Chemistry: analytical and immunological testing – Optical result – With fluorescence or luminescence
Reexamination Certificate
1999-02-18
2001-06-26
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Optical result
With fluorescence or luminescence
C422S052000, C422S051000, C422S068100, C422S082080, C422S186000, C436S159000, C436S180000, C436S046000
Reexamination Certificate
active
06251685
ABSTRACT:
TECHNICAL FIELD
The invention relates to microelectronic array systems for performing analysis of molecular biological material such as DNA and RNA. More particularly, the invention relates to methods and systems for reading out data from microelectronic array systems.
BACKGROUND ART
Microelectronic arrays having electronically addressable microlocations, or features, are being used to carry out in-parallel multiple sample DNA hybridization analysis on a source DNA sample. The microelectronic arrays are fabricated on semiconductor substrates using semiconductor processing techniques. An exemplary microelectronic array system for performing molecular biological diagnosis is disclosed in U.S. Pat. No. 5,632,957, entitled “Molecular Biological Diagnostic Systems Including Electrodes,” issued to Heller et al. (hereinafter Heller). Microelectronic arrays such as Heller allow charged molecules to be actively moved and concentrated at designated microlocations within an array. In one application, DNA probes are located at specific microlocations and then target DNA molecules are electronically directed to specific probes in order to promote hybridization of the target DNA with the probe DNA. By utilizing electronically addressable microelectronic arrays to concentrate DNA, hybridization rates are significantly accelerated over prior passive hybridization techniques.
There are many techniques available for detecting the extent of DNA hybridization that has occurred on DNA arrays, whether microelectronic or not. A common technique involves incorporating fluorescent markers, or labels, into the target DNA that is provided to hybridize with the probe DNA. After hybridization, each microlocation can be contacted with light, for example laser light, in order to activate any fluorescent labels that are present at a particular microlocation. Fluorescent light given off from the fluorescent material located at the microlocations is measured and related to DNA concentrations.
In most detection techniques that require outside sources of energy, such as laser light energy, to activate a label, the activation energy is applied to the array one microlocation at a time. Specifically, in a laser-activated system, the laser systematically steps through each microlocation, or pixel, applying laser light to each location individually. Laser-based readout systems are disclosed in U.S. Pat. No. 5,631,734, entitled “Method and Apparatus for Detection of Fluorescently Labeled Materials,” issued to Stern et al. and U.S. Pat. No. 5,653,939, entitled “Optical and Electrical Methods and Apparatus for Molecule Detection,” issued to Hollis et al. Although sequentially reading out microlocations works well when the number of microlocations is small, when the number of microlocations in a microelectronic array is large, the readout time required to individually read each microlocation can be significant.
In view of the advancements involved with electrically addressable microelectronic arrays and in view of the limitations involved with sequentially activating microlocations on any DNA array with an outside source of energy such as laser light, what is needed is a readout method that takes advantage of the addressing control capability of microelectronic arrays in order to more efficiently read out desired biological data.
SUMMARY OF THE INVENTION
A method for reading out data from microlocations of a microelectronic array involves activating multiple microlocations in parallel and simultaneously detecting the responses from the activated microlocations to determine concentrations of molecular biological material at each microlocation. In a preferred embodiment, the microelectronic array includes electronically addressable electrodes at each microlocation which can be individually activated via a control system. An electrochemiluminescent detection technique is used to detect the presence and the concentration of hybridized molecular biological material that is located at each microlocation. Electrochemiluminescent material is utilized because it gives off light when in the presence of an electrical field. With an addressable microelectronic array, electrical fields can be applied to various combinations of microlocations simultaneously to allow readout of several microlocations in parallel. This is in contrast to the laser-based readout approach which applies activation energy sequentially to each microlocation by impacting each microlocation one after another with a single laser beam. Reading out multiple microlocations simultaneously in accordance with the invention can produce significant time savings in large arrays.
A preferred system for implementing the readout method of the invention includes a microelectronic array and a complementary detection system. The microelectronic array consists of multiple microlocations that are formed on a semiconductor substrate using integrated circuit fabrication technology. The microlocations are electronically addressable locations where manipulation of biological molecules occurs, such that each of the microlocations represents an area of defined, or constant, biological material. Preferably, the microlocations are configured in arrays of linear columns and rows, however, other configurations are possible. The number of microlocations in an array may be from 2 to about 16,000,000, preferably from about 100 to 100,000, and the size of each microlocation may be from 5 &mgr;m
2
to about 1 mm
2
, usually about 100 &mgr;m
2
to about 200 &mgr;m
2
. Each microlocation is individually connected to contact pads which enable the electronic array to be connected to control systems that can individually control the microlocations. In an alternative embodiment, each microlocation has leads that are connected to address decoders such that each microlocation does not have a dedicated contact pad requiring connection to a control system.
There are different known techniques available to manipulate and concentrate biological material at designated microlocations. The specific technique utilized is not critical to the invention.
A preferred detection system includes a detector that is located directly above, and in close proximity with, the microlocations of an array. The detection system may also include optics as needed to provide adequate light collection. In a preferred embodiment, the optics include an objective lens that captures light generated from the microlocations and directs the light to the detector. The detector may be a spatially resolving detector such as a CCD array or single-element detector such as a single photo cell. Optical filters and/or additional lenses may be placed between the first lens and the detector to further improve light detection.
The preferred readout method includes activating multiple microlocations in parallel to initiate an electrochemiluminescent reaction. Some techniques of activating multiple microlocations in parallel are more preferred than others. Two categories of parallel readout are referred to as partially parallel readout and fully parallel readout. Various preferred examples of partially parallel readout techniques are briefly described below in addition to a fully parallel readout technique. Preferably, activated microlocations are distributed such that light generated from the microlocations can be individually distinguished using a detection system that has only moderate spatial resolution. That is, adjacent activated microlocations should be separated by enough space on the array that a detection system with moderate resolution can distinguish the light being emitted from each microlocation. An example of a partially parallel readout technique involves activating alternating columns of microlocations simultaneously. Activating alternating columns of microlocations reduces interference (e.g., crosstalk) that could be encountered between adjacent columns of microlocations. If more space is needed between columns, then every M
th
column (where M>2) can be activated, instead of every other column. The column-by-column approach ca
Dorsel Andreas Nikolaus
Gordon Gary B.
Kronick Mel N.
Agilent Technologie,s Inc.
Gakh Yelena
Warden Jill
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