FIELD OF THE INVENTION

The present disclosure generally relates to highly sensitive electrochemical biosensors based on organic electrochemical transistors (OECTs), and methods for the use thereof.

BACKGROUND OF THE INVENTION

Present technology for the analysis of biomarkers is based on molecular biology methods, including western blotting, gel electrophoresis, mass spectrometry, enzyme-linked immunosorbent assays and reverse transcription polymerase chain reaction (RT-PCR), all of which are normally time-consuming and laborious.

For example, western blotting is a general method to specifically detect the existence of a single protein in a mixture. A semi-quantitative estimation of a protein can be derived from the size and color intensity of a protein band on the blot membrane. Protein gel electrophoresis is a method for the separation and analysis of the proteins in a fluid or an extract. Its selectivity is based on the different electrophoretic mobilities of proteins, which are caused by the length and charge of different polypeptide chains. The two aforementioned methods both have disadvantages including: the length of time required for the procedures, low selectivity and low sensitivity. These methods are not suitable for early disease detection since the biomarker concentration change is very small.

Mass spectrometry is an analytical technique that ionizes chemical species and sorts the ions based on their mass-to-charge ratio. In a typical mass spectrometry procedure, the sample is ionized first and these ions are then separated according to their mass-to-charge ratio. The ions are detected by a mechanism capable of detecting charged particles, such as an electron multiplier. Results are displayed as spectra of the relative abundance of detected ions as a function of the mass-to-charge ratio. The atoms or molecules in the sample can be identified through a fragmentation pattern. Mass spectrometry requires complex and expensive instruments and experienced researchers to operate the instruments and to analyze the results. It also involves a devastating sample preparation process and therefore it is not suitable for the analysis of living cells.

An enzyme-linked immunosorbent assay is a test that uses antibodies and color change to identify a substance. It requires a complex detection process with limited sensitivity, which affects its suitability for clinical application.

RT-PCR is a technique commonly used to qualitatively detect gene expression in molecular biology. In RT-PCR, the RNA samples react with reverse transcriptase to produce complementary DNA (cDNA). Exponential amplification cycles are then conducted to amplify cDNA fragments so that they can be identified by available substrates. The process is very complex and has low selectivity.

There is therefore a need to develop a highly sensitive, low-cost, and simple method/device to detect specific analytes within a sample.

SUMMARY OF THE INVENTION

The present invention meets at least one of the needs mentioned above by providing devices and methods for the highly sensitive detection of analytes within a sample.

In a first aspect, the present invention provides an electrochemical biosensor comprising a plurality of electrodes, comprising:

a gate electrode comprising a first agent capable of specifically binding to an analyte;

a source electrode;

a drain electrode;

a channel comprising an organic semiconductor between the source electrode and the drain electrode;

a plurality of nanoparticles comprising an electrochemically active enzyme and a second agent capable of specifically binding to an analyte in a sample; and

a substrate for the electrochemically active enzyme,

wherein the gate electrode and the channel are separated by an electrolyte, and wherein the electrolyte contacts the gate electrode and the channel.

In some embodiments of the first aspect, the first agent and/or the second agent is an antibody.

In additional embodiments, the first agent is a monoclonal antibody capable of specifically binding to the analyte.

In further embodiments, the first agent is a nucleic acid probe which is capable of specifically binding to the analyte.

In some embodiments, the second agent is a polyclonal antibody capable of specifically binding to the analyte.

In additional embodiments, the electrochemically active enzyme is horseradish peroxidase and the substrate for the electrochemically active enzyme is hydrogen peroxide.

In some embodiments, the organic semiconductor comprises a film coating on the drain and source electrodes.

In further embodiments of the present invention, the organic semiconductor comprises poly (3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS).

In some embodiments, at least one of the plurality of electrodes and/or the plurality of nanoparticles further comprises gold.

In additional embodiments, the gate electrode comprises grooves comprising the first agent capable of specifically binding to an analyte.

In some embodiments, the nanoparticles have a diameter of under 90 nm, under 80 nm, under 70 nm, under 60 nm, under 50 nm, under 40 nm, under 30 nm, under 20 nm, under 10 nm, or under 1 nm.

In further embodiments, the channel has a width of 5 mm and a length of 0.1 mm.

In additional embodiments, the gate electrode has a surface area that is under 100 times, under 90 times, under 80 times, under 70 times, under 60 times, under 50 times, under 40 times, under 30 times, under 20 times, under 10 times, under 5 times or under 2 times the surface area of the channel.

In some embodiments, the sample is a cell lysate.

In further embodiments, the sample is whole cells.

In additional embodiments, the analyte is protein.

In some other embodiments, the analyte is nucleic acid.

In some embodiments, the analyte is microRNA (miRNA).

In a second aspect, the present invention also provides a method for detecting an analyte in a sample, the method comprising contacting the sample in the electrochemical biosensor according to the first aspect of the present invention to produce a measurable signal.

In some embodiments, the detecting further comprises contacting a control sample in the electrochemical biosensor according to the first aspect of the present invention to produce a measurable signal and comparing the measurable signal of the sample with the measurable signal of the control sample.

In some additional embodiments, the analyte is a biomarker and the detecting comprises the diagnosis and/or prognosis of a disease and/or medical condition.

In some further embodiments, the disease and/or medical condition is cancer.

In some embodiments, the cancer is breast cancer.

In additional embodiments, the biomarker is human epidermal growth factor receptor 2 (HER2).

DEFINITIONS

Certain terms are used herein which shall have the meanings set forth as follows.

As used in this application, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “nanoparticle” also includes a plurality of nanoparticles unless otherwise stated.

As used herein, the term “comprising” means “including”. Variations of the word “comprising”, such as “comprise” and “comprises” have correspondingly varied meanings. Thus, for example, a nanoparticle “comprising” may consist exclusively of or may include one or more additional components.

As used herein, the term “plurality” means more than one. In certain specific aspects or embodiments, a plurality may mean 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or more, and any numerical value derivable therein, and any range derivable therein.

As used herein, the term “between” when used in reference to a range of numerical values encompasses the numerical values at each endpoint of the range.

As used herein, the term “gate electrode”, also known in the field as the reference electrode, refers to the electrode that controls the flow of electrical current between the source and drain electrodes.

As used herein, the terms “source electrode” and “drain electrode” refer to the electrodes that transmit and receive the electrical current respectively across the organic semiconductor.

