Method for identifying biochemical and chemical reactions and micromechanical processes using nanomechanical and electronic signal ident

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BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for measuring characteristic micromotions created by chemical, mechanical, optical, or electrical processes using scanning probe microscopy or laser interferometry. In particular, a stationary mode atomic force microscope is used to monitor microscopic dynamic processes, such as the replication of DNA for DNA sequencing.

2. Description of Related Art

The development of scanning probe microscopes in the 1980s, such as the scanning tunneling microscope and the atomic force microscope, provided the opportunity to locate and identify microscopic sites with atomic resolution. A wide variety of sites can be observed: small biological molecules that perform sophisticated biological functions, atomic sites on the surface of materials where corrosion, gasification, or catalytic reactions take place, and atomic sites where stress-induced fracture occurs.

The scanning tunneling microscope (STM) has a fine conducting probe that is held close to the surface of a site. Electrons tunnel between the site and the probe, producing an electrical signal. The probe is moved slowly across the surface and raised and lowered so as to keep the signal constant. A profile of the surface is produced, and a computer-generated contour map of the surface is generated. The technique is capable of resolving individual atoms, but requires conductive materials for image formation.

The atomic force microscope (AFM) also has a small probe that is held on a spring-loaded or flexible cantilever in contact with the surface of a site. The probe is moved slowly across the surface, and the tracking force between the tip and the surface is monitored. Forces as small as 10.sup.-13 N can be measured. The probe is raised and lowered so as to keep this force constant, and a profile of the surface is produced. Typically, a laser beam is bounced off the cantilever to monitor its position with angstrom-scale precision. Scanning the probe over the site at a constant force gives a computer-generated contour map of the surface. This instrument is similar to the STM, but uses mechanical forces rather than electrical signals. The AFM can resolve individual molecules and, unlike the STM, can be used with non-conducting samples, such as biological specimens.

A number of research groups, including ones at Lawrence Livermore National Laboratory, have attempted, with limited success, to use the scanning probe microscopes to sequence DNA by resolving individual nucleotide bases in tunneling images. Sequencing the human genome is one of the major scientific goals in the United States and in the world today. Dramatic improvements in human health and well-being will be possible by understanding the ordering of the billions of base pairs contained in DNA. Major research projects, like the Human Genome Project, have a need for improved techniques that will significantly reduce the analysis time of DNA fragments. The demands on the sequencing technology are even greater than the Genome Project because scientists also want to understand the DNA of different animals, plants, and microorganisms as quickly as possible.

Presently, the most common approach for sequencing the human genome is electrophoresis, which uses an electric field to separate fragments of DNA as they migrate through a sieving matrix (a gel). The DNA fragments are produced in a number of ways. The most widely employed method uses restriction enzymes, which act as molecular scissors, to sever the DNA at precise locations, producing a unique family of fragments for each enzyme used. The necessary number of DNA fragments are produced by biochemical reactions such as the DNA polymerase chain reaction (PCR), which can make millions of copies of a given DNA sequence. Unfortunately, these conventional electrophoresis techniques are relatively slow and costly, and time estimates for mapping or sequencing the entire human DNA molecule using these techniques range from decades to centuries.

Likewise, the use of STM and AFM in the conventional scanning or visualizing mode to sequence DNA presents problems. The normal process of raster scanning the microscope tip across the DNA molecules causes the molecules to move. In addition, the poor conductivity of the DNA precludes STM observation of the bases as attached to the phosphate backbone. The difficulty with the conventional AFM approach is that the radius of curvature of even the best AFM tips interferes with the identification of DNA bases.

U.S. Pat. No. 5,106,729 by Lindsay et al. describes a method for determining and visualizing the base sequence of DNA and RNA with a scanning probe microscope. The method replaces the oxygen in the DNA with a metal-sulfur complex, and passes the probe over the complexed polymer to measure and record the differences in electrical conductivity at preselected increments along the scanning path. Lindsay acknowledges the limitations of STM and AFM, caused by the distortion of the soft molecules as the tip touches them. Lindsay attempts to solve this problem by using the metal complex to enhance the electrical contrast in the STM.

However, an urgent need exists for a DNA sequencing technique that is faster than the conventional electrophoresis techniques, and that does not require biochemical labeling or complexing the DNA molecule in order to image the base sequence. This invention addresses these challenges in particular, and introduces a general technique for detecting and measuring micromotions caused by an unlimited variety of chemical, mechanical, optical, and electrical processes.

SUMMARY OF THE INVENTION

This invention is a method for detecting microchemical reactions or micromechanical processes by measuring the characteristic signals emitted during a time sequence of the reactions using stationary mode operation of scanning probe microscopes. The signals may be detected directly, or may be transmitted through a medium and received indirectly. The method can be applied to any kind of reaction or process that produces vibrations, including biomechanical, biochemical, and inorganic reactions, and electrical, optical, and mechanical processes. The method greatly extends the range of information that is gathered with a scanning probe microscope.

