WO2013058646A1 - Système permettant d'identifier la présence de polynucléotides d'intérêt dans un échantillon - Google Patents

Système permettant d'identifier la présence de polynucléotides d'intérêt dans un échantillon Download PDF

Info

Publication number
WO2013058646A1
WO2013058646A1 PCT/MY2011/000244 MY2011000244W WO2013058646A1 WO 2013058646 A1 WO2013058646 A1 WO 2013058646A1 MY 2011000244 W MY2011000244 W MY 2011000244W WO 2013058646 A1 WO2013058646 A1 WO 2013058646A1
Authority
WO
WIPO (PCT)
Prior art keywords
layer
sample
operating circuit
sensing platform
polynucleotides
Prior art date
Application number
PCT/MY2011/000244
Other languages
English (en)
Inventor
Sidek OTHMAN
Abd Manaf ASRULNIZAM
Hamiza Hamzah IRNI
Korakkotil Kunhi Mohd SHUKRI
Original Assignee
Universiti Sains Malaysia
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universiti Sains Malaysia filed Critical Universiti Sains Malaysia
Publication of WO2013058646A1 publication Critical patent/WO2013058646A1/fr

Links

Classifications

    • 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
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3276—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a hybridisation with immobilised receptors

Definitions

  • the present invention relates to a system for detecting polynucleotides.
  • the present invention relates to a system for identifying the presence of polynucleotides of interest from a sample that utilizes a sensing platform composed of electrodes fabricated of gold, nickel and copper layers.
  • Nucleotide hybridization is an important technique as it contributes greatly to a variety of fields including clinical diagnosis, food safety monitoring, forensic application and drug screening.
  • DNA and RNA are polynucleotides with different biological function. Detection and analysis of a specific nucleotide sequence is made possible through this technique where the sequence similarity among polynucleotides of different species and the amounts of sequence repetition within a polynucleotide could be determined.
  • the target polynucleotide sequence is identified by hybridization probe such as oligonucleotide probe that is single-stranded that forms a double helix structure with its complementary nucleic acid upon hybridization. The close relationships of two nucleic acids are measured base on the amount of sequence complementarity.
  • Electrochemical polynucleotide sensor utilizes a transducer to detect nucleotide immobilization and hybridization. Initially, there is a large amount of electron flows on the electrode surface. However, later during immobilization, the single-stranded nucleic acid immobilizes on an electrode surface of the sensor causing less electron flows and hence the value of current measured drops. The electron flow is further decreased during the hybridization process where helical structures of the polynucleotides are formed. Thus the current value of the hybridization process could be determined.
  • DNA electrochemical sensor is one of the widely used analytical devices that employ biological recognition properties for detecting DNA hybridization due to its high reliability in providing better detection specificity, sensitivity and low cost.
  • 2010300899 which is about an active CMOS sensor array for electrochemical biomolecular detection system that comprises an integrated circuit; at least one working electrodes on the integrated circuit, wherein the working electrodes are configured to receive one or more biomolecular probes with a desired potential maintained through one or more reference electrodes and the working electrodes are configured to form a portion of one or more corresponding potentiostats; and a digitizing circuit on the integrated circuit configured to measure a signal indicative of a biomolecule sensing operation in real time.
  • the DNA sensor used is an array which is fabricated on silicon where an analog-to-digital converter is manufactured on the silicon through CMOS process. Another patent is U.S. Patent No.
  • nucleic acid detection sensor that comprises a plurality of nucleic acid chain fixed electrodes to which a probe nucleic acid chain is fixed, and a counter electrodes which is arranged opposite to the nucleic acid chain fixed electrode, and a current following between the counter electrode and the nucleic acid chain fixed electrode.
  • the potentiostat is connected to a computer to display the measurement results.
  • the main aspect of the present invention is to provide a system for identifying presence of polynucleotides of interest from a sample through electrical response generated during the absence of nucleotide, nucleotide immobilization and nucleotide hybridization.
  • Another aspect of the present invention is to provide a portable system for identifying presence of polynucleotides of interest from a sample.
  • Still another aspect of the present invention is to provide a system for identifying presence of polynucleotides of interest from a sample that is easy to fabricate and set up.
  • Yet another aspect of the present invention is to provide a system for identifying presence of polynucleotides of interest from a sample with high efficiency, sensitivity, specificity and economical in terms of sensor fabrication and readout circuitry development.
