Abstract:
The present invention relates generally to monitoring of biochemical amplification reactions using electromagnetic radiation, and more particularly to an apparatus for optical monitoring of isothermal and thermally-cycled amplification reactions using radiation ranging from the ultraviolet region through the infrared regions of the electromagnetic spectrum. Moreover, the method discussed herein could be similarly applied to any process that results in biochemical amplification, regardless of the specific technique employed.

Description:
FIELD OF INVENTION 
     The present invention relates to the field of biomolecule amplification reactions, and more particularly to an apparatus for optical monitoring of biomolecule amplification reactions using electromagnetic radiation. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  illustrates a perspective view of an exemplary embodiment of nucleic acid amplification and monitoring apparatus with visual output capability. 
         FIG. 2  illustrates a perspective view of an alternate embodiment of nucleic acid amplification and monitoring apparatus with an external visual output. 
         FIG. 3  illustrates an exemplary embodiment of a tissue sample tube in a tissue sample chamber of nucleic acid amplification and monitoring apparatus. 
         FIG. 4  illustrates an exploded view of an exemplary embodiment of a tissue sample chamber with optical assembly for nucleic acid amplification and monitoring apparatus. 
         FIG. 5  illustrates an exemplary embodiment of a heated tissue sample chamber of nucleic acid amplification and monitoring apparatus. 
         FIG. 6  illustrates a block diagram of an exemplary embodiment of nucleic acid amplification and monitoring system. 
     
    
    
     GLOSSARY 
     As used herein, the term “nucleic acid” refers to a biological polymer of nucleotide bases, and may include but is not limited to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), micro RNA (miRNA), and peptide nucleic acid (PNA). 
     As used herein, the term “nucleic acid amplification” refers to a process by which a limited quantity of nucleic acid undergoes a biochemical reaction in which a larger quantity of nucleic acid is generated. 
     As used herein, the term “tissue sample” refers to a quantity of cellular or sub-cellular material extracted from a prokaryotic or eukaryotic organism, or other cellular or sub-cellular material, which potentially contains nucleic acid. 
     As used herein, the term “tissue sample chamber” refers to a receptacle structurally capable of holding or containing a tissue sample tube or other removable component which contains a tissue sample and facilitates insertion and removal of a tissue sample from a tissue sample chamber. 
     As used herein, the term “output” refers to any discernible audio or visual signal known in the art. For example, a visual output may be a computer interface, touch screen, liquid crystal display (LCD) screen, personal communication device display, or any other visual component on which data or output may be displayed and viewed by a user. An output may be integrally constructed with other components of nucleic acid amplification monitoring apparatus into a single device, or may be located on another device to which a signal is sent from the nucleic acid amplification and monitoring apparatus. 
     As used herein, the term “quantitative result” refers to a numerical value which is related to the concentration of nucleic acid being measured. 
     As used herein, the term “qualitative result” refers to a binary result which indicates presence or absence of a specified nucleic acid sequence. 
     As used herein, the term “photon” refers to a unit particle of electromagnetic energy. 
     As used herein, the term “conduit” refers to a passageway of any shape, which serves to direct the path of photons, and which may contain one or more optical components. 
     As used herein, the term “modulator” refers to a component which may cause the amplitude, intensity, frequency, or other property of a signal to vary. 
     As used herein, the term “demodulator” refers to a device which extracts an information-bearing signal from a modulated signal. 
     As used herein, the term “signal processor” refers to a device which performs mathematical operations on a signal. A demodulator is one example of a signal processor. 
     As used herein, the term “excitation beam” refers to energy which is used to stimulate a sample. 
     As used herein, the term “emission beam” refers to energy which is produced by a sample when appropriately excited. 
     BACKGROUND 
     Biomolecules, such as deoxyribonucleic acid (DNA) and other nucleic acids, play an important role in cellular functioning as well as control of processes at the tissue, organ, system, and organism level. DNA is a biomolecule formed from two helically-arranged strands; each strand is a biopolymer formed from a linear sequence of four fundamental nucleotide units or bases—adenine, cytosine, guanine and thymine, abbreviated respectively as A, C, G and T. Each base on one strand is hydrogen bonded to exactly one complementary base (A with T and G with C) on the opposite strand, thereby forming a nucleotide base pair. DNA found in a human cell typically consists of approximately three billion base pairs. The specific sequence of A, C, G and T bases which forms the DNA molecule within the cells of an individual is referred to as that individual&#39;s genotype, which can vary from person to person. Subsections of the DNA biomolecule may serve to encode for specific proteins; these subsections may consist of several thousands (to millions) of base pairs, and are referred to as genes. 