The approach of this invention is to use the scanning probe microscope in a conventional way to locate or image a site where a process of interest occurs. The scanning probe is then used in a stationary mode to position a microdetector on or near the site. The probe detects and processes the signals (micromechanical, acoustic, electronic, optical, chemical) that are emitted directly from the site or that are transmitted (indirectly) through the surrounding medium. This latter approach is a type of acoustic detection. A micromechanical detector, such as the tip of an atomic force microscope (AFM) or a scanning tunneling microscope (STM), when held stationary can detect characteristic pulsations, configurational changes, charge fluctuations, or phonon emissions associated with a local chemical or mechanical event. An advantage of the AFM is its routine use in liquid environments, including reacting environments; however, the AFM has only been used in the scanning mode, and not in the stationary mode as described in this invention.

The invention is based on the direct micromechanical, acoustic, electronic, or chemical reception of information and identification of a time sequence of chemical or micromechanical reactions. A micromotion detector directly or indirectly monitors a process by reading the force waves (e.g., direct or acoustic pulses) that originate at a reaction site and are transmitted directly or through a fluid or other medium surrounding or coupled to the process. The detection efficiency of the process depends on the source transmitting the mechanical motions with sufficient intensity and fidelity that the detector receives a signal that can be distinguished from the background noise. In the case of detection through a surrounding medium, the coupling to the medium and the medium transmission are important properties. Other detectors may monitor electronic or chemical signals generated during this process.

Many important biological and inorganic processes occur at acoustic rates (.about.10-100 reactions per site per second), and these reactions may lead to new molecular structures. The creation of new structures generates structural or fluid motions in the environment that are characteristic of the structure being created. The present method can identify a characteristic signature created by the presence or absence of normal or abnormal processes in a living organism, in a cell, or in a subunit of a cell. The method can receive information on catalytic processes, or on the extraction of information from a chemical template (possibly leading to a new structure). The invention can be used to monitor micromechanical processes such as fracture, or biomechanical functions such as breathing or other metabolic processes in very small creatures. These events are detectable using the present method when the events occur at suitable rates, typically between 10-1000 Hz, and if they occur with suitable amplitudes.

The method is applied to structures as small as a single molecule, and therefore can provide information about biochemical processes such as egg fertilization, viral attack on a cell, the operation of mitochondria, and other biochemical sequences occurring during the normal operation of cellular structures. Of particular interest is the monitoring of genetic processes, such as DNA unwinding during cell replication, reading DNA in situ during normal or abnormal workings of a cell, enzyme processing of DNA, RNA processing, and reading messenger RNA (which generates proteins). Catalytic reactions, including biochemical catalysts (enzymes) and inorganic catalysts (e.g., zeolite catalysis of hydrocarbons), can be studied using the present method. Biological events can be measured in situ or separate from their natural host, as long as the techniques allow the desired process to continue and satisfy measurement criteria.

In addition to the motion measuring device and the structure that catalyzes or promotes the formation of a new molecular species, a method to couple the vibrations to the detector is required. This may be a substrate to support the structure, with direct or indirect contact of the micromechanical detector to the structure promoting the chemical process. In the case of measuring cellular processes such as viral invasion, simple contact to the cell surface will suffice. In the case of detecting enzymatic promotion of a chemical or biochemical reaction, such as DNA copying, direct contact with the enzyme surface may be used.

The approach provided by the present invention has the potential to sequence DNA at a rate ten to one hundred times faster than the presently used gel methods. Dramatic reductions in time and cost for sequencing DNA would result, which will significantly improve the progress of the Human Genome Project. This new technique can also be applied to a wide variety of other enzyme studies, as well as to micro-acoustic detection in non-destructive testing and seismology. In addition, more (if not all) of the sequence structure of the DNA molecule may be accessible with this method, which is not true of present sequencing methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C are schematic diagrams showing the positioning of a cantilever-tip assembly on or near a site of interest according to the present invention.

FIG. 2 shows schematically the method for monitoring the processing of DNA by polymerase directly using the tip of the probe.

FIG. 3 shows the method for indirectly monitoring polymerase processing of DNA through a surrounding medium.

FIG. 4 is a schematic diagram of the method for indirectly monitoring the processing of DNA by detecting micromotions of an intermediary site that is attached to the processing enzyme.

DETAILED DESCRIPTION OF THE INVENTION

General Description

The present invention is a method for detecting motions on a microscopic level that are created by a variety of reactions and processes, including micromechanical, microchemical, microbiological, micro-optical, and microelectrical. This method uses a scanning probe microscope in a stationary mode to gather information at a molecular scale and smaller. The method measures vibrations created by local mechanical events, such as microcracking, or by chemical processes, such as catalysis, DNA replication, or cellular viral attack.