  • the embodiment of the present invention describes a system for identifying presence of polynucleotides of interest from a sample comprising a sensing platform having a working electrode (102), a reference electrode (103) and a counter electrode (104) which are electrically connected to a connection port (101) and that the electrodes (102, 103, 104) have a top and a bottom layer respectively made of gold (111) and nickel (112), wherein surface of each layer of gold (111) is anchored with one or more oligonucleotide probes to readily hybridize with a substantially complementary polynucleotide in the sample; an operating circuit (105) provided with a receptive port to adaptably communicate with the sensing platform by mating with the connection port (101) and configured to measure a signal indicative of hybridization of the sensing platform; a display (108) connected to the operating circuit to acquire and show electrical readings of the operating circuit (105); wherein presence of polynucle
  • (112) is in between the layer of gold (111) and a base layer (109) made of copper
  • Figure 1 shows a schematic diagram of the system for identifying presence of polynucleotides of interest from a sample.
  • Figure 2 shows a cross section view of the electrodes.
  • Figure 3 shows a diagram of the operating circuit and the sensing platform.
  • the present invention discloses a system for identifying presence of polynucleotides of interest from a sample comprising a sensing platform having a working electrode (102), a reference electrode (103) and a counter electrode (104) which are electrically connected to a connection port (101) and that the electrodes (102, 103, 104) have a top and a bottom layer respectively made of gold (111) and nickel (112), wherein surface of each layer of gold (111) is anchored with one or more oligonucleotide probes to readily hybridize with a substantially complementary polynucleotide in the sample; an operating circuit (105) provided with a receptive port to adaptably communicate with the sensing platform by mating with the connection port (101) and configured to measure a signal indicative of hybridization of the sensing platform; a display (108) connected to the operating circuit to acquire and show electrical readings of the operating circuit (105); wherein presence of polynucleotides of interest in the sample is identified by detecting the signal upon hybridization onto the probes; characterized
  • Detection of polynucleotide by the present invention for analyzing its gene sequence is based on nucleotide hybridization which could be determined through measuring electrical response such as current generated during the hybridization process.
  • the sample Prior to the detection and identification of the polynucleotides through hybridization, the sample is pretreated to denature the double-stranded polynucleotides to single- stranded polynucleotides. The denaturation process unwinds and separates the double- stranded polynucleotides into single-stranded strands through breaking the hydrogen bonds between the bases. This could be achieved by heating the double-stranded polynucleotides.
  • the single-stranded polynucleotide is left to cool down while oligonucleotide probes are prepared by anchoring one or more immobilized oligonucleotide probes onto the gold layer (111) of the electrodes (102, 103, 104). A drop of the sample is applied to the oligonucleotide probes for hybridization to occur with a coverslip covering the mixture of the sample and the probes. If the single-stranded polynucleotides are complementary with the oligonucleotide probes, hybridization occurs.
  • Post hybridization washing procedures include removing the coverslip and washing the surface of these electrodes (102, 103 and 104) where the hybridized polynucleotides are anchored on with buffered saline solutions. All these electrodes fabricated on the sensing platform are detachable so that it can be removed from the sensing platform while being washed.
  • each electrode (102, 103, 104) After drying the washed surface of these electrodes (102, 103 and 104), a drop of redox mediator such as ferricyanide/ferrocyanide, ferrocene or methylene blue is applied onto the electrodes (102, 103, 104). The system is then turned on to measure the current generated during the hybridization process.
  • the sensing platform containing the three electrodes (102, 103, 104) serves as a sensor for detecting and identifying the polynucleotides of interest in the sample. Referring to Figure 2, the cross section view of each electrode (102, 103, 104) shows that the materials used in the electrode (102, 103, 104).
  • each electrode (102, 103, 104) which is the outer layer surface is a gold layer (111) where the oligonucleotide probes are anchored on.
  • Gold is selected as the outer layer surface of the electrode due to it being the most stabilized metal for the thiolated-attached of nucleotides during immobilization as proven in previous research.
  • the thickness of the gold layer (111) could be approximately 200 to 400nm, preferably 300nm.
  • Below the top layer (111) is a thin layer of nickel which acts as a barrier metal to avoid the tendency of copper (113) atoms from the base layer (109) to diffuse through the gold (111) atoms on the top surface causing tarnishing of its surface and formation of an oxide or sulfide layer.
