WO2021206539A1 - Dispositif de profilage et d'identification d'un oligonucléotide - Google Patents

Dispositif de profilage et d'identification d'un oligonucléotide Download PDF

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Publication number
WO2021206539A1
WO2021206539A1 PCT/MY2021/050026 MY2021050026W WO2021206539A1 WO 2021206539 A1 WO2021206539 A1 WO 2021206539A1 MY 2021050026 W MY2021050026 W MY 2021050026W WO 2021206539 A1 WO2021206539 A1 WO 2021206539A1
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WO
WIPO (PCT)
Prior art keywords
oligonucleotide
sample
electrode
profiling
voltage
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Application number
PCT/MY2021/050026
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English (en)
Inventor
Vengadesh A/L PERIASAMY
Souhad M.A DARAGHMA
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Universiti Malaya
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Publication date
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Publication of WO2021206539A1 publication Critical patent/WO2021206539A1/fr

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    • 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
    • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
    • 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

Definitions

  • the present invention relates to a device for profiling and identifying oligonucleotide.
  • Oligonucleotides are one of the most important tools in modern-day molecular biology. The many potential applications of oligonucleotides include site-directed mutagenesis, protein engineering and recombinant DNA technology. Oligonucleotides have become a cornerstone in molecular biology, synthetic biology and biotechnology due to oligonucleotides having identical structure and chemical qualities to DNA.
  • oligonucleotide profiling is done by a polymerase chain reaction, PCR or chromatography.
  • An example is disclosed by a PCT Patent Publication No. WO 2009117031 which relates to a method of nucleotide profiling for microorganism identification.
  • the method includes the steps of extracting a nucieotide sequence template from the sample, performing nucieotide sequence amplification on the template using PCR to create an amplified sample, performing less than four nucleotide- specific chemical cleavage reactions to obtain nucieotide sequence fragments, performing size separation on the fragments by gel electrophoresis, detecting the fragments' separation, generating a profile based on the detection, and comparing the profile to a database to identify the sample.
  • the present invention provides a device (100) for profiling and identifying a sample of an oligonucleotide.
  • the device (100) comprises a first electrode (110) and a second electrode (120) disposed on a substrate (130), and a source measure unit (140) connected to the first electrode (110) and the second electrode (120).
  • the source measure unit (140) is configured to supply and measure voltage and current in order to obtain a plurality of electronic properties of a Schottky diode formed by the sample of oligonucleotide deposited over the first (110) and second (120) electrodes.
  • FIG. 1 shows a diagram of a device (100) for profiling and identifying oligonucleotide in accordance with a preferred embodiment of the present invention.
  • FIGS. 2(a-b) show graphs of measured current against voltage supplied for four samples of different oligonucleotide sequences.
  • FIG. 3 shows a graph of resistance against voltage supplied for four samples of different oligonucleotide sequences.
  • a device (100) for profiling and identifying a sample of an oligonucleotide The device (100) profiles the oligonucleotide based on its electronic properties, wherein the electronic properties refer to electronic parameters of a Schottky diode formed by the oligonucleotide. Such electronic parameters include, but not limited to turn-on voltage, ideality factor, barrier height, series resistance, shunt resistance, knee-voltage and breakdown voltage. Based on the electronic properties measured and obtained from the amino acid, the device is able to identify the oligonucleotide of the sample by referencing to a database having a list of known oligonucleotides and its respective electronic properties.
  • the device (100) comprises of a first electrode (110) and a second electrode (120) disposed on a substrate (130), and a source measure unit (140).
  • the first electrode (110) and the second electrode (120) are two metal layers fabricated side-by-side on an FR4 substrate, wherein the two metal layers are of the same material suitably gold or aluminium.
  • the first electrode (110) is connected to a positive terminal of the source measure unit while the second electrode (120) is connected to a negative terminal of the source measure unit (140).
  • the source measure unit (140) is configured to supply and measure voltage and current simultaneously in order to obtain the electronic properties of the Schottky diode formed by the oligonucleotide. It would be apparent by a person skilled in the art that the source measure unit (140) may be substituted with an arrangement of multiple set of equipment for supplying and measuring voltage and current independently.
  • a method for profiling and identifying a sample of an oligonucleotide is provided hereinbelow. Initially, the sample of the oligonucleotide is deposited onto the first (110) and second (120) electrodes fabricated on the substrate (130), wherein the amount of the sample should be sufficient to cover over both electrodes (110, 120).
  • the source measure unit (140) induces a voltage signal over its terminals which consequently causes an oligonucleotide-metal junction to be formed by the electrodes (110, 120) and the sample, wherein the oligonucleotide-metal junction constitutes a Schottky diode. While the voltage signal is applied, the source measure unit (140) obtains the electronic properties of the Schottky diode formed by the electrodes (110, 120) and the oligonucleotide.
  • the electronic properties are obtained from current-voltage characteristics of the Schottky diode in a forward bias mode and a reverse bias mode, wherein the electronic properties in the forward bias mode include the measurements of turn-on voltage, series resistance, shunt resistance, ideality factor and barrier height, while the electronic properties in the reverse bias mode include measurements of knee-voltage, breakdown voltage and breakdown current.
  • the voltage signal supplied in the forward bias mode is in a range of OV to 3V and the voltage signal supplied in the reverse bias mode is in a range of OV to -3V.
  • the electronic properties obtained are considered as an electronic profile of the sample of the oligonucleotide.
  • the electronic profile of the sample is then compared with a database having a list of known oligonucleotides and its respective electronic profiles. If the electronic profile of the sample matches one of the electronic profiles in the database, the sample is identified as the particular oligonucleotide that relates to the matched electronic profile in the database.
  • a printed circuit board or PCB as shown in FIG. 1 was fabricated.
  • the PCB was a single-sided FR4 1.6 mm designed with two electrodes and two connecting pads, wherein each electrode was connected to one of the connecting pads by a copper track with a thickness of approximately 36 pm.
  • the electrodes and connecting pads were with electroplated nickel-gold plates having a nickel thickness layer of approximately 4 to 5 pm and a gold thickness layer of approximately 0.049 to 0.052 pm.
  • the copper tracks were covered by epoxy solder mask to allow only the electrodes and the connecting pads being exposed.
  • the PCB was pre-treated by immersing in acetone for 60 s, rinsing using deionized water, immersing in isopropanol for 60 s, rinsing using deionized water, and drying using Nitrogen gas.
  • the PCB was connected to a source measure unit via the connecting pads.
  • the source measure unit was used to supply a voltage signal and measure a current signal produced over the electrodes.
  • oligonucleotide sequences in lyophilized form were solubilized in Tris-EDTA or TE buffer, aliquoted and stored at -20°C.
  • the aliquots of stock solutions were diluted in DNase-free sterile ultrapure water to a required concentration. The concentration and the integrity of stock solutions were evaluated by measuring the absorbance of DNA at 260 nm using a Nanodrop system. All samples were diluted again to a concentration of 50 ng/pL.
  • FIG. 2a shows a graph of the measured current against the voltage supplied within a range of 0 to 2 V.
  • the graph shows a clear differentiation between purines, Poly A and Poly G, and pyrimidines, Poly C. and Poly T. This is due to its structure which was evidently exhibited by the electronic properties of the samples.
  • the measured current against the voltage supplied was further scrutinized within a voltage range of 0 to 1 .5 V for all samples as shown in FIG. 2b. Based on the graph of FIG. 2b, a characteristic “hump” voltage value was found distinctively for each sample.
  • the characteristic “hump” voltage values were 0.65 V, 0.6 V, 0.5 V and 0.72 V for Poly A, Poly T, Poly G and Poly C, respectively
  • a graph of resistance against the voltage supplied for all samples was plotted as shown in FIG. 3. Based on the graph of FIG. 3, a characteristic shunt resistance which is the highest resistance shown by each sample was extracted. The values of the characteristic shunt resistance were distinctive for different oligonucleotide sequences. The shunt resistance values were 40.22 MW, 63.50 MW, 61.06 MW and 52.34 MW for Poly A, Poly T, Poly G and Poly C, respectively.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • General Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un dispositif (100) destiné à profiler et identifier un échantillon d'un oligonucléotide. Le dispositif (100) profile l'oligonucléotide sur la base de ses propriétés électroniques, les propriétés électroniques se rapportant à des paramètres électroniques d'une diode Schottky formée par l'oligonucléotide. Le dispositif (100) comprend une première électrode (110) et une seconde électrode (120) disposées sur un substrat (130), et une unité de mesure de source (140).
PCT/MY2021/050026 2020-04-11 2021-04-12 Dispositif de profilage et d'identification d'un oligonucléotide WO2021206539A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
MYUI2020001869A MY193934A (en) 2020-04-11 2020-04-11 A device for profiling and identifying an oligonucleotide
MYUI2020001869 2020-04-11

