claim 1, characterized in that the definition of the target specific response is adapted based on the measured responses of scanned locations (11, 12).

9. The method according to claim 1, characterized in that the size of the scanned sample (10) and the whole examination duration are chosen such that about 100 to 1000 occupied binding sites (15) are detected.

10. An apparatus for the detection of occupied binding sites (15), which contain at least one labeling element, in a sample (10), the apparatus comprising

a scanning unit (20) that is adapted to scan the sample (10) with a spot (26) of radiation, wherein the extension (d) and the scanning speed of the spot (26) are such that approximately always at most one occupied binding site (15) is present within the currently examined spot area during the corresponding examination duration;

a detection unit (30) that is adapted to detect a target specific response from locations (11) examined by a spot (26) of radiation generated by the scanning unit;

an evaluation unit (40) that is adapted to memorize locations (11) with a target specific response as candidates for an occupied binding site (15) and to classify such a candidate as detected occupied binding site (15) if it shows a predetermined response behavior in two or more scans.