     In some cases, a complete determination of the sequence of bases in a DNA molecule—referred to as “DNA sequencing”—defines an individual&#39;s genotype, and can be helpful in assessing the propensity for acquiring disease or metabolizing medications, for example. More often, however, knowledge of the sequence within a small subset of the DNA molecule is very useful. For example, confirmation of a specific sequence for a specific gene may be sufficient to assess a predisposition for the development of a certain cancer. The determination of the presence of specific subsection sequences of DNA is referred to as molecular diagnostics; in vitro molecular diagnostics is the term applied when this determination is accomplished using a sample of tissue extracted from an organism. 
     Molecular diagnostic techniques are typically based on the use of primer molecules, which are short, specific sections of single-stranded DNA used to identify the presence of specific genes or specific sequences within DNA molecules. Primer molecules may, for example, be gene-specific, disease-specific, or organism-specific. Primers and associated reagents are typically mixed with sample of unknown DNA in solution, and the presence of a specific type of DNA is determined through an amplification reaction: if a given DNA sequence is present in the unknown sample, it can be amplified and detected as the reaction proceeds. A widely-used form of amplification is based on the polymerase chain reaction (PCR) technique. 
     Detection of amplification products (referred to as amplicons) or monitoring of the amplification process itself may be accomplished in numerous ways. One of the most common techniques detects a fluorescence optical signal which increases in intensity during the amplification process if the target DNA sequence is present in an unknown sample. Traditional detection approaches for monitoring the progress of an amplification process use optical sensors (e.g., photodiodes or phototransistors) for sensing the fluorescent light. 
     The ability of a monitoring system to detect the fluorescence signal ultimately impacts the sensitivity with which low concentrations of DNA can be detected. In many cases the initial quantity of DNA may be extremely small. One problem known in the prior art is the inability to detect very small quantities of DNA due to susceptibility to noise sources inherent in any monitoring system, including optical noise, detector noise, and electrical signal processing noise. 
     It is desirable to have an apparatus capable of both amplifying nucleic acid under optimal conditions and monitoring the amplification while rejecting undesired signals introduced by sources of noise. 
     It is desirable to have an apparatus capable of amplifying and monitoring nucleic acid with improved sensitivity for nucleic acid detection. 
     SUMMARY OF THE INVENTION 
     The present invention is an apparatus for amplifying and monitoring the amplification of nucleic acid. A tissue sample is placed into a tissue sample chamber and an excitation beam from a light excitation component is directed onto the tissue sample. One or more properties of the excitation beam are modulated using a modulator. Electromagnetic energy emitted from the tissue sample is collected and directed onto a photodetector, which produces an electrical signal in response to the collected light. The electrical signal is then demodulated by a signal processor. The nucleic acid amplification and monitoring apparatus also includes a power source, and an output which allows the amplification of the nucleic acid in the tissue sample to be monitored. 
     DETAILED DESCRIPTION OF INVENTION 
     For the purpose of promoting an understanding of the present invention, references are made in the text to exemplary embodiments of an apparatus for amplifying and monitoring the amplification of nucleic acid, only some of which are described herein. It should be understood that no limitations on the scope of the invention are intended by describing these exemplary embodiments. One of ordinary skill in the art will readily appreciate that alternate but functionally equivalent components, materials, and layouts may be used. The inclusion of additional elements may be deemed readily apparent and obvious to one of ordinary skill in the art. Specific elements disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one of ordinary skill in the art to employ the present invention. 
     It should be understood that the drawings are not necessarily to scale; instead, emphasis has been placed upon illustrating the principles of the invention. In addition, in the embodiments depicted herein, like reference numerals in the various drawings refer to identical or near identical structural elements. 
     Moreover, the terms “substantially” or “approximately” as used herein may be applied to modify any quantitative representation that could permissibly vary without resulting in a change in the basic function to which it is related. 
       FIG. 1  shows a perspective view of an exemplary embodiment of nucleic acid amplification and monitoring apparatus  100 . In the embodiment shown, nucleic acid amplification and monitoring apparatus  100  is comprised of outer housing  5  with tissue sample port  12  through which tissue sample chamber  10  ( FIG. 5 ) is accessed, and visual display  7 . Outer housing  5  encases tissue sample chamber  10  and other components including, but not limited to a light excitation component, an excitation collimating lens, an excitation filter, an emission filter, an emission collimating lens, a temperature sensor, temperature control circuitry, a modulator, and a signal processor. 
