WO2013058646A1

WO2013058646A1 - A system for identifying presence of polynucleotides of interest from a sample

Info

Publication number: WO2013058646A1
Application number: PCT/MY2011/000244
Authority: WO
Inventors: Sidek OTHMAN; Abd Manaf ASRULNIZAM; Hamiza Hamzah IRNI; Korakkotil Kunhi Mohd SHUKRI
Original assignee: Universiti Sains Malaysia
Priority date: 2011-10-19
Filing date: 2011-12-15
Publication date: 2013-04-25
Also published as: MY173426A

Abstract

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 in that the layer of nickel (112) is in between the layer of gold (111) and a base layer (109) made of copper (113) where the sensing platform and operating circuit are fabricated on.

Description

A SYSTEM FOR IDENTIFYING PRESENCE OF POLYNUCLEOTIDES OF

INTEREST FROM A SAMPLE

FIELD OF INVENTION

The present invention relates to a system for detecting polynucleotides. In more particular, 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.

BACKGROUND OF THE INVENTION

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.

While nucleotide hybridization could be detected via a few ways such as optical, acoustic and piezoelectric, 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. One example of a related prior art is U.S. Patent No. 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. 7491311 which discloses a 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. In this patent, the potentiostat is connected to a computer to display the measurement results.

With the use of arrays as the biosensor and having the sensor system to be connected to a computer for analysis and exhibiting the measurement results, the aforementioned prior art lacks portability which causes inconvenience to users. There is a need to develop a system for identifying presence of polynucleotides of interest from a sample that addresses this problem which eliminates the use of CPU and monitor screen or other bulky devices. Furthermore, it is also advantageous to produce a system for identifying presence of polynucleotides with high efficiency, sensitivity and specificity, yet economical in terms of sensor fabrication and readout circuitry development.

SUMMARY OF INVENTION 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.

At least one of the preceding aspects is met, in whole or in part, by the present invention, in which 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 polynucleotides of interest in the sample is identified by detecting the signal upon hybridization onto the probes; characterized in that the layer of nickel

(112) is in between the layer of gold (111) and a base layer (109) made of copper

(113) where the sensing platform and operating circuit are fabricated on.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

DETAILED DESCRIPTION OF THE INVENTION

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 in that the layer of nickel (112) is in between the layer of gold (111) and a base layer (109) made of copper (113) where the sensing platform and operating circuit are fabricated on.

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. 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. After the denaturation process, 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.

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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). The top surface of 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. With this nickel (112) layer sandwiched between the gold (111) and copper (113) 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 9μη , preferably 7μηι. A layer of copper (113) could be added as an extra layer below the bottom layer which is nickel

(112) . However, this copper (113) layer is not necessary as the base layer (109) where the sensing platform having the electrodes are fabricated on is already made of copper

(113) . 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). On the other hand, the counter electrode

(104) contributes a driving current resulted from the oxidation and reduction reactions. The electrodes (102, 103, 104) are electrically connected to the 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

(105) that comprises two operational amplifiers (106a, 106b) and at least one resistor (107). An example of the operating circuit can be referred to Figure 3. The receptive port of the operating circuit connects to the connection port (101) to communicate with the sensing platform. Based on the cyclic voltammetry measurement method, 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 (μΑ) could then be measured. In an immobilization situation where there are single-stranded nucleotides such as oligonucleotides probes, the movement of electrons is interrupted by the immobilization of these oligonucleotides probes. The lesser electron flows, the lesser the value of current generated. However, as hybridized polynucleotides form barriers on the gold layer (111) surface, there are lesser electron flows on the gold layer (111) surface as compared to the immobilization situation, leading to an even lower current flow generated. Hence, if there is a reduced current flow in the working electrode (102) in relative to the reference electrode (103) which is the signal generated by the operating circuit (105), the occurrence of hybridization could be confirmed, indicating that the immobilized oligonucleotide probes have been hybridized and therefore the presence of the polynucleotides of interest from the sample could be identified. The current value measured should correspond to the predetermined current values obtained from experiments. To further explain, if 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.

In a preferred embodiment, 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. As mentioned above, 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.

Although the description above contains many specifications, it is understood that the embodiments of the preferred form are not to be regarded as a departure from the invention and it may be modified within the scope of the appended claims.

Claims

2. A system according to claim 1 , wherein the sample is pretreated prior to hybridization.

3. A system according to claim 1 , wherein the connection port ( 101 ) is a USB port.

4. A system according to claim 1, wherein the signal is reduced current flow in the working electrode (102) in relative to the reference electrode (103) during immobilization and hybridization.

5. A system according to claim 1, wherein the sensing platform and operating circuit (105) are fabricated on a FR 4-based printed circuit board as the base material (109).

6. A system according to claim 1, wherein the display (108) is a multimeter.

7. An apparatus according to claim 1 , wherein the system is portable.

8. An apparatus according to claim 1, wherein the system further comprising an electrical power source (110).