Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/487—Physical analysis of biological material of liquid biological material
  • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
  • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6869—Methods for sequencing
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/453—Cells therefor

Definitions

• the invention relates to an arrangement for nucleic acid sequencing by tunnel current analysis.
• nucleic acid sequencing Numerous methods for nucleic acid sequencing are known in the literature. These include methods of so-called sequencing by synthesis (English: sequencing-by-synthesis). Here, a component which is detected by means of enzyme cascades during installation of a suitable nucleo ⁇ TIDS is released. Furthermore, nucleic acid sequencing in nanopores is known. It is advantageous that in this method, neither a marking of the DNA strand nor ei ⁇ ne complex reaction cascade is needed.
• DNA strands pass a biological or artificial (so-called solid state) nanopore.
• Individual bases of the nucleic acid strand can be ananalysiert by a change in pore resistance in Pas ⁇ Sieren the DNA through the nanopore.
• the DNA is placed in a conductive fluid. A voltage is applied to the fluid, so that an electric current flows.
• this stream changes. This change is dependent on the base passing the pore so that the base can be analyzed.
• tunnel current can be measured across the pore (sequencing-by-tunneling), with the tunneling current being dependent on the base that is in the pore.
• Tunnel current methods advantageously have a better one
• electrodes are very complex and time-consuming to manufacture.
• a sufficiently precise arrangement of the nanopore to the electrodes or a capacitor such that tunnel currents can be measured with high accuracy, is currently hardly technically feasible and their production is disadvantageously associated with high labor and time ⁇ effort.
• the object of the present invention is to provide an arrangement for the analysis of nucleic acid sequences and a method for producing the arrangement, which include the above-mentioned
• the arrangement for nucleic acid sequencing by tunneling current analysis comprises at least two according to the invention elekt ⁇ driven conductive particles having a diameter of 1 nm to 100 nm, in particular with a diameter of 1 nm to 10 nm. Further, it comprises at least two electrically insulating yield particles with a diameter of 1 nm to 100 nm, in particular from 1 nm to 10 nm.
• the arrangement comprises white ⁇ terhin at least two first electrodes to contact the electrically conductive particles. The first electrodes and the particles are on a substrate. Erfindungsge ⁇ Telss at least four particle are arranged substantially quadra ⁇ schematically planar, whereby the conductive particles and insulating particles are opposed to each diagonal.
• a gap is formed in the middle of the four square-planar particles arranged.
• the size of this gap is in the range of nm depending on the size and shape of the particles.
• the gap is in the analysis of nucleic ⁇ leinklaresequenzen a solid-state nanopore.
• the Anord ⁇ voltage makes it possible to represent a defined tailored nanopore.
• depressions are produced in a substrate.
• the first electrodes each with at least one electrically conductive particles in direct electrical contact.
• a current can flow through the electrically conductive particles.
• the arrangement of the first electrodes is advantageously such that the conductive particles form the capacitor on which a tunneling current can be measured for analysis when there is a DNA or RNA strand in the nanopore.
• the arrangement of the nanopore to the capacitor ⁇ is then advantageously such that tunneling currents can be measured with high accuracy, since the nanopore and capacitor are locally very close to each other.
• the forming Kondesatorspalt and Nanopore are identical.
• the arrangement comprises at least two orthogonal to the first electrodes arranged second electrode the.
• Electrodes are advantageously used for the targeted movement of a DNA strand through the particles.
• Particularly suitable for this purpose are electrodes which are used in gel electrophoresis in order to move the DNA / RNA through the gel.
• the substrate comprises recesses arranged in the grid.
• the diameter of the depression is in particular between 10 nm and 1 ⁇ m.
• the Vertie ⁇ levies represent a fixing unit for the particles.
• the substrate is a CMOS chip. This is built up in layers, with insulating and conductive
• the CMOS chip typically already includes a device for powering the electrodes and measuring a current between the first electrodes.
• the particles are spherical particles.
• spherical particles arrange themselves in a spherical packing.
• the diameters of the resulting gaps can be advantageously calculated. Tailor-made nanopores for nucleic acid sequencing can thus be formed.
• the electrically conductive particles comprise gold.
• the electrically conductive particles are typically substantially the same size.
• the Kugelpa ⁇ ckung is regular.
• the electrically insulating particles comprise polystyrene.
• the electrically conductive particles are firmly connected to each other.
• This fixed connection can advantageously be produced by means of a galvanization process.
• this fixed connection can by means of an electrically conductive coating, which is generated in particular by means of electroless plating of metal, it ⁇ follow.
• the electrical contact between the electrically conductive particles, which are each less than lnm away from each other, is thus advantageously ensured.
• the wells are filled with at least 100 electrically conductive and electrically insulating particles.
• the recesses which are already filled with electrically conductive and electrically insulating particles are selected, which have exactly one arrangement according to claim 1, ie with a nanopore.
• the tunnel current which flows through the nanopore when a DNA / RNA flows, is measured without the superimposition of further tunnel currents.