     In the embodiment shown, visual display  7  is a touch screen with display capability for viewing and controlling nucleic acid amplification and includes a plurality of controls  9   a ,  9   b ,  9   c ,  9   d ,  9   e . In the embodiment shown, visual display  7  has temperature control  9   a , amplification initiation control  9   b , calibration control  9   c , power control  9   d , and display adjustment control  9   e  which controls options for a graphical, numeric, quantitative, or qualitative measurement representation and display of data. In the embodiment shown,  9   c  calibration control is used to calibrate the system using a known sample so that the system reflects accurate nucleic acid concentration and/or to gauge the accuracy and performance of the particular device. 
     In various embodiments, visual display  7  may include more or fewer or varying types of controls. In other embodiments, controls  9   a ,  9   b ,  9   c ,  9   d ,  9   e  may be physical controls, such as levers, buttons, switches, and dials; electronic signals; voice activated or timed controls; and/or a remote or local user interface. In other embodiments, visual display  7  may be eliminated and replaced with an audio output signal, such as a synthesized voice signal. In still other embodiments, visual display  7  may be eliminated and replaced with means for generating an electronic representation of the data, which may be subsequently stored or transmitted to a receiver capable of decoding said electronic representation of the data. 
     In the embodiment shown, visual display  7  is integrally constructed with nucleic acid amplification and monitoring apparatus  100 ; however, in various other embodiments visual display  7  may be a visual display on another device to which a signal is sent from nucleic acid amplification and monitoring apparatus  100 . 
     Also visible in  FIG. 1  is a tissue sample enclosed in tissue sample tube  8  (of a type known in the art for storing tissue samples). Tissue sample tube  8  is inserted through tissue sample port  12  into tissue sample chamber  10  ( FIG. 5 ). In the embodiment shown, tissue sample tube  8  is a transparent tube which allows light to be absorbed by or emitted from a sample and subsequently detected for measurement. 
     In the embodiment shown, nucleic acid amplification and monitoring apparatus  100  is portable and hand-held; however, in other embodiments, nucleic acid amplification and monitoring apparatus  100  may be any size. 
       FIG. 2  illustrates an alternative embodiment of nucleic acid amplification and monitoring apparatus  100  which is integrated with external visual display  15 . In the embodiment shown, nucleic acid amplification and monitoring apparatus  100  includes a plurality of tissue sample ports  12  and external visual display  15  is capable of displaying readings for multiple tissue samples. 
     In the embodiment shown, nucleic acid amplification and monitoring apparatus  100  includes a means for transmitting diagnostic data derived from signal processor  40  ( FIG. 4 ) to external device  88  to be displayed on a single display device, or over a local or wide area network. In the embodiment shown, multiple samples are analyzed in a laboratory using nucleic acid amplification and monitoring apparatus  100 , and diagnostic data is transmitted over the internet for review on external visual display  15  by technician  55 . 
       FIG. 3  illustrates an exemplary embodiment of tissue sample tube  8  in tissue sample chamber  10  of nucleic acid amplification and monitoring apparatus  100 . In the embodiment shown, tissue sample chamber  10  is a receptacle structurally capable of holding tissue sample tube  8  having a measurable amount of tissue sample  51 . In an exemplary embodiment, tissue sample  51  contains nucleic acid, standard reagents, and a fluorescent molecule (of a type known in the art for monitoring nucleic acid amplification processes), which produces fluorescent light when illuminated by an appropriate wavelength of excitation light. 
     Also visible in  FIG. 3  are light excitation component  17 , excitation collimating lens  21 , excitation filter  24 , emission filter  42 , emission collimating lens  36 , photodetector  37 , light insulating cover  14 , and temperature sensor  71 . 
     Light excitation component  17  is a component which illuminates, excites, or initiates photonic activation of a fluorescent molecule in tissue sample  51 . Light excitation component  17  produces excitation beam  26  ( FIG. 4 ) which is directed to tissue sample tube  8  and tissue sample  51 . Before excitation beam  26  reaches tissue sample  51 , excitation beam  26  passes through excitation collimating lens  21  and excitation filter  24 . Excitation collimating lens  21  aligns the photons in excitation beam  26  and excitation filter  24  filters excitation beam  26 , limiting the bandwidth of the excitation photons reaching tissue sample  51 . 
     Excitation beam  26  excites the fluorescent molecule in tissue sample  51 , which produces a fluorescent signal proportional to the concentration of the target nucleic acid molecule, creating emission beam  45  ( FIG. 4 ), which has a wavelength greater than that of excitation beam  26 . Emission beam  45  passes through emission filter  42  and emission collimating lens  36  before reaching photodetector  37 . Emission collimating lens  36  aligns the fluorescent photons in emission beam  45  and emission filter limits the bandwidth of emission beam  45 . 
