Nanogold biochip for detecting Alzheimer's disease marker as well as preparation method and application of nanogold biochip

[0079] Example 1

[0080] Preparation and protein testing of Aβ40 nano gold biocontrol

[0081] 1, preparation of nano-gold biocontrol

[0082] (1) Golden nanoparticles by wet method on the glass substrate to obtain a nano-gold chip;

[0083] (2) Print the capture antibody of a concentration of 1.0 μg / ml of the concentration of 1.0 μg / ml on the nano-gold chip by using a micro-point gauge.

[0084] (3) Further print chicken IGY antibody on the chip is a nano-gold biocontrol;

[0085] 2, capture antibody and fluorescent molecule Irdye800 coupling

[0086] (4) The captured antibody of the Aβ40 and the fluorescent molecule IrDye 800 were mixed according to the molar ratio of 1:10, the reaction was half an hour, and then mixed uniform, and the reaction was continued for half an hour;

[0087] (5) After the reaction is completed, the amount of PBS is added to make the reaction system volume reaches 500 μl;

[0088] (6) Add NAP-5 column with 10 mL of PBS, wash the column. When the solution in the column is completely dripped, the mixed solution of step (5) is added to the column;

[0089] (7) After the drop is complete, 500 μl of PBS is added. At this time, it is collected, i.e., the capture antibody of the Irdye800 marked Aβ40, which is subjected to it, and is stored in -20 ° C refrigerator.

[0090] 3, get standard curve

[0091] (8) The prepared nano-gold biocontrol is placed in the chip tank, and 100 μl of 1% BSA is added to the reaction hole, and 40 min is closed at room temperature, and the sealing liquid is poured;

[0092] (9) The Aβ40 protein standard is diluted into a 20 ng-0.1 ng concentration gradient protein solution, and the capture antibody of the IrDye800 labeled Aβ40 obtained by step (7) is 100 times (diluted 50-200 times), and will be different The protein solution of the concentration gradient is mixed with the diluted trap antibody, and then 100 μl of the mixed solution is added dropwise in the reaction hole, shake 30s on the oscillator, mix well, then placed in an incubator 37 ° C to mix mixing 1H;

[0093] (10) After the reaction was completed, the reaction hole was cleaned twice with 1% BSA, and then washed once with PBS, and finally remove the chip from the reaction tank, put it in deionized water, remove impurities, and finally dry with a small centrifuge. The chip is placed in a chip scanner scanning the fluorescent image;

[0094] (11) Calculate the fluorescence value, the fluorescence value is proportional to the concentration of the target protein, and the standard curve of the concentration of the target protein is calculated based on fluorescence image analysis.

[0095] 4, Alzheimer's symbol detection

[0096] (12) The prepared nano-gold biocontrol is placed in the chip tank, and 100 μl of 1% BSA is added to the reaction hole, and the room temperature is closed for 40 min, and the sealing liquid is poured;

[0097](13) The Aβ40 protein of the approved measurement is mixed with the captured antibody of the IrDye800 labeled Aβ40 obtained by the diluted step (7), and then adding 100 μl of the mixed solution in the reaction hole, shocking 30s on the oscillator, mixing , Then placed in an incubator at 37 ° C shock mixing for 1 h;

[0098] (14) After the reaction was completed, the reaction hole was cleaned twice with 1% BSA, and then washed once with PBS, and finally remove the chip from the reaction tank, put it in deionized water, remove impurities, and finally dry with a small centrifuge The chip is placed in a chip scanner scanning the fluorescent image;

[0099] (15) The fluorescence value is calculated based on fluorescence image analysis, and the concentration of the Aβ40 protein to be tested is calculated into the standard curve.

[0100] The fluorescent image of the Aβ40 and the standard curve such as Figure 4 The shown, where Y = 0.39x + 0.51, R 2 = 0.9658.

[0101] Example 2

[0102] Preparation and protein detection of Aβ42 nano gold biocontrol

[0103] Preparation of A [beta] 42 nano-gold biocontrol and preparation method of the Aβ40 nano gold biocontrol in Example 1 was to print the capture antibody of Aβ42 in the nano-gold chip to obtain a Aβ42 nano-gold biocontrol;

[0104] A [beta] 42 protein detection of Aβ42 protein was detected in Example 1 was a standard curve of Aβ42 by Aβ42 protein standard, and the protein to be tested was Aβ42 protein.

[0105] The fluorescent image of the Aβ42 and the standard curve such as Figure 5 Disted, where Y = 0.55x + 0.34, R 2 = 0.994.

[0106] In summary, the present invention proposes a method for detecting a nano-gold biocontrol of Alzheimer's markers, and the gold nanoparticles are deposited on the chip substrate, so that the chip surface distributes many discontinuous, Golden nanoparticles with uniform spacing, and control the size of the gold nanoparticles at 20-50 nm, the spacing is controlled at 5-50 nm, forming a nano-gold chip on the substrate to form a nano-gold chip. The capture antibody that detects the Alzheimer's symbol is then fixed to the nano-gold chip, and the capture layer is formed to obtain a nano-gold biocontrol. Among them, the gold nanoparticle gap causes a local electric field to increase the fluorescent molecules of the gold nanoparticles, and the surface plasma of the gold nanoparticles is resonant to the fluorescent molecule, and the fluorescence amplification is realized, and the detection signal is improved. In addition, gold nanoparticles can also increase the specific surface area, increasing the binding site for capturing antibodies of the Alzheimer's markers. Not only that, but the near-infrared fluorescent molecules employed by the present invention can effectively avoