TW200827714A

TW200827714A - Electrochemical sensing of biomolecules using synthetic polymer probes

Info

Publication number: TW200827714A
Application number: TW95148085A
Authority: TW
Inventors: Tse-Chuan Chou; Rick John Frank; xin-yi Lin; Pei-Zhen Zhou
Original assignee: Tse-Chuan Chou
Priority date: 2006-12-20
Filing date: 2006-12-20
Publication date: 2008-07-01

Abstract

A method for the detection and quantification of bio-molecules such as proteins, which employs electrodeposited, micro-contact printed, or spin-coated polymers as sensing materials in cyclic volumetric or amperometric protocols, is given. The sensing materials can be composed of: aminophenylboronic acid, or a composition comprising functional monomers and crosslinkers such as polyethylene glycol 400 and methacrylic acid, formulated as thin films on glass or metallic supports. The sensing of proteins is achieved by enzyme linked immunosorbent assay (ELIZA), amperometry, or by analysis of the cyclic voltammetric profile of the bound proteins in the solvent media of the cyclic voltammetric test cell when subject to a varying potential. In the case of ELIZA detection the extent of protein adhesion to the materials fabricated for their sensing is measured by a colourimetric procedure, whereby the intensity of a coloured species produced is proportional to the amount of adhering protein. In the case of the amperometric and cyclic voltammetric determinations the measurement of protein adhesion is determined as a function of the passage of electrical current through the solution in the cyclic voltammetry cell.

Description

200827714 九、發明說明：【發明所屬之技術領域】生物材料、電化學、生物感測、分析化學【先前技術】電化學感測、酵素免疫法。【發明内容】200827714 IX. Description of the invention: [Technical field of invention] Biomaterials, electrochemistry, biosensing, analytical chemistry [Prior Art] Electrochemical sensing, enzyme immunoassay. [Summary of the Invention]

An original method for detecting the re-binding of proteins to molecularly imprinted polymer films, in an electrochemical cell, by cyclic voltammetry or amperometry is provided. The electrochemical cell contains a working electrode with an imprinted polymer film forming its outer layer, a counter electrode (which can be made of platinum) and a reference electrode such as a Ag/AgCl couple. The workingAn original method for detecting the re-binding of proteins to molecularly imprinted polymer films, in an electrochemical cell, by cyclic voltammetry or amperometry is provided. The electrochemical cell contains a working electrode with an imprinted polymer film forming its outer layer, a counter electrode (which can be made of platinum) and a reference electrode such as a Ag/AgCl couple.

electrolyte is a buffer, which may be phosphate buffer or lOxphosphate buffered saline solution with the pH adjusted to generate a positive charge, or negative charge, on the protein* A potential is applied to the working electrode, which may be a continuously varying potential that is cycled between certain predetermined values thereby facilitating a current flow, which is dependent on the attraction of the proteins for the working electrode at a given potential. Or the potential may be 7 200827714 a fixed potential at a voltage that is relatively negative to the positive charge on the proteins, or alternatively the potential may be a relatively positive charge to the negative charge on the analyte proteins, such that the proteins are attracted to the working electrode, thereby causing a measurable change in the passage of current.The electrolyte is a buffer, which may be phosphate buffer or lOxphosphate buffered saline solution with the pH adjusted to generate a positive charge, or negative charge, on the protein* A potential is applied to the working electrode, which may be a continuously varying potential that Is cycled between certain predetermined values of facilitating a current flow, which is dependent on the attraction of the proteins for the working electrode at a given potential. Or the potential may be 7 200827714 a fixed potential at a voltage that is relatively negative to the positive Charge on the proteins, or alternatively the potential may be a relatively positive charge to the negative charge on the working proteins, such that the proteins are attracted to the working electrode, thus causing a measurable change in the passage of current.

The above may be illustrated with reference to lysozyme. In a pH 5.5 solution of lOxphosphate buffered saline solution lysozyme will carry a positive charge, therefore the application of a relatively or formally negative charge will induce the migration of said proteins to the working (cathodic) electrode. Lysozyme is an inherent non-conductor of electricity. Therefore as the concentration of protein in the buffer solution is increased the conductivity of the electrode will decrease as a function of the bound protein (Fig. 4). In the illustrated example the sensing material is amioophenylboronic acid. Other sensing polymers, able to form selective binding structures with proteins of interest, may be used as the working electrode’s surface coating. Such coating may optionally be formed on another polymer, acting as a support, for example polypyrrole· 8 200827714单词 lysozyme will carry a positive charge, therefore the application of a relatively or formally negative charge will induce the migration of said proteins to the working (cathodic) The singularity of the electrolyte in the buffer solution is increased the conductivity of the electrode will decrease as a function of the bound protein (Fig. 4). In the illustrated example the sensing Other sensing polymers, able to form selective binding structures with proteins of interest, may be used as the working electrode's surface coating. Such coating may optionally be formed on another polymer, acting as a support, for example polypyrrole· 8 200827714

The shape of such electrodes may be flat or rod-shaped while the detection apparatus may be operated at temperatures ranging from 0° to 40° C，or higher，assuming that the bio-molecules such as protein that is being detected is stable at the working temperature within this range.The shape of such electrodes may be flat or rod-shaped while the detection apparatus may be operated at temperatures ranging from 0° to 40° C, or higher, assuming that the bio-molecules such as protein that is being detected is stable at the Working temperature within this range.