     Excitation filter  24  and emission filter  42  isolate excitation and emission light, respectively, preventing photons from excitation beam  26  from reaching photodetector  37 . Excitation filter  24  and emission filter  42  have different wavelength ranges and vary depending on the light excitation component and the fluorescent molecule used. 
     In an exemplary embodiment, excitation filter  24 , excitation collimating lens  21 , and light excitation component  17  are supported within excitation conduit  23 . Emission filter  42 , emission collimating lens  36 , and photodetector  37  are supported within emission conduit  31 . 
     Light excitation component  17  may be any type of coherent or non-coherent light source known in the art, including but not limited to, a solid-state laser, diode laser, gas laser, dye laser, light emitting diode (LED), superluminescent diode (SLD), non-coherent lamp, or any other light source known in the art. In embodiments in which light excitation component  17  is an LED, excitation collimating lens  21  may or may not be used. Excitation collimating lens  21  and/or excitation filter  24  may also be omitted with other types of light sources, such as a solid-state laser, which emits a characteristically very narrow wavelength, eliminating the need for excitation filter  24 . 
     In various embodiments, excitation beam  26  and emission beam  45  ( FIG. 4 ) have a wavelength ranging from 10 nm to 10 μm. 
     In the embodiment shown, excitation filter  24  and emission filter  42  are filters known in the art. In various other embodiments, excitation collimating lens  21  and emission collimating lens  36  are replaced by an alternative collimating component known in the art, such as a collimating tube, collimating aperture, or aperture set. 
     In the embodiment shown, light insulating cover  14  insulates tissue sample tube  8  and tissue sample  51 , preventing tissue sample  51  from being illuminated from light external to nucleic acid amplification and monitoring apparatus  100 . Light insulating cover  14  may or may not be heated; however, heating of light insulating cover  14  may be desirable for preventing sample evaporation. 
     In the embodiment shown, temperature sensor  71  measures the temperature inside tissue sample chamber  10 . The measured temperature is used to control the temperature of tissue sample  51  via temperature control circuitry  70  ( FIG. 6 ). 
     In the embodiment shown, photodetector  37  converts photons in emission beam  45  to a measurable voltage or current signal. 
       FIG. 4  illustrates an exploded view of an exemplary embodiment of sample chamber with optical assembly for nucleic acid amplification and monitoring apparatus  100 . Also visible in  FIG. 4  are modulator  29 , printed circuit board  65 , amplifier  62 , and signal processor  40 . 
     In the embodiment shown, modulator  29  is a mechanical shutter (e.g., chopper wheel) that modulates excitation beam  26 . In other embodiments, the mechanical shutter is omitted and replaced by an electronic circuit which modulates the light intensity produced by light excitation component  17 , thereby modulating excitation beam  26 . In various embodiments, modulator  29  may produce a periodic waveform or an aperiodic waveform to modulate excitation beam  26 . 
     In the embodiment shown, emission beam  45  is converted into a voltage or current signal by photodetector  37 . The signal from photodetector  37  is amplified by amplifier  62  and processed by signal processor  40 . 
     In the embodiment shown, signal processor  40  is a device which operates on the signal from photodetector  37  to substantially separate the information-bearing component of the signal, which substantially expresses the modulation impressed by modulator  29 , from the noise component, which remains substantially unmodulated at the frequency of modulator  29 , thereby improving the signal-to-noise ratio of the resulting measurement. One type of signal processor, for example, forms the mathematical product of the amplified photodiode signal and a reference signal proportional to the modulation signal. Signal processor  40  may be any type of multiplier known in the art, including but not limited to, a demodulator, mixer, digital multiplier, and balanced demodulator. 
     Signal processor  40  produces a voltage or current which is related to the concentration of nucleic acid in tissue sample  51 , and the aforementioned current or voltage is used to generate a display on visual display  7  ( FIG. 1 ). 
     In various embodiments, amplifier  62  may be a logarithmic amplifier, a non-linear amplifier, a voltage amplifier, a current amplifier, a power amplifier, a transimpedance amplifier, or a transadmittance amplifier. 
     In various embodiments, nucleic acid amplification and monitoring apparatus  100  may further include a physical memory component for storing sample data. 
