Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
  • G01N21/6458—Fluorescence microscopy

Definitions

• the present invention relates to a physico-chemical analysis method with associated spectrometer for molecules and sequence analysis of molecular samples.
• Nucleic acid sequencing was the central problem in genetic biochemistry.
• the previously established chemical-analytical processes process a maximum of around 20 bases per second and still require around six years for a human genome.
• the DNA molecule of the longest human chromosome with about 3 * 10 base pairs is around 10 cm long. Other species sometimes have longer chromosomes.
• the diploid genome of a human body cell, as a single strand, is about 4 meters long and (' around 2 nanometers wide.
• the primary identity period, which, if at all, is only about 1.5% larger than that of the crystallographic c-axis of natural graphite with 0.3348 nm is 0.34 nm and the secondary is 3.4 nm.
• each nucleotide connected to it is the information-carrying part of a nucleic acid, and in contrast to the rest of the molecule, such bases can be determined optically as chromophores by spectral analysis.
• the two base genera of puric acids can be removed from nucleic acids in a highly selective manner by means of an acid-catalytic polymer-analogous chemical reaction at p H 3. This is also possible enzymatically, for example with the help of a nucleosidase. What remains is adenine- and guanine-free apuric acid.
• the sequence analysis of the primary and complementary strand, each with the remaining two base genera cytosine and thymine and uracil, complements Chargaff again for complete genetic information.
• the excitation of individual atomic elements with the tip of a scanning tunneling microscope to emit luminescence has already been achieved / EP 0 801 310 /.
• Biradical one of which is particularly reactive. Furthermore, a change in the polarization between the excitation beam and the emission suggests that energy transfer has taken place. This means, above all at higher temperatures, that the glow mostly comes from a position other than the one just excited.
• Pentium 4 microprocessor is now operated at an operating frequency of 2.66 GHz, which is currently a monthly increase of +130 MHz.
• the present invention solves the technical problem of specifying a method which allows an exact, reliable and economical sequence and molecular analysis to be carried out in a very short time.
• a key area of application is the key technology of genome analysis, for example for the purpose of molecular diagnostics.
• a second relates to the reading of miniaturized stored data or pits, for example in the field of extremely compact molecular storage density, and a third relates to the field of molecular trace analysis, for example for the detection of hidden explosives and intoxicants.
• this invention is useful in polymer analysis, for example for the exact determination of the tacticity of macromolecules and the block length in copolymers and also for high-precision, also macroscopic, length measurements with an atomic-molecular step size.
• the content of DE 101 23 443 is also part of this invention.
• a thin light beam is generated in one of several ways, of which at least one dimension corresponds to the diameter of a molecule. That is the "molecular spotlight”.
• This can be a convergent beam with a small focal spot, a thin parallel beam and a preferably linearly polarized beam through a "picospalt". That is, a gap which is about 1 to 10 mm long and about as wide as the position distance of the sample. For nucleic acids it is 340 picometers.
• the electric field vector oscillates in particular in the longer direction of the "picospalt", so that no diffraction phenomena occur.
• It can be manufactured e.g. by drilling and milling with an electron beam, if necessary, place several thin platelets with a gap on top of each other, as well as from plane-parallel separated and polluted corrosion-resistant metal pieces, which are provided with a molecularly thin spacer around the gap.
• inventive method for sequence and molecular analysis is accordingly characterized by the features of independent claims 1), 7), 23), 24), 25), 27), 32) and 38).
• Advantageous refinements and developments are the subject of the claims which are directly or indirectly dependent thereon.
• the spectrometer according to the invention is given by claims 38) and 39).
• the inventive method for sequence analysis is characterized in that
• step B) the individual sequentially consecutive positions within the sample, e.g. in the location determined in step A), are exposed to an electromagnetic excitation signal with the aid of a “molecular spotlight”, the one dimension corresponding to the position distance of the sample, in order to generate a response signal which is designed in particular as a characteristic light intensity such as transmission or luminescence,
• the respective response signal is detected, evaluated and stored, a detector device being used whose detection sensitivity is e.g. is in the range of a photon or electron,
• the detection, the data processing and the advance to the next position are carried out within a relatively short time, in particular under one microsecond to under one nanosecond per position of the sample, and
• steps B) to E) can be repeated at further positions within the sequence of the sample, preferably at their information-carrying parts.