  • the gold (111) layer can be prevented from peeling off from the copper (113) layer.
  • the nickel layer (112) could be as thin as 5 to 9 ⁇ , preferably 7 ⁇ .
  • a layer of copper (113) could be added as an extra layer below the bottom layer which is nickel
  • the gold layer (111) having the hybridized polynucleotide anchored on its surface works as a transducer responding to the excitation signal generated when oxidation and reduction reactions occur on the surface of the gold layer (111), allowing flow of electrical current.
  • the reference electrode (103) serves as a reference to measure the electrical potential of the working electrode (102) whereby a desired potential is maintained by the reference electrode (103).
  • the counter electrode serves as a reference to measure the electrical potential of the working electrode (102) whereby a desired potential is maintained by the reference electrode (103).
  • connection port (101) which is preferably a USB port.
  • the presence of the polynucleotides of interest from the sample is identified by detecting the signal which is the reduced current flow in the working electrode (102) in relative to the reference electrode (103). This could be done by the operating circuit
  • the receptive port of the operating circuit connects to the connection port (101) to communicate with the sensing platform.
  • the first operational amplifier (106a) serves to compare the potential between the working electrode (102) and the reference electrode (103). Difference of the potential is produced at the counter electrode (104).
  • the second operational amplifier (106b) acts as a converter to measure the current between the working electrode (102) and the counter electrode (104).
  • the measurement is then shown on the display (108) which could be a multimeter for the user to read.
  • the redox mediators enable redox reactions which are reduction and oxidation to occur, resulting in the movement of electrons that produces current.
  • the current preferably in micro ampere ( ⁇ ) could then be measured.
  • ⁇ micro ampere
  • the measured current value corresponds to a known predetermined current value resulted from a hybridization process using the same system, it can be confirmed that hybridization has occurred and the polynucleotide of interest can be identified. However, if hybridization fails to occur, the measured current value should correspond to a known predetermined current value determined from an immobilization process using the same system, which is a value higher than the current value obtained from the hybridization process.
  • the sensing platform and operating circuit are fabricated on a FR 4-based printed circuit board as the base layer (109) as indicated in Figure 1.
  • the base layer is preferred to be fabricated of copper. This is because the FR 4-based printed circuit board made of copper possess good mechanical strength property and is easy to cut and fabricate.
  • One of the main features of the present invention, besides enabling effective detection and identification of polynucleotides is its portability. It is preferred that the system is portable to provide convenience to the user.
  • An electrical power source (110) can be fabricated on the FR-4 board (109) to provide power to the system.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un système d'identification de la présence de polynucléotides d'intérêt dans un échantillon. Le système de l'invention comprend : une plate-forme de détection dotée d'une électrode de travail (102), d'une électrode de référence (103) et d'une contre-électrode (104) qui sont électriquement connectées à un port de connexion (101), les électrodes (102, 103, 104) comportant une couche supérieure et une couche inférieure composées respectivement d'or (111) et de nickel (112), une ou plusieurs sondes oligonucléotidiques étant ancrées dans la surface de chaque couche d'or (111) pour permettre une hybridation avec un polynucléotide sensiblement complémentaire se trouvant dans l'échantillon ; un circuit d'exploitation (105) doté d'un port de réception pour communiquer de manière adaptable avec la plate-forme de détection par accouplement au port de connexion (101) et conçu pour mesurer un signal indiquant l'hybridation de la plate-forme de détection ; et un écran d'affichage (108) connecté au circuit d'exploitation pour acquérir et présenter les lectures électriques du circuit d'exploitation (105). La présence de polynucléotides d'intérêt dans l'échantillon est identifiée par la détection du signal généré lors d'une hybridation sur les sondes. Le système de l'invention est caractérisé en ce que la couche de nickel (112) se trouve entre la couche d'or (111) et une couche de base (109) composée de cuivre (113) sur laquelle la plate-forme de détection et le circuit d'exploitation sont fabriqués.
PCT/MY2011/000244 2011-10-19 2011-12-15 Système permettant d'identifier la présence de polynucléotides d'intérêt dans un échantillon WO2013058646A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
MYPI2011700155 2011-10-19
MYPI2011700155A MY173426A (en) 2011-10-19 2011-10-19 A system for identifying presence of polynucleotides of interest from a sample