       FIG. 5  illustrates an exemplary embodiment of the wiring configuration for heated tissue sample chamber  10  of nucleic acid amplification and monitoring apparatus  100 . In the embodiment shown, tissue sample tube  8  with tissue sample  51  is passed through tissue sample port  12  into tissue sample chamber  10 , which is heated electrically by heating coil  75 . Temperature control circuitry  70  ( FIG. 6 ) controls the temperature of heating coil  75 . 
     In various embodiments, tissue sample chamber  10  may be heated by another means, such as an electronic resistance heater, Peltier heater, chemical heater, or a photonic heater, or may be operable at room temperature or ambient temperature without the need to be heated. In still other embodiments, the temperature of tissue sample chamber  10  may be maintained at a fixed temperature, regulated, cycled, varied, controlled, and/or intermittently adjusted. For example, the temperature of tissue sample chamber  10  may be chemically controlled. In the embodiment shown, tissue sample chamber  10  is maintained at an appropriate temperature at which a nucleic amplification reaction can proceed. 
     In the embodiment shown, nucleic acid may be amplified using thermocycling amplification (e.g., polymerase chain reaction (PCR), quantitative PCR (Q-PCR), multiplex-PCR, asymmetric PCR). In still other embodiments, nucleic acid may be amplified using isothermal amplification using a technique known in the art (e.g., loop-mediated isothermal amplification (LAMP), helicase-dependent amplification, PAN-AC, recombinase polymerase amplification (RPA), nicking enzyme amplification reaction). 
       FIG. 6  illustrates a block diagram of an exemplary embodiment of nucleic acid amplification and monitoring system  200 . Nucleic acid amplification and monitoring system  200  is comprised of modulated photonic source driver circuitry  68 , light excitation component  17 , excitation filter  24 , tissue sample  51  in tissue sample tube  8 , temperature-control circuitry  70 , heating coil  75 , emission filter  42 , photodetector  37 , amplifier  62 , signal processor  40 , visual display  7 , and power supply/power conditioning electronics  90 . 
     In the embodiment shown, modulated photonic source driver circuitry  68  modulates the intensity of excitation beam  26  (not shown) over time, temperature control circuitry  70  controls the temperature of tissue sample tube  8  and ensures that tissue sample tube  8  is heated to a temperature which allows the nucleic acid amplification reaction to proceed, and power supply/power conditioning electronics  90  provide power to nucleic acid amplification and monitoring system  200 . 
     In the embodiment shown, light excitation component  17  emits excitation beam  26  (not shown). Excitation beam  26  is powered and modulated by photonic source driver circuitry  68 , and filtered to a given wavelength range by excitation filter  24 . Excitation beam  26  excites fluorescent molecules in tissue sample  51  in tissue sample tube  8  producing emission beam  45 , which is proportional to the concentration of the target nucleic acid molecule in tissue sample  45 . Emission beam is collected, passed through emission filter  42 , and directed onto photodetector  37  and amplifier  62 . Photodetector  37  converts photons in emission beam  45  to a measurable voltage or current signal and signal processor  40  multiples the temporal waveform of the detected emission beam  45  intensity by the input modulation waveform. The average value of the voltage at the output of signal processor  40  is proportional to the fluorescence intensity, and accordingly, the concentration of the target nucleic acid solution in tissue sample  51 . 
     In various other embodiments, nucleic acid amplification and monitoring system  200  further includes excitation collimating lens  21  and emission collimating lens  36 . 
     Nucleic acid amplification and monitoring system  200  may be used to obtain both qualitative and/or quantitative results. For example, if the fluorescence signal increases as the amplification process proceeds, then the target nucleic acid is present. The increase in fluorescence signal also causes an increase in the signals from photodetector  37  and amplifier  62 , which cause an increase in the average output from signal processor  40 . The increasing electrical signals are proportional to the concentration of nucleic acid being measured, allowing the concentration of the target nucleic acid to be calculated. 
     In various embodiments, modulated photonic source driver circuitry  68  may produce a periodic waveform or an aperiodic waveform to modulate excitation beam  26 . 
     Nucleic acid amplification and monitoring system  200  is simultaneously highly sensitive to the fluorescence signal emitted from tissue sample  51  and insensitive to noise signals (e.g., ambient light), which interfere with measurement. The modulation and signal processor processes improve signal-to-noise ratio and increase sensitivity, allowing for measurements of small quantities of nucleic acid, which would otherwise yield a fluorescence signal obscured by noise. In addition, nucleic acid amplification and monitoring system  200  provides the optimal reaction conditions for nucleic acid amplification while simultaneously monitoring for fluorescence and rejecting optical noise signals during the reaction process.