The present invention relates to the art of effecting and monitoring nucleic acid accumulation. More particularly, the present invention relates to a microfabricated chip including a waveguide formed with an optical microcavity in which nucleic acid accumulation can take place, which can be used to monitor nucleic acid accumulation. The invention further relates to a device constructed for accepting the chip and for effecting the monitoring of nucleic acid accumulation in the cavity therein. Yet, the invention further relates to a system which includes the chip and the device and to a method for effecting and monitoring nucleic acid accumulation using same.
Nucleic acid amplification methods, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA) and related techniques have become a central technology in genetic engineering and molecular biology. These methods enable the detection of single copy genes and/or DNA fragments with a very high degree of sensitivity, due to the extreme amplification of the signal obtained. As a result, these methods are gradually becoming critical methods in genetic diagnostics as well.
Yet, while implemented, nucleic acid amplification methods call for a series of steps which are tedious, time consuming and require highly trained personnel in order to be effectively executed. It is, therefore, not surprising that efforts are undertook to streamline nucleic acid amplification methods by rendering such methods more amenable to automation.
Should nucleic acid amplification technology become rapid, easy to perform and simple and sensitive to readout results, then it would immediately be applicable to the enormous, world-wide practice of genetic diagnostics.
Immobilized DNA has been known for years and is routinely used in nucleic acid blotting technology. Immobilization of short oligonucleotides has also been demonstrated and standartized. Recent technologies have demonstrated the immobilization of oligonucleotides to micro fabricated chips, to provide templates for hybridization and new techniques for mass sequencing on DNA chips. Thus, the concept and chemistries of using immobilized oligonucleotides is not new.
The combination of DNA amplification and micro fabricated chip technology is also not new. Here the basic concept has been to exploit the attributes of conducting the PCR reaction in microchambers, therefore benefiting from very small volumes of reagents to be used. This obviously has advantages when sample is rare. See, for example, Shoffner et al. (1996), xe2x80x9cChip PCR I. Surface passivation of microfabricated silicon-glass chip for PCRxe2x80x9d, Nucleic Acids Research. Vol. 24, No. 2, 375-379; and Burns et al. (1996), xe2x80x9cMicrofabricated structures for integrated DNA analysisxe2x80x9d, Proc. Natl. Acad. Sci. USA, Vol. 93, 5556-5561.
U.S. Pat. No. 5,498,392, for example, discloses devices for amplifying a preselected polynucleotide in a sample by conducting a polynucleotide polymerization reaction. The devices comprise a substrate microfabricated to define a sample inlet port and a mesoscale flow system, which extends from the inlet port. The mesoscale flow system includes a polynucleotide polymerization reaction chamber in fluid communication with the inlet port which is provided with reagents required for polymerization and amplification of a preselected polynucleotide. In one embodiment the devices may be utilized to implement a polymerase chain reaction (PCR) in the reaction chamber (PCR chamber). The PCR chamber is provided with the sample polynucleotide, polymerase, nucleoside-triphosphates, primers and other reagents required for the polymerase chain reaction, and the device is provided with means for thermally controlling the temperature of the contents of the reaction chamber at a temperature controlled to dehybridize double stranded polynucleotide, to anneal the primers, and to polymerize and amplify the polynucleotide.
However, devices such as the device described in U.S. Pat. No. 5,498,392, although capable of supporting miniaturized amplification reactions, fail in providing monitoring means for monitoring such reactions in an automatable fashion, preferably in real-time.
There is thus a widely recognized need for, and it would be highly advantageous to have miniaturized chip, device, system and method for effecting and monitoring nucleic acid accumulation.
It is an object of the present invention to provide chip, device, system and method for effecting nucleic acid accumulation and monitoring thereof, preferably in real-time.
It is another object of the present invention to provide chip, device system and method for effecting nucleic acid accumulation via amplification and/or hybridization and monitoring thereof, preferably in real-time.
It is yet another object of the present invention to provide chip, device, system and method for effecting nucleic acid accumulation via polymerase or ligase chain reaction and monitoring thereof, preferably in real-time.
Thus, according to one aspect of the present invention there is provided a nucleic acid accumulation analyzing chip comprising an optical waveguide having a radiation input port and a radiation output port, the optical waveguide being formed with at least one optical microcavity along its optical path, at least one oligonucleotide being immobilized to the optical waveguide in the microcavity, such that when the at least one oligonucleotide is contacted with reaction reagents under conditions allowing a nucleic acid accumulation reaction to take place, accumulated nucleic acid is detectable by providing radiation at the radiation input port of the optical waveguide and monitoring radiation signal modulation at the radiation output port of the optical waveguide.