Publications (1)

Publication Number Publication Date
WO2021206539A1 true WO2021206539A1 (fr) 2021-10-14

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WO (1) WO2021206539A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050053964A1 (en) * 2000-11-15 2005-03-10 Minerva Biotechnologies Corporation Oligonucleotide identifiers
WO2009117031A2 (fr) * 2007-12-18 2009-09-24 Advanced Analytical Technologies, Inc. Système et procédé pour le profilage de séquences nucléotidiques pour l’identification d’échantillons
WO2017189930A1 (fr) * 2016-04-27 2017-11-02 Quantum Biosystems Inc. Systèmes et procédés de mesure et de séquençage de biomolécules
US20180180567A1 (en) * 2015-06-23 2018-06-28 Bgi Shenzhen Microwell electrode and method for analysis of a chemical substance

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050053964A1 (en) * 2000-11-15 2005-03-10 Minerva Biotechnologies Corporation Oligonucleotide identifiers
WO2009117031A2 (fr) * 2007-12-18 2009-09-24 Advanced Analytical Technologies, Inc. Système et procédé pour le profilage de séquences nucléotidiques pour l’identification d’échantillons
US20180180567A1 (en) * 2015-06-23 2018-06-28 Bgi Shenzhen Microwell electrode and method for analysis of a chemical substance
WO2017189930A1 (fr) * 2016-04-27 2017-11-02 Quantum Biosystems Inc. Systèmes et procédés de mesure et de séquençage de biomolécules

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TALEBI SARA; DARAGHMA SOUHAD; SUBRAMANIAM S. RAMESH T.; BHASSU SUBHA; PERIASAMY VENGADESH: "Exploring the Electronic Properties of Ribonucleic Acids Integrated Within a Schottky-Like Junction", JOURNAL OF ELECTRONIC MATERIALS, SPRINGER US, NEW YORK, vol. 48, no. 11, 14 August 2019 (2019-08-14), New York, pages 7114 - 7122, XP036896924, ISSN: 0361-5235, DOI: 10.1007/s11664-019-07530-x *

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