• a preferred embodiment of the method according to the invention is characterized in that the tips of tunnel or atomic microscopes are used to fix the sample.
• steps A) and B) can be carried out simultaneously, i.e. the measuring beam designed as a “molecular spotlight” serves on the one hand to determine the location and the size of a position and on the other hand it also causes characteristic absorption and Rayleigh scattering and the photoluminescence of the sample.
• the two steps A) and B) are temporal and carried out locally one after the other
• step B) can be carried out in a further pass.
• the spontaneous luminescence is preferably suppressed. This happens both with the stimulated emission and with a quencher, i.e. a substance that accelerates the quenching of the luminescence.
• Typical radical scavengers such as carbon tetrachloride, CCI 4 , polyphenols such as hydroquinone and gases such as methane, CH 4 are suitable for this.
• Electromagnetic radiation is suitable for excitation, i.e.: radio waves, microwaves, infrared (FR), visible radiation, ultraviolet (UV), X-rays and ⁇ radiation, but also material radiation, such as electron beams, also in the form of alternating current and electrical fields.
• a system excitation and a system response in the area of optical radiation, that is from UV to FIR, are preferred.
• the ionizing electromagnetic radiation types are applied so elastically, in particular by flat angles and radiation near the edge, that only an energy component required for characteristic transitions is transmitted to the sample, which is below the total energy 5 of the exciting photon.
• the optical excitation signal is designed so as to be characteristic of intensity, wavelength, spectral resolution, coherence length, polarization, phase and the duration of pulse and pause so that it is maximally absorbed by the part of the molecule to be excited. In particular, this takes place in a characteristic wavelength range and with a characteristic polarization, which is typical for the chromophore of the position of a sample just examined.
• a preferred embodiment of the method according to the invention is characterized in that a stimulated emission is carried out in step B). This can also be done with an atomic molecular spatial resolution and in an internal environment, e.g. in the absence of oxygen,
• cooling is carried out. This is done by flowing with a fluid, also supercritical, coolant, such as water, in particular at high speed.
• coolant such as water
• cooling can be achieved by contact with a cooling finger, which can also be designed as a wire.
• the material used for this is preferably characterized by a relatively high
• Thermal conductivity and surface cleanliness This is, for example, copper, silver and gold with an ion beam cleaned surface.
• cooling can also be carried out alone or in combination with a special laser beam with high spectral resolution. This is all the more effective if such radiation is carried out simultaneously from several different directions.
• a scanning microscope can also be used for process steps A) or B).
• the sample is water-free, undergoes rotation about its longitudinal axis, and deflection and collection optics increase the quantum yield of the detection.
• the working time for the system excitation, for the detection, the advance to the subsequent position and for the associated data processing preferably takes less than a nanosecond.
• a particularly preferred embodiment which is used in particular for the case of simultaneous excitation of a plurality of, in particular adjacent, positions, is distinguished in that the emission is evaluated via the appropriate use of difference spectra. This is also possible with the aid of "optical spreading".
• the position to be analyzed 5 is located directly in the focal point of a parabolic mirror. Adjacent sequences are outside of it. According to the rules of geometric beam optics, their luniinescence is not reflected parallel to the optical axis. A relative Long light path over several kilometers, for example with rolled-up fiber optic cables or plane-parallel special mirrors, enables spreading and thus also a lateral separation from the focal rays.
• a further preferred embodiment uses several detector devices for the evaluation of the luminescence. This consists of the detector and upstream analysis units to refine the detection.
• the detector can be a photomultiplier, pulse height or multi-channel analyzer, a CCD camera, also pelet-cooled, or other fast and highly detection-sensitive photodetectors based on semiconductors, such as an avalanche
• an extra folying device which acts on the excitation signals that are emitted. This consists of electric or magnetic field lenses as well as the "molecular spotlight” with an adjusted light intensity.
• the space quantum yield is much better with the aid of a deflection and collection optics.