According to another aspect of the present invention there is provided a nucleic acid accumulation analyzing system comprising (a) a nucleic acid accumulation analyzing chip including an optical waveguide having a radiation input port and a radiation output port, the optical waveguide being formed with at least one optical microcavity along its optical path, at least one oligonucleotide being immobilized to the optical waveguide in the microcavity; (b) a radiation source being in optical communication with the radiation input port; and (c) a radiation detector being in optical communication with the radiation output port; such that when the at least one oligonucleotide is contacted with reaction reagents under conditions allowing a nucleic acid accumulation reaction to take place, accumulated nucleic acid is detectable by providing radiation at the radiation input port of the optical waveguide via the radiation source and monitoring radiation signal modulation at the radiation output port of the optical waveguide via the radiation detector.
According to yet another aspect of the present invention there is provided a nucleic acid accumulation analyzing device comprising (a) a housing being formed with an acceptor for accepting a nucleic acid accumulation analyzing chip including an optical waveguide having a radiation input port and a radiation output port, the optical waveguide being formed with at least one optical microcavity along its optical path, and at least one oligonucleotide being immobilized to the optical waveguide in the microcavity; (b) a radiation source being engaged by the housing, the radiation source being in optical communication with the radiation input port of the nucleic acid accumulation analyzing chip when engaged in the acceptor; and (c) a radiation detector being engaged by the housing, the radiation detector being in optical communication with the radiation output port of the nucleic acid accumulation analyzing chip when engaged in the acceptor; such that when the at least one oligonucleotide is contacted with reaction reagents under conditions allowing a nucleic acid accumulation reaction to take place, accumulated nucleic acid is detectable by providing radiation at the radiation input port of the optical waveguide via the radiation source and monitoring radiation signal modulation at the radiation output port of the optical waveguide via the radiation detector.
According to still another aspect of the present invention there is provided a method of monitoring nucleic acid accumulation comprising the steps of (a) providing an optical waveguide being formed with an optical microcavity containing at least one oligonucleotide being immobilized thereat; (b) effecting nucleic acid accumulation by contacting the at least one oligonucleotide with reaction reagents under conditions allowing a nucleic acid accumulation reaction to take place; and (c) monitoring nucleic acid accumulation by passing radiation through the optical microcavity and monitoring a signal modulation of the radiation, associated with nucleic acid accumulation.
According to further features in preferred embodiments of the invention described below, the optical waveguide is of Si3N4.
According to still further features in the described preferred embodiments a support layer is provided supporting the optical waveguide.
According to still further features in the described preferred embodiments the optical microcavity splits the waveguide, such that a bottom of the optical microcavity is effected by the support layer.
According to still further features in the described preferred embodiments the support layer is of SiO2.
According to still further features in the described preferred embodiments a foundation layer is provided for supporting the support layer.
According to still further features in the described preferred embodiments the foundation layer is of doped silicon and serves as a resistor layer being in heat transfer relation with the optical microcavity. The resistor layer serves for providing the optical microcavity with heat for generating the conditions allowing the nucleic acid accumulation reaction to take place.
According to still further features in the described preferred embodiments a base layer is provided for supporting the foundation layer.
According to still further features in the described preferred embodiments the base layer is of silicon.
According to still further features in the described preferred embodiments the optical microcavity has a volume in a range of about 1 to about 10 cubic micrometers.
According to still further features in the described preferred embodiments the at least one oligonucleotide is immobilized in the optical microcavity in a surface density of between about 103 oligonucleotide molecules per square micrometer and about 106 oligonucleotide molecules per square micrometer.
According to still further features in the described preferred embodiments the nucleic acid accumulation reaction is selected from the group consisting of polymerase chain reaction, ligase chain reaction, nucleic acid sequence based amplification and nucleic acid hybridization.
According to still further features in the described preferred embodiments the radiation source provides a coherent radiation.
According to still further features in the described preferred embodiments the radiation source provides radiation in the visible range.
According to still further features in the described preferred embodiments the radiation source includes a laser source.
According to still further features in the described preferred embodiments the radiation detector serves for detecting radiation intensity modulation associated with nucleic acid accumulation.
According to still further features in the described preferred embodiments the radiation detector serves for detecting radiation phase modulation associated with nucleic acid accumulation.
According to still further features in the described preferred embodiments the radiation source and the a radiation detector are engaged in a nucleic acid accumulation analyzing device, the device is formed with an acceptor for receiving the nucleic acid accumulation analyzing chip.
The present invention successfully addresses the shortcomings of the presently known configurations by providing chip, device, system and method for effecting nucleic acid accumulation and monitoring thereof in real-time and in a fully automated fashion.