Publications (1)

Publication Number Publication Date
WO2013058646A1 true WO2013058646A1 (fr) 2013-04-25

Family

ID=48141152

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/MY2011/000244 WO2013058646A1 (fr) 2011-10-19 2011-12-15 Système permettant d'identifier la présence de polynucléotides d'intérêt dans un échantillon

Country Status (2)

Country Link
MY (1) MY173426A (fr)
WO (1) WO2013058646A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001083674A1 (fr) * 2000-05-03 2001-11-08 Gau Jen Jr Systeme d'identification biologique dote d'une puce de detection integree
US20050003399A1 (en) * 1998-06-23 2005-01-06 Gary Blackburn Binding acceleration techniques for the detection of analytes
US20080087554A1 (en) * 2006-05-24 2008-04-17 Antara Biosciences Inc. Electrochemical detection device
EP2180315A1 (fr) * 2007-08-01 2010-04-28 Nipro Corporation Trousse de mesure de stress et procédé de mesure de stress

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050003399A1 (en) * 1998-06-23 2005-01-06 Gary Blackburn Binding acceleration techniques for the detection of analytes
WO2001083674A1 (fr) * 2000-05-03 2001-11-08 Gau Jen Jr Systeme d'identification biologique dote d'une puce de detection integree
US20080087554A1 (en) * 2006-05-24 2008-04-17 Antara Biosciences Inc. Electrochemical detection device
EP2180315A1 (fr) * 2007-08-01 2010-04-28 Nipro Corporation Trousse de mesure de stress et procédé de mesure de stress

Also Published As

Publication number Publication date
MY173426A (en) 2020-01-23

Similar Documents

Publication Publication Date Title
CA2858036C (fr) Transducteurs a nano-interstice a electrode au diamant
JP6030618B2 (ja) 検体の検出
Xu et al. An ultrasensitive electrochemical impedance sensor for a special BRCA1 breast cancer gene sequence based on lambda exonuclease assisted target recycling amplification
CN103597094B (zh) 利用电活性水解探针（e-tag探针）监测实时聚合酶链式反应（pcr）的方法和装置
US9804120B2 (en) Systems and methods for multiplexed electrochemical detection
US20170211136A1 (en) Sensing strategies and methods for nucleic acid detection using biosensors
JP2009524046A (ja) バイオセンサセル及びバイオセンサアレイ
WO2013100949A1 (fr) Transducteurs à nano-intervalle comportant des sites d'immobilisation superficielle sélective
Mohammadi et al. Spectrophotometric and electrochemical determination of MicroRNA-155 using sandwich hybridization magnetic beads
CN112378971A (zh) 一种CRISPR/Cas13a驱动的催化可再生电化学生物传感器及其应用
Zari et al. Label-free DNA biosensor for electrochemical detection of short DNA sequences related to human papilloma virus
Erdem et al. ZNA probe immobilized single-use electrodes for impedimetric detection of nucleic acid hybridization related to single nucleotide mutation
Zambry et al. A label-free electrochemical DNA biosensor used a printed circuit board gold electrode (PCBGE) to detect SARS-CoV-2 without amplification
CN108445067A (zh) 一种双信号无酶的信号放大rna纳米生物传感器、制备方法及其应用
Choi et al. Hybridization by an electrical force and electrochemical genome detection using an indicator-free DNA on a microelectrode-array DNA chip
JP2012501174A (ja) インピーダンス分光法を用いたｄｎａの測定
WO2013058646A1 (fr) Système permettant d'identifier la présence de polynucléotides d'intérêt dans un échantillon
EP1964933A1 (fr) Capteur électrochimique, kit comprenant ledit capteur et procédé pour sa fabrication
Tabata et al. Label-free Nucleic Acid Amplification Detection using Electrochemical Sensors for Liquid Biopsy
KR100789651B1 (ko) 일회용 바이오센서
JP3887212B2 (ja) 塩基配列検出用チップ、ハイブリダイゼーションモニタリングシステム、ハイブリダイゼーションモニタリング方法
KR20070031957A (ko) 나노크기 전자 검출 시스템 및 그의 제조 방법
Dou et al. Target-induced reconfiguration of DNA probes for recycling amplification and signal-on electrochemical detection of hereditary tyrosinemia type I gene
US10983088B2 (en) Coulometric microfluidic sensors using a silver band electrode, and methods thereof
KR101941771B1 (ko) 반도체 2d 결정 물질을 이용한 dna의 전기화학적 검출방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11874436

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 04.09.2014)

122 Ep: pct application non-entry in european phase

Ref document number: 11874436

Country of ref document: EP

Kind code of ref document: A1