• This is, for example, a parabolic mirror, a part of it can also suffice, in the focal point of which the luminous position of the sample is preferably arranged.
• these 5 reflectors can also already contain the grating lines required for spectral decomposition, preferably of the echelet type, that is to say optimized in terms of efficiency, in particular on the focus of the wavelength of the radiation to be analyzed. If only the light intensity is measured, a sphere with a radius of 20 cm, for example, is sufficient, the inner surface of which is lined with relatively small detectors such as photodiodes.
• a sphere with a radius of, for example, 5 cm is used with the light source in the center.
• the spherical surface are exactly vertical, that is to say radial, the one ends of optical fibers which collimate the emission of the detector device from the other ends located at a common point
• optical fibers of different lengths are then preferably made thin and flexible, so their diameter is in the micrometer range.
• Their cross section is preferably planar and perpendicular to the length and is oriented towards full use of the spherical surface. That means, like a soccer ball, pentagons and hexagons alternate. Fewer optical fibers are required if the inner surface of the sphere that remains free is reflective
• ⁇ is trained, e.g. as an aluminum mum ball.
• the radiation collected in this way is directed onto a detector device, in particular after a cross-sectional reduction with convergence, better with a pair of parabolic mirrors to maintain collimation.
• Optical fibers can also serve as sample carriers, e.g. as a tube or hollow fiber filled with the sample, as a full fiber with the sample in a preferably central gap and with a sample fixed in casting resin 5.
• Light sources are e.g. thermal, colorful and coherent emitters. One is required
• Dye lasers are suitable for wide spectral ranges. Their spectral resolution can be improved) e.g. through tuning according to Fabry-Perot or Etalon as well as mode selection and mode coupling with electronic readjustment to a sharp spectral line.
• For pulsed excitation of the sample e.g. electronic breakers, flash lamps and choppers are used.
• Reflectors can also be partially transparent, e.g. for preferably one-sided light passage, e.g. 1%.
• a chopper separates temporally separate processes such as reflection and scattered light, stimulated emission, spontaneous fluorescence and phosphorescence, also to improve the signal / noise ratio.
• the electron spin reversal stimulated by a characteristic microwave radiation is separated from their
• Characteristic J-R absorptions or Raman scatterings can also be measured, especially the stretching and bending vibrations of the base residues on the oxygen of the first carbon atom from the sugar.
• the chemical improvement of the fluorescence intensity can also be achieved by photochemical derivatization with an organic reagent for the duration of the excited state.
• Hydroquinone for example, is suitable for this.
• the contact between the sample and the support can also be achieved using an adhesive, e.g. a household adhesive. It is also possible to extend the sample at one end. This happens with another polymer molecule that is chemically bound to it, for example via a condensation reaction.
• Non-polar solvents such as ether or hexane, a glassy alcohol-ether matrix according to Spolsky at low temperature or high vacuum are also suitable as the chemical environment of the sample, with the advantage that the glass bulb of the detector tube, which may interfere with its reflection, can be omitted.
• Data storage is more compact if only one valid signal is saved at several measurements in the same position.
• Disturbing length fluctuations are achieved through a relatively high constancy of the working temperature through thermostatting, shading against external heat sources and use of materials with a particularly low expansion coefficient as well as a correspondingly low-vibration mounting of the spectrometer. This happens, for example, as with the tower microscope with a special suspension or how for a seismograph with a particularly heavy mass.
• "Low-leveP 'measuring station typical) precautions are the steel shielding to the outside, for example by the usual several cm thick lead walls, and within the spectrometer through the use of low-radioisotope materials.
• the gain selectivity e.g. is operated in the lowest temperature range at temperatures around -196 ° C 3 to below 1 K (-272 ° C),
• the measuring beam is also matched to a maximum and selective absorption as possible according to the respective characteristic intensity, wavelength, spectral resolution, coherence length, polarization, phase and the duration of the pulse and pause, and
• a preferred embodiment of the method according to the invention is characterized in that a gas measuring cell, optionally with a heatable wall, is used in step I).
• the light path in the gas measuring cell covers the entire Irmen cross-sectional area. The multiple total reflection of the measuring beam, even with several measuring beams, makes this possible, for example.
• the non-irradiated partial areas should not be significantly larger than one
• measuring beam Either its surface moves through the measuring volume or the gas flows through it. Instead of round e.g. also be square. This happens before the optical downsizing, if necessary after the measuring beam has been expanded, e.g. to 5 cm, with the help of an appropriately shaped panel.
• the measuring cell itself can also be square and reflective.
• this gas must be replaced by a gas, such as noble gas, for example argon, which has a weaker absorption therein.
• a gas such as noble gas, for example argon, which has a weaker absorption therein.
• Interfering particles such as aerosols etc. are separated beforehand, eg electrostatically or by filtration.
• a molecule can also be examined optically in a capillary tube. Enrichment of the substance or group of substances sought is also advantageous.
• the characteristic strong absorption bands of organic nitro compounds in the IR spectrum at 1650, 1290 and 850 cm " and medium intensities are suitable for detecting explosives
• a significantly longer measuring time is available for determining a molecule. This can be less than a second, for example, but can also take up to an hour.
• a copy of the sample likewise a synthetic sample for molecular data storage, can be synthesized with more intense and faster-emitting, but preferably fluorescent, luminophores and then advantageously sequenced.
• all components should be ideally transparent, absorbent and reflective, that is, each with a high degree of efficiency.
• a “picospalt” is also suitable for determining the position of the position under investigation within the sequence of a sample, which is also arranged in the beam path after the sample and is provided with an aperture to protect against radiation from adjacent positions.
• the transmission of the excitation beam also works in this sense.
• the photons specific for sequence positions are directed to a preferably pelet-cooled CCD camera after spectral decomposition in order to achieve the shortest possible measurement times, under one microsecond per sequence position.
• a fast electronic buffer with time-delayed data output that is to say a transient memory, is used.
• the parallel operation of several microprocessors is also possible.
• Characteristic features of the invention in this method are that locally selective optical excitation of a sequence position of the sample using the “molecular Spotlights "takes place and for transmission and fluorescence measurements a detection of the resulting response signal is carried out in the range of highest possible detection sensitivity and shortest measuring time specific to the location and genus.
• the variant of the exciter spectroscopy technique can also be used for this.
• the invention also relates to a spectrometer for performing the above-mentioned methods.
• a spectrometer for performing the above-mentioned methods.
• devices or units which enable the method steps mentioned above to be carried out in a corresponding manner.
• luminescence can also be excited with the aid of both types of radiation. This is preferably done optically, but also with electrons. In principle, electric fields are also suitable for this alone, in particular in the form of alternating fields, and a sufficiently high temperature, which is, however, above the decomposition point of most organic substances.
• Double-stranded molecules are preferred, in particular thermally, melted into single-stranded molecules and then individually examined. There is a possibility that the molecules to be sequenced e.g. be measured in a capillary with a light collecting effect.
• Fabry-Perot plates can be used to bring even higher spectral resolution.
• the photons which are characteristic of a position of the sample and which, as is known, can originate from various excited species are made as short as possible to achieve this
• a relatively short detection time e.g. below one microsecond to nanosecond
• Figure 1 is a schematic representation of a spectrometer for absorption and fluorescence measurements for conducting a molecular and sequence analysis with a "molecular spotlight”. It consists of: a light source (76), an attachment lens (96), e.g. a condenser, one
• FIG. 2 shows a freely movable arrangement of the sample carrier (22) consisting of two tunnel microscope tips (24 + 28) with the sample (12/40) hanging between them. The position just measured is then marked with "X”.
• the excitation beam (97), the transmission (98) and the associated detel device (14) also follow.
• the radiation for the stimulated emission (34), the response from luminescence (30) also follows Rayleigh scattering and stimulated emission (36) and the associated detector device (14.)
• the x coordinate runs in the longitudinal direction of the sample, the y coordinate runs perpendicular to it and the z coordinate runs perpendicular to both.
• the position coordinates of the sample are determined and stored, or the excitation to emit a luminescence (30) takes place. Furthermore, both a separate second fine pass can be carried out and an excitation signal (20) can be emitted via a second “molecular spotlight” that follows immediately after it.
• FIGS. 1 and 2 there is a spectrometer that generates a “molecular spotlight” and can direct it onto the fixed sample. It has a detector device (14), a deeply cooled electronic amplifier (86) and a control and evaluation unit. and storage means (18) followed by an output unit for the data.
• the diameter and thus the relatively high lateral resolution of the excitation beam (20/97) in the order of magnitude of typical positional distances within the sample is therefore 0.34 nm.
• the sample is taken from a swab of the cheek mucosa from a person's oral cavity. This is followed by enzymatic digestion with lysis of the cell wall and nucleus, followed by isolation, separation and purification in one operation by accelerated gel permeation chromatography with a particularly short column, with negative pressure on the removal side and with centrifugal force. Duration about 1 A to 1 hour, also automated. If the spectral purity of the sample is insufficient, this is followed by repeated cleaning if necessary, i.e. phenol extraction and precipitation from aqueous solution using isopropanol or ethanol. The double strands are then thermally separated into two complementary single strands by heating to over 65 to 90 ° C. in water. Possibly. add some glycerin or a mixture of sodium chloride and citrate.
• PH 3 is adjusted at room temperature by adding phthalate-hydrochloric acid buffer. Possibly. ) reheat. Subject the mixture to gel permeation chromatography again or pour it into a dish, let it dry, rinse with pure water, let it dry again. Electrostatically, if necessary with biotin, fix the sample at two tips and place it in the holding device. Pull the tips apart without tearing the sample and place them in the measuring beam.
• the transmission, fluorescence and possibly the scattered light must be measured. To do this, start with one photon per second and then increase the intensity to saturation. With an emission stimulated with any lateral resolution, the saturation intensity is several orders of magnitude higher. The determinations become more precise when the photons that are superfluous for a measurement burden the detection. This can be achieved by the selective selection of a characteristic polarization; a correspondingly high spectral resolution of the excitation radiation is also advantageous.
• the phase also influences the success of the absorption of a light quantum. This can be moved cheaply by triggering the flash lamp or chopper. A matching extension of the light path is also beneficial. Heat dissipation contacts and the mostly multiple use of laser cooling dissipate disruptive heat.
• Piezoelectric also precision mechanical or thermohydraulic, actuators move the sample holder relative to the spectrometer position by position to the last position after each successful measurement. After the limited maximum deflection of the actuator has been reached, the moving front part is fixed there, the rear starting part is set back to a minimum deflection, is then tracked, fixed and the fixing of the front part is released again.
• the actuators move a reflector that directs the measuring beam from the first to the last sequence position of the semicircular sample.
• a second reflector which is also moved, to a fixed detector. When measuring fluorescence, the sample is moved.
• the complementary strand is sequenced in the same way and then the appropriate complementary direction and the complete sequence are determined with the aid of EDP.
• the result is then output as a complete sequence protocol or only in the form of special deviations as well as molecular genetic diagnosis and prognosis with therapy suggestions saved on a data carrier. This happens, for example, on a magnetic tape, a memory chip or a burned CD-ROM.
• Characteristic features of the invention in this analysis method are that local excitation takes place in the atomic-molecular order of magnitude and at the same time a detection of the resulting specific response signal of the sequence position in the range of the highest possible detection sensitivity is carried out selectively from the location of the excitation.
• This enables simple, automatable, fast, inexpensive, direct and sequence-accurate analyzes of (macro) molecules and their parts as well as trace molecules.
• a human genome can be sequenced within a very short time, from less than a month to a quarter of an hour, at a cost that is compatible with health insurance.

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Citations (33)

• 2002
  • 2002-11-20 DE DE10297995T patent/DE10297995D2/de not_active Expired - Lifetime
  • 2002-11-20 WO PCT/DE2002/004275 patent/WO2004046709A1/de not_active Application Discontinuation
  • 2002-11-20 AU AU2002363822A patent/AU2002363822A1/en not_active Abandoned

Patent Citations (33)

Non-Patent Citations (6)

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