Systems and methods for detecting and analyzing polymers
First Claim
1. A detection system for analyzing a polymer having three or more distinct fluorophores, each fluorophore having an excitation wavelength and a corresponding emission bandwidth, the detection system comprising:
- a polymer interrogation zone constructed and arranged to accept the polymer;
at least three different emitters, each of the emitters constructed and arranged to emit an excitation signal substantially at an excitation wavelength of a corresponding one of the at least three fluorophores such that each fluorophore emits a distinct emission signal within its respective emission bandwidth and each distinct emission signal has an emission maximum separated by at least 60 nm from any other of the emission maximums; and
a detector constructed and arranged to distinctly detect the emission signal from each of the at least three fluorophores.
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Abstract
A detection system and methods for improving the ability of the detection system to recognize labels that are disposed on a polymer. Embodiments of the invention include schemes for selecting emitters and labels used within the system in a manner that allows an increase in the number of distinct labels that can be used together in a system. In other embodiments, the detection system and methods are directed to identifying portions of a detection signal that may be associated with extra labels residing within a detection zone. In other embodiments, the detection system and methods relate to using wide field imaging detectors while reducing out of focus noise contributions to detection signals of the system. Still, other embodiments relate to the use of linear array detectors to detect labels
82 Citations
114 Claims
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1. A detection system for analyzing a polymer having three or more distinct fluorophores, each fluorophore having an excitation wavelength and a corresponding emission bandwidth, the detection system comprising:
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a polymer interrogation zone constructed and arranged to accept the polymer;
at least three different emitters, each of the emitters constructed and arranged to emit an excitation signal substantially at an excitation wavelength of a corresponding one of the at least three fluorophores such that each fluorophore emits a distinct emission signal within its respective emission bandwidth and each distinct emission signal has an emission maximum separated by at least 60 nm from any other of the emission maximums; and
a detector constructed and arranged to distinctly detect the emission signal from each of the at least three fluorophores. - View Dependent Claims (6)
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2. A detection system for analyzing a polymer having three or more distinct fluorophores, each fluorophore having an excitation wavelength and a corresponding emission bandwidth, the detection system comprising:
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a polymer interrogation zone constructed and arranged to accept the polymer;
at least three different emitters, each of the emitters constructed and arranged to emit an emission signal substantially at an excitation wavelength of a corresponding one of the at least three fluorophores such that each fluorophore emits a distinct emission signal within its respective emission bandwidth that does not overlap any other of the distinct emission signals at normalized intensities above 70%; and
a detector constructed and arranged to detect the distinct emission signal from each of the at least three fluorophores. - View Dependent Claims (3, 4)
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5. A detection system for analyzing a polymer having three or more distinct fluorophores, each fluorophore having an excitation wavelength and a corresponding emission bandwidth, the detection system comprising:
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a polymer interrogation zone constructed and arranged to accept the polymer;
at least three different emitters, each of the emitters constructed and arranged to emit an excitation signal substantially at an excitation wavelength of a corresponding one of the at least three fluorophores such that each fluorophore emits a distinct emission signal within its respective emission bandwidth, each of the at least three emitters and corresponding at least three fluorophores selected from a group consisting of;
an emitter emitting an excitation signal substantially at a wavelength of 488 nm and a corresponding fluorophore having an emission maximum substantially located at 512 nm, an emitter emitting an excitation signal substantially at a wavelength of 532 nm and a corresponding fluorophore having an emission maximum substantially located at 575 nm, an emitter emitting an excitation signal substantially at a wavelength of 633 nm and a corresponding fluorophore having an emission maximum substantially located at 665 nm, and an emitter emitting an excitation signal substantially at a wavelength of 750 nm and a corresponding fluorophore having an emission maximum substantially located at 775 nm or 806 nm; and
a detector constructed and arranged to detect the distinct emission signal from each of the at least three fluorophores.
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7-18. -18. (canceled)
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19. A method of detecting emissions of three or more distinct fluorophores bound to a polymer, the method comprising:
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selecting three or more fluorophores, each of the fluorophores characterized by an excitation wavelength and a corresponding emission bandwidth, the emission bandwidth of each of the fluorophores not overlapping the emission bandwidth of any other of the fluorophores at normalized intensities above 70%;
attaching the three or more fluorophores to the polymer in a sequence specific manner;
illuminating each of the fluorophores with an excitation signal within the excitation wavelength of the corresponding fluorophores, thereby causing each of the fluorophores to emit an emission signal within the emission bandwidth of the fluorophore;
detecting and analyzing the emission signal of each of the fluorophores.
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20-21. -21. (canceled)
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22. A method for analyzing a polymer, the method comprising:
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selecting at least three distinct fluorophores and at least three different excitation signals each emitted from a corresponding emitter, each of the at least three fluorophores having an excitation wavelength and a corresponding emission bandwidth, each of the excitation signals having an excitation wavelength of a corresponding one of the at least three fluorophores such that each fluorophore emits a distinct emission signal having an emission maximum within its respective emission bandwidth, each of the at least three excitation signals and corresponding at least three fluorophores selected from a group consisting of;
an excitation signal having a wavelength of 488 nm and a corresponding fluorophore having an emission maximum substantially located at 512 nm, an excitation signal having a wavelength of 532 nm and a corresponding fluorophore having an emission maximum substantially located at 575 nm, an excitation signal having a wavelength of 633 nm and a corresponding fluorophore having an emission maximum substantially located at 665 nm, and an excitation signal having a wavelength of 750 nm and a corresponding fluorophore having an emission maximum substantially located at 775 nm or 806 nm;
binding the at least three fluorophores to a polymer in a sequence specific manner;
illuminating each of the fluorophores with the corresponding emitter; and
detecting and analyzing the emission signal of each of the fluorophores.
