WO2021206539A1

WO2021206539A1 - A device for profiling and identifying an oligonucleotide

Info

Publication number: WO2021206539A1
Application number: PCT/MY2021/050026
Authority: WO
Inventors: Vengadesh A/L PERIASAMY; Souhad M.A DARAGHMA
Original assignee: Universiti Malaya
Current assignee: Universiti Malaya
Priority date: 2020-04-11
Filing date: 2021-04-12
Publication date: 2021-10-14
Anticipated expiration: 2022-10-11
Also published as: MY193934A

Abstract

The present invention relates to 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. 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).

Description

A DEVICE FOR PROFILING AND IDENTIFYING AN OLIGONUCLEOTIDE

FIELD OF INVENTION

The present invention relates to a device for profiling and identifying oligonucleotide.

BACKGROUND OF THE INVENTION

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.

Conventionally, 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.

These methods used often face with difficulties when the starting template is limited in quantity and quality leading to inaccuracy. Besides, the process is time- consuming. Hence, it would be advantageous to develop a technology that leverages on the properties of oligonucleotides that could extend beyond or overcome conventional nucleic acid detection methods limitations which also allows to minimize the amount of sample and profiling time use.

SUMMARY OF INVENTION

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.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

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.

DESCRIPTION OF THE PREFERRED EMBODIMENT

A preferred embodiment of the present invention will be described hereinbelow with reference to the accompanying drawings. In the following description, well-known functions or constructions are not described in detail since they would obscure the description with unnecessary detail.

Referring to FIG. 1, there is shown 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). Preferably, 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).

Thereon, 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. Preferably, 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.

The following examples are given to demonstrate in-depth of the device (100) and method thereof for identifying and profiling a sample of an oligonucleotide. The examples used herein are intended merely to facilitate an understanding of ways in which embodiments herein may be practised and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be considered as limiting the scope of the embodiment herein.

Examples of Experiments are illustrated as follow:

Fabrication of the electrodes

A printed circuit board or PCB as shown in FIG. 1 was fabricated. In particular, 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.

Experimental setup

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.

Profiling of Oligonucleotide Specimen

For preparing stock solutions, oligonucleotide sequences in lyophilized form were solubilized in Tris-EDTA or TE buffer, aliquoted and stored at -20°C. For preparing samples, 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. There were four samples prepared which are a sequence of a single nucleotide adenine (Poly A), thymine (Poly T), guanine (Poly G) and cytosine (Poly C), wherein each sample has a length of 20 nucleotides.

10 pL of one of the samples was deposited onto the electrodes using a micropipette and incubated for 5 min to allow self-assembly and stabilization of the molecules. Thereon, measurements were carried out at a room temperature of 23°C and humidity of 70% RH by applying a voltage of 0 V to 3 V and measuring the corresponding current. For each sample, experiments were iterated for multiple times to show high reproducibility of the resulted data. The data was then analysed and l-V profiles were plotted accordingly.

Results and Discussion

Based on the measurements obtained, each sample of the oligonucleotide sequences reveals distinctive electronic profiles based on its natural characteristics. 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

In addition to that, 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.

While embodiments of the invention have been illustrated and described, it is not intended that these embodiments illustrated and described all possible forms of the invention. Rather, the words used in the specifications are words of description rather than limitation and various changes may be made without departing from the scope of the invention.

Claims

CLAIM

1. A device (100) for profiling and identifying a sample of an oligonucleotide comprising a first electrode (110) and a second electrode (120) disposed on a substrate (130), characterised in that: a) a source measure unit (140) is connected to the first electrode (110) and the second electrode (120), wherein 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.