Color-changing eye drops for early screening alzheimer's disease and application thereof

Abstract

The invention relates to color-changing eye drops for early screening Alzheimer's disease and an application thereof. The color-changing eye drop for early screening Alzheimer's disease comprises 1 pg/ml to 10 ng/ml of A.sub.42 aptamer-gold nanoparticles (AuNPs) and a pharmaceutically acceptable carrier or vehicle thereof, which can be used to mix with a tear sample of a test subject to analyze a color change of the color-changing eye drop for determining the concentration proportion of A.sub.40:A.sub.42 contained in the tear sample of the test subject.

Claims

1. A method for early screening Alzheimer's disease by use of a color changing eye drop, comprising: (a) preparing A.sub.42 aptamer containing amino acid sequence of SEQ ID NO:1, (b) bonding the prepared A.sub.42 aptamer with gold nanoparticles (AuNPs) to obtain a complex of A.sub.42 aptamer-gold nanoparticles (Aptamer-AuNPs), (c) formulating a color-changing eye drop containing Aptamer-AuNPs in a range of 1 pg/ml-10 ng/ml and a pharmaceutically acceptable carrier thereof, (d) mixing approximately 10 l of the color-cha aging eye drop with approximately 10 l of a tear sample of a test by a technique selected from a group containing mixing in a centrifuge tube, using a test strip, dropping the color-changing eye drop into at least one eye of the test subject, and combination thereof, for a reaction time of approximately 10 minutes, (e) analyzing a color of the color-changing eye drop mixed with the tear sample; and (f) if the color of the mixture of the color-changing eye drop and the tear sample has changed, a concentration proportion of A.sub.40:A.sub.42 contained in the tears ample of the test subject in a range 9:1-1:1.

2. The method for early screening Alzheimer's disease by use of a color-changing eye drop as claimed in claim 1, further comprising: in said step (e), anal zing the color of the color-changing drop mixed with the tear sample by spectrophotometry, and in said step (f), indicating a possible presence of the Alzheimer's disease if said concentration proportion of the A.sub.40:A.sub.42 has excessively high A.sub.42 concentration corresponding to a deviation of said proportion A.sub.40:A.sub.42 from 9:1 in the range from 9:1 to 1:1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 is a schematic diagram showing a color change of color-changing eye drops mixed with a tear sample containing A.sub.42;

(3) FIG. 2 is a schematic diagram showing an application of color-changing eye drops to a test strip for detection.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(4) The present invention relates to color-changing eye drops for early screening Alzheimer's disease and an application thereof. The color-changing eye drop for early screening Alzheimer's disease disclosed herein comprises 1 pg/ml to 10 ng/ml of A.sub.42 aptamer-gold nanoparticles (AuNPs) and a pharmaceutically acceptable carrier or vehicle thereof. Preferably, the A.sub.42 aptamer comprises the amino acid sequence of SEQ ID NO:1.

(5) Additionally, the method for early screening Alzheimer's disease by use of the color-changing eye drop as described above comprises the steps of: mixing the color-changing eye drop with a tear sample of a test subject; and analyzing a color change of the color-changing eye drop for determining a concentration proportion of A.sub.40:A.sub.42 contained in the tear sample of the test subject.

(6) Hereinafter, an exemplary embodiment of the present invention will be described in detail with reference to the accompanying drawings.

(7) The present invention used the existing electrochemical impedance spectroscopy (EIS) to analyze the ratio of A.sub.40:A.sub.42 concentration. Prior to the establishment of a calibration curve of the A.sub.40 and a calibration curve of the A.sub.42, the specificity of the A.sub.40 antibody and the specificity of the A.sub.42 antibody were first confirmed. Then, various solutions of 100 pg, 1 ng, 10 ng, 100 ng, 1 g, and 10 g were prepared by using commercially available A.sub.40 protein and A.sub.42 protein dissolved in PBS solvent. A tear sample was separately dropped into a wafer containing A.sub.40 antibodies and a wafer containing A.sub.42 antibodies to obtain the impedance difference between the A.sub.40 protein and the A.sub.42 protein, and the impedance difference was substituted into the calibration curves of A.sub.40 and A.sub.42, respectively, thereby obtaining the concentration proportion of A.sub.40 and A.sub.42 in the tear sample. After the parameter of the A.sub.40:A.sub.42 ratio were confirmed, the parameter was used as a base to prepare aptamer with specific range of color change.

Example 1: Preparing A.SUB.42 .Aptamer-Gold Nanoparticles (AuNPs)

(8) 1. Preparation of A.sub.42 Aptamer

(9) In general, A.sub.42 antibodies only specifically bind A.sub.42 in the sample without binding to A.sub.40. However, the traditional antibodies are quite expensive and difficult to store, and the procedure for manufacturing antibodies into detection reagents or test kits is complicated. Therefore, the present invention established an A.sub.42 aptamer to replace the traditional antibody, so as to achieve specific binding to A.sub.42 as well as A.sub.40 and improve the convenience of preservation.