A scanning probe microscope, such as the AFM or STM, is typically used to image or visualize a physical structure by scanning. However, the present method uses the instrument in a stationary mechanical mode, whereby the probe's tip measures characteristic motions (vibrations) or acoustic signals (frequencies) created or emitted by a specific site. Using the probe in a stationary mode allows one to "listen" to the locally-emitted micromotions at sites on a micrometer to nanometer scale; consequently, this technique is referred to as "nanostethoscopy". The stationary mode minimizes the problems associated with conventional scanning probe microscopes; in particular, this mode eliminates lateral sample distortion or deformation.

The micromotions created by a chemical structure or process can be detected by the probe in at least three ways. The probe can be used to detect movements of a structure of interest directly. That is, the probe tip physically touches the structure that will produce characteristic movements and measures the motions as the structure undergoes some kind of process (chemical, biological, mechanical, electrical, optical). These motions may include the change in configuration of an enzyme, movement of an inorganic catalyst, expansion of a microcrack, or the expansion and contraction of a material caused by temperature or electrical changes.

Second, the probe can be used to detect micromotions indirectly, by receiving signals that are transmitted from a site through a medium, such as a fluid or a solid. The motions in the medium propagate away from the source for a distance to the detector and are caused by the same kinds of processes described above. The detector may also measure time-varying motions caused by incoming or outgoing chemical products in a reaction.

Third, an intermediary site may be attached to a site of interest, and the probe can be used to detect the physical movements of the intermediary site, which in turn are caused by motions of the primary site. Instead of using a scanning probe microscope, the technique of laser interferometry could be used to detect the motions of the intermediary site. For example, an enzyme could be attached to a membrane, and the enzyme will recognize input products or a template and then fabricate a product or products. The enzyme motions cause vibrations in the surrounding liquid and in the membrane, which serves as a reflector to interferometrically detect the motions. In another example, a gold ball could be attached to an enzyme, which would move as the enzyme moved. A light signal could detect the motions interferometrically.

Specific Description

The present method is illustrated schematically in FIGS. 1A-1C. A specific site of interest is first located using a location detector, such as a tip of an AFM or a STM probe. The site may be attached to a substrate for support. Attachment to a substrate is typical for biological and chemical applications. After the site is located, a motion detector is positioned at the site. This detector may be the same AFM or STM tip used as the location detector, but the mode is changed to keep the tip stationary to detect micromotions emitted from the site. Alternatively, a separate, second detector may be used as a motion detector to measure time-varying qualities at the site (movements in vertical motion, acoustic or sound waves) or changes in the system (comparing motion at two times).

FIG. 1A shows the tip 10 of an AFM or STM cantilever 12 positioned on a structure site 14 of interest, which may be attached to a substrate 16 for support. The cantilever 12 moves vertically as a function of time (z(t)) as the site 14 undergoes chemical, mechanical, optical, or electrical changes. The physical movements of the cantilever 12 are converted into an electrical signal, which is compared to the signal obtained when the structure site 14 is not undergoing the process of interest.

FIG. 1B shows the tip 18 of the cantilever 20 positioned at a distance from the structure site 22. The site 22 is surrounded by a medium (gas, liquid, or solid) that transmits acoustic or sound waves 24 caused by micromotions at the site 22. The site 22 may change physical configuration, or undergo chemical reactions or changes in electrochemical potential, or experience mechanical, optical, or electrical transformations, which generates pressure waves 24 that propagate through the medium across the distance between the site 22 and the tip 18 (detector). The deflections of the tip 18 are converted electronically to measure characteristic vibrations front the structure site 22.

FIG. 1C illustrates a third way to measure distinctive vibrations caused by activity at a selected site. An intermediary site 26, such as a membrane as shown or a gold sphere, is attached to the primary site 28. A counter-mass, or another substrate, or another probe tip 36 as shown is typically in contact with or attached to the back of the primary site 28 for support. The tip 30,32 of the cantilever 34 is positioned at or near the intermediary site 26 to detect micromotions that originate at the primary site 28 and then are transmitted to the intermediary site 26.

As in FIG. 1A, the tip 30 may detect vibrations by sitting directly on the intermediary site 26. Alternatively, the movements of the primary site 28 may be conveyed to the intermediary site 26, as in FIG. 1B. The physical movements of the intermediary site 26, in turn, create acoustic waves that are transmitted through the surrounding medium to the tip 32, and thus the motions of the primary site 28 are detected indirectly. Another alternative is to use the intermediary site 26 as a reflector for laser interferometric detection of movements caused by vibrations in the primary site 28.

The present method has a wide variety of possible applications, as summarized below.

A. Cellular Properties

Any vibrational phenomenon associated with normal or abnormal cell functioning can be monitored using the present techniqu