Although the present invention and its advantages have been described in detail above, those skilled in the art should understand that they can make various changes，substitutions and alterations herein without departing from the spirit and scope of the invention as defined by the appended claims· 【實施方式】此專利主張發展一套結合電化學、微流體學及分子模版技術應用在生物分子的感測上，同時可利用此技術，發展在各種尺寸的感測器上並可整合高科技1C設計。【圖式簡單說明】 FIG. 1 shows the electrical response of an imprinted polymer electrode (made with myoglobin as the template to direct polymer formation) and a non-imprinted electrode having the same polymer coating. FIG. 2 shows the overall schematic arrangement of the protein detection cell and ancillary equipment. 9 200827714 FIG 3 shows a schematic diagram of the microcontact imprinting process leading to the formation of imprinted sites in a polymer matrix. FIG. 4 shows the selectivity of the micro-contact imprinting method applied to the myoglobin.Although the present invention and its advantages have been described in detail above, those skilled in the art should understand that they can make various changes, substitutions and alterations herein without departing from the spirit and scope of the invention as defined by the appended claims. Implementation] This patent advocates the development of a combination of electrochemical, microfluidics and molecular stencil technology for the sensing of biomolecules. At the same time, this technology can be used to develop sensors of various sizes and integrate high-tech 1C. design. 1 shows the electrical response of an imprinted polymer electrode (made with myoglobin as the template to direct polymer formation) and a non-imprinted electrode having the same polymer coating. FIG. 9 shows the selectivity of the micro-contact imprinting method applied to the Myoglobin.

FIG. 5 shows the reduction of electrical conductivity using polymerised aminophenylboronic acid when challenged with increasing amounts of protein. FIG. 6 shows the selectivity of a micro-contact formed polymer imprinted with myoglobin and re-bound in human serum and urine. Where bar 3 depicts rebinding in human serum and bar 4 shows rebinding in human urine. FIG. 7 shows the incorporation of a sensing chip，bearing a polymer as described above, into a micro-fluidic system. 10 200827714 【主要元件符號說明】5 shows the selectivity of a micro-contact formed polymer imprinted with myoglobin and re-bound in human serum and urine. FIG. 6 shows the selectivity of a micro-contact formed polymer imprinted with myoglobin and re-bound in human serum and urine. 7 shows the incorporation of a sensing chip, bearing a polymer as described above, into a micro-fluidic system. 10 200827714 [Description of main component symbols]

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Claims

200827714 X. The patent application scope: 1. A method for forming a sensor for the detection of bio_molecules and proteins, comprising the steps of: (a) forming a polymer layer on an electrode; (b) forming a polymer layer on an electrode; (c) applying the electrode to an electrochemical cell; (d) a a a buffer solution to the electrochemical cell to facilitate electrical conductivity;

(e) applying a voltage to the electrochemical cell; (f) introducing a protein into the said electrochemical cell; and (g) measuring the current passed through the polymer layer in the electrochemical cell.

2. The method of claim 1 where the electrode is a screen-printed electrode.

3. The method of claim 1 where the electrode is a conductive or metal electrode such as a platinum, gold or silver electrode; or substituted an electrode made from another material.

4. The method of claim 1 where the polymer is a functionalised polymer·

5. The method of claim 4 where the fimctioealised polymer is present together with a non-ftmctionalised or cross-linking polymer·

6. The method of claim 4 where the polymer is poly-aminophenylboronic acid aed aey other polymer formed from a functional monomer and cross-linker. 7· The method of claim 4 where the polymer is a methacrylic acid polymer· 12 200827714

8. The method of claim 6 where the polymer is applied to an existing substrate polymer layer already applied to the screen-printed electrode.

9. The method of claim 8 where the existing substrate polymer layer is polypyrrole.

10. The method of claim 1 where the polymer layer is formed by microcontact printing.

11 .The method of claim 1 where the polymer layer is formed by electro-deposition.

12·The method of claim 1 where the polymer layer is formed by spin-coating.

13. The method of claim 1 where the deposited polymer layer is a molecularly imprieted polymen

14. The method of claim 1 where the electrode consists of a working electrode, a counter electrode and a reference electrode or any combination of these three electrodes·

15. The method of claim 1 where the electrical potential is supplied in a continuous cyclical maoeer.

16. The method of claim 1 where the electrical potential is supplied at a constant potential.

17. The method of claim 1 where the bio-molecule is a protein, or any electro-active or electro-inactive biomolecule.

18. The methods of claims 15 and 16 where the supplied electrical potential is of a suitable negative, or positive, value to attract bio-molecules, such as protein molecules in solution, to the working electrode. 13 200827714

19. The method of claim 1 where the protein is lysozyme, cytochrome c, ovalbumin, C reactive protein or any other protein in a native or denatured conformation.

20. The method of claim 19 where the protein is any protein, or glycated protein, that by virtue of its pi value in buffer solution is attracted to the working electrode at the potential applied.

21. The method of claim 1 where the buffer is phosphate buffer.

22. The method of claim 1 where the buffer is any dilute phosphate buffered saline.

23. The method of claim 20 where the pH of the buffer is adjusted so as to induce an electrical charge on the bio-molecule, which may be a protein molecule, opposite to that on the working electrode.

24. The sensor of claim 1 incorporated into a sensing device.

25. The sensing device of claim 24, which is equivalent a micro-sensing device. XI. Schema: 14