(10) In this example, systematic evolution of ligands by exponential enrichment (SELEX) was used to prepare an A.sub.42 aptamer as set forth in SEQ ID NO: 1.

(11) 2. Bonding A.sub.42 Aptamers with Gold Nanoparticles (AuNPs)

(12) Gold (Au) nanoparticles has excellent biocompatibility and surface plasmon resonance. Therefore, gold nanoparticles (AuNPs) are commonly used in biomedical detection, disease diagnosis and gene detection.

(13) In this example, A.sub.42 aptamer-gold nanoparticles (AuNPs) were prepared by reference to journals published by Wen Yun et al. (European Food Research and Technology 238(6):989-995) and published by Lin, Xiaoyan et al. (Chem Sci. 2017 May 1; 8(5):3905-3912).

(14) Primarily, sodium citrate was added as a reducing agent to chloroauric acid (HAuCl.sub.4) to prepare gold nanoparticles. A 100 mL solution containing 0.01 g of chloroauric acid was refluxed, and 2.5 mL of a 1% sodium citrate solution was added to the chloroauric acid solution while stirring the chloroauric acid solution to form a mixed solution. Then, the mixed solution was boiled for 30 minutes, cooled to room temperature, and stored in a dark glass bottle at 4 C. for use. A.sub.42 aptamers was added to 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution for 30 minutes at room temperature for reaction, and then 100 nM reaction A.sub.42 aptamers, 0.01% tween 20 and 200 nM PEG-thiol were added to 50 nM gold nanoparticles and reacted at room temperature for 1.5 hours. After the reaction, the mixture was centrifuged at 12,000 rpm for 30 minutes, and the supernatant includes a complex of A.sub.42 aptamer-gold nanoparticles (Aptamer-AuNPs).

Example 2: Using Aptamer-AuNPs for Detection

(15) In this example, the color-changing eye drops were used to detect the color change of a tear sample of a normal test subject. The tear sample were collected by using a strip paper that prevents tears from sucking back, and the tear collection strip was placed in a centrifuge tube and centrifuged at a speed of 13,000 rpm to obtain about 10 l of the tear sample. Then, 10 l of tear sample was mixed with 10 l of Aptamer-AuNPs solution for reaction for 10 minutes. According to the report of the Alzheimer's Association, the ratio of A.sub.40:A.sub.42 in normal human cerebrospinal fluid is 9:1, whereas the ratio of A.sub.40:A.sub.42 in AD patients is 1:1. When the ratio of A.sub.40 to A.sub.42 in the tear sample is not 9:1, the color of the Aptamer-AuNPs will be changed, which can be used to preliminarily determine the symptom of Alzheimer's disease. Furthermore, the degree of change in the color of Aptamer-AuNPs with the ratio of A.sub.40:A.sub.42 from 9:1 to 1:1 can be further measured by spectrophotometry.

(16) Referring to FIG. 1, the color-changing eye drops of the present invention exhibit a color change (blue) after the addition of A.sub.42-containing tear sample. Accordingly, the color-changing eye drops can be added to the tear sample isolated from the test subject, or directly dropped into the eyes of the test subject, to rapidly screen whether the test subject has the symptom of Alzheimer's disease based on color change, e.g. excessively high A.sub.42 concentration.

(17) Referring to FIG. 2, the color-changing eye drops of the present invention can also be dropped onto a test strip, and then the tear sample of the test subject is absorbed by the test strip to detect whether a color change occurs. As shown in FIG. 2, the color change of the test strip indicates that the normal test subject contains A.sub.42 in the tear sample. If the test subject has a high concentration of A.sub.42 that changes the general ratio of A.sub.40:A.sub.42 of 9:1, the color change of the test strip will be more obvious and can be determined that there is a symptom of Alzheimer's disease.

(18) Compared with the technique available now, the present invention has the following advantages:

(19) 1. The present invention uses the A.sub.42 aptamer-gold nanoparticles to specifically recognize A.sub.42, which solves the problem that the traditional antibodies are quite expensive, not easy to store, and difficult to be made into detection reagents or detection kits.

(20) 2. The present invention quickly and easily detects whether a sample has a high concentration of A.sub.42 by color change and does not require expensive instruments for detecting or analyzing the results.

(21) 3. The invention is made into an eye drop type, so that people can observe the color change after the eye drops are dripped into the eye in daily life for preliminary monitoring whether the signs of Alzheimer's disease appear. Therefore, the present invention can be conveniently used to initially monitor the occurrence of the symptom of Alzheimer's disease, and only when the test results show that people have the symptom of Alzheimer's disease, they need to go to the hospital for further examination.