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23-33. -33. (canceled)
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34. A detection system for analyzing a polymer, the detection system comprising:
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a first detection zone disposed in a first area of a microchannel and adapted to detect a polymer having first and second polymer portions to create a first detection signal when the polymer is in the first zone;
a second detection zone disposed in a second area of the microchannel that is different from the first area, the second detection zone adapted to detect the second polymer portion to create a second detection signal when the second portion is in the second area;
a data processor adapted to comparing the first and second detection signals and identify components that are not common to both the first and second detection signals.
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35-46. -46. (canceled)
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47. A method of analyzing a polymer in a detection system, the method comprising:
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passing a plurality of polymers comprising a first polymer portion and a second polymer portion through a first detection zone to create a first detection signal, the first detection zone disposed in a first area of the microchannel;
passing the second portion of polymers through a second detection zone to create a second detection signal, the second detection zone disposed in a second area of the microchannel;
identifying components of the first detection signal that are associated with the second polymer portion by comparing the first detection signal with the second detection signal; and
detecting components of the first detection signal associated with the second polymer portion to analyze the polymers.
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48-59. -59. (canceled)
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60. A detection system to detect a label disposed on a polymer, the detection system comprising:
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a channel constructed and arranged to receive the polymer carried in a carrier fluid, the channel having a sample area defined by upper and lower channel surfaces separated from one another by a channel height less than about 0.500 microns;
an emitter constructed and arranged to illuminate the sample area with an excitation signal to excite the label in the sample area, thereby causing the label to emit an emission signal; and
a detector constructed and arranged to detect a detection signal from a detection zone disposed within the sample area, the detection signal including the emission signal, the detection zone being disposed at least partially within the sample area.
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61-75. -75. (canceled)
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76. A method to detect a label disposed on a polymer, the method comprising:
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providing a carrier fluid containing the polymer;
providing a channel constructed and arranged to receive the carrier fluid, the channel having a sample area defined by upper and lower channel surfaces separated from one another by a channel height less than about 0.500 microns;
exciting the label with an excitation signal, causing the label to emit an emission signal; and
detecting the emission signal.
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77-91. -91. (canceled)
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92. A detection system to detect a label disposed on a polymer, the detection system comprising:
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a channel adapted to allow a carrier fluid containing a polymer to pass in a flow direction through the channel and through a detection zone within the channel;
an emitter constructed and arranged to emit an excitation signal as a sheet of light into the detection zone; and
a detector constructed and arranged to detect a detection signal emanating from the detection zone from a direction substantially orthogonal to the sheet of light, the detection signal including an emission signal from the label when the label is present in the zone and excited by the excitation signal.
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93-105. -105. (canceled)
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106. A method for detecting a label on a polymer having the label, the method comprising:
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providing a carrier fluid containing the polymer;
providing a channel having a detection zone located within the channels;
flowing the carrier fluid through the channel in a flow direction and through a detection zone in the channel;
emitting an excitation signal as a sheet of light into the detection zone; and
detecting, with a detector, an emission signal from the label when the label is present in the zone and excited by the excitation signal, the detector located in a direction substantially orthogonal to the sheet of light.
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107-110. -110. (canceled)
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111. A detection system for detecting a first and second distinct labels on a polymer, the detection system comprising:
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a detection zone adapted to receive the polymer for detection;
an emitter for exciting each of the first and second distinct labels on the polymer when in the detection zone, causing each of the first and second distinct labels to emit a first and second emission signal, respectively;
an mirror adapted to substantially separate the first and second emission signals from one another; and
a wide field detector adapted to receive the first and second emission signals on spatially separate portions of a detection surface.
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112. A method of detecting a first and a second distinct label on a polymer, the method comprising:
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providing a detection zone;
placing the polymer and the label into the detection zone;
emitting an excitation signal for exciting the first and second distinct labels, causing the first and second labels to emit a first and second emission signal, respectively;
substantially separating the first and second emission signals from one another; and
detecting the first and second emission signals on a spatially separated portions of a detector.
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113. A detection system for analyzing a polymer having a label, the detection system comprising:
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a channel adapted to provide a carrier fluid containing a polymer through the channel in a flow direction;
a detection zone lying within the channel, the detection zone comprising a substantially linear array, the array arranged in a direction substantially orthogonal to the flow direction;
an emitter constructed and arranged to emit an excitation signal into the detection zone, the excitation signal comprising a sheet of light extending into the detection zone; and
a detector constructed and arranged to detect an emission signal from the label when the label is present in the detection zone and excited by the excitation signal.
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114. (canceled)
Specification