• the electrically conductive particles which have mutually less than lnm distance are coated with egg ⁇ ner electrically conductive layer such that they are in electrical contact with each other.
• the electrically conductive particles are coated by means of electroless deposition of metal.
• FIG. 1 schematically shows a particle arrangement 13 in a plan view from above.
• FIG. 1 shows schematically the structure of the grid assembly 1 from the side.
• the grid arrangement 1 comprises the particle arrangement 13.
• FIG. 3 schematically shows a depression 3 with two first electrodes 4 in plan view from above with an electric current 10.
• the particle arrangement 13 shown schematically in FIG. 1 comprises two conductive particles 8, two insulating particles 9, and DNA 6.
• the particles are in section shown in plan view from above, that is, the illustrated round areas show the sectional area of a particle at its maximum diameter.
• the helical DNA 6 is to se ⁇ hen from above.
• the conductive particles 8 are made of gold.
• the insulating particles 9 are made of polystyrene. Alternatively, the insulating particles 9 are made of latex.
• the diameter 11 of the particles 8 and 9 is 5 nm.
• the gap which forms Zvi ⁇ rule to the particles, provides an active nanopore 7 for DNA sequencing. It has a pore diameter of 12 2 nm.
• Figure 2 shows a side view of the grid assembly 1 with, for example three recesses 3.
• the substrate 2 comprises the special ⁇ silicon.
• the recess diameter 14 is ty-
• the recess diameter 14 in this exemplary embodiment is 35 nm.
• a first electrode 4 is located on each of the two lateral walls of the depression 3.
• the first electrodes 4 are expediently supplied with voltage.
• the first electrodes 4 may extend over portions of the side walls, as in this example. Alternatively, the first electrodes 4 may extend over the entire area of the side walls.
• a second electrode 5 is arranged in each case.
• Electrodes 4 and 5 are shown by way of example with reference to the central recess 3 of FIG.
• the substrate is typically a CMOS chip. On this CMOS chip, the wells are arranged in a grid. One of the second electrodes 5 and the two first electrodes 4 are applied to this substrate, ie the CMOS chip.
• the CMOS chip has very good analog electronic self ⁇ properties which guarantee at a precise measurement of the tunneling current. In particular, via an analog-to-digital conversion and a fast multiplexing method, it is possible to read a plurality of electrodes for measuring the tunneling current. The contacting of the electrodes takes place in particular by means of the uppermost metallization of the CMOS chip.
• the depressions 3 are introduced by means of an etching technique in silicon oxide or silicon nitride.
• the depression 3 is filled with a mixture of conductive particles 8 and insulating particles 9, the composition of the mixture being divided essentially in half and the particles being distributed randomly in the depression.
• the second electrodes 5 are supplied with voltage, so that the DNA 6 is transported into the recess 3, similar to the transport of the DNA in an electrophoresis arrangement .
• Figure 3 section through one of the wells 3 is shown.
• an electric current 10 is produced in the particle arrangement 13.
• the conductive particles 8 flows a tunnel current, the conductive particles 8 themselves carrying the capacitors represent.
• the tunneling current depends on the base of DNA 6, which passes through the active nanopore 7.
• the base may be analyzed in the active nanopore 7 ⁇ the.
• the conductive particles 8, which directly adjoin the active nanopore 7, form the capacitor, which allows a short pore length. This significantly improves the base resolution of DNA sequencing.
• the current between the second electrode 5 moves the DNA 6 through the active nanopore 7, so that a base after the walls ⁇ ren the active nanopore happened 7 and the nucleic acid sequence ⁇ is analyzed.
• the second electrodes 5 are not supplied with voltage during the measurement of the tunneling current. Alternatively, a constant voltage supply of the second electrodes 5 can take place, if this does not disturb the measurement of the tunneling current.
• the depressions 3 are first filled with an electrolyte solution. Subsequently ⁇ ° d an AC voltage to the particle and
• Electrolyte solution filled well 3 is applied and measured the resistance.
• the resistance is characteristic of the arrangement of the non-conductive and conductive particles 8 and 9.
• a depression 3 comprises a plurality of particle arrangements 13 in which DNA 6 is present at the same time, this results to several tunnel currents in a depression 3. These tunnel currents can then not be discriminated so that this depression 3 can not be evaluated.
• a grid comprises 1000 depressions 3, so that nevertheless sufficient depressions 3 with exactly one particle arrangement 13 are available for DNA analysis.
• a galvanization can take place.
• the conductive particles 8 of gold are connected to each other by so-called electroless plating.
• this contacting can be done really galvanic.
• the conductive particles are thus fixed and an electrical contact between the directly touching conductive particles 8 ensured.
• RNA sequences can also be analyzed.
• sequence analysis of short RNA fragments in particular miRNAs (micro-RNAs) or mRNAs (messenger RNA), is possible.

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• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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• 2012
  • 2012-09-27 DE DE102012217603.9A patent/DE102012217603A1/de not_active Ceased
• 2013
  • 2013-09-18 JP JP2015533535A patent/JP6033448B2/ja not_active Expired - Fee Related
  • 2013-09-18 EP EP13771414.3A patent/EP2880183B1/de not_active Not-in-force
  • 2013-09-18 WO PCT/EP2013/069384 patent/WO2014048816A1/de not_active Ceased
  • 2013-09-18 IN IN1996DEN2015 patent/IN2015DN01996A/en unknown
  • 2013-09-18 CN CN201380050219.6A patent/CN104704130A/zh active Pending
  • 2013-09-18 US US14/431,896 patent/US9804146B2/en not_active Expired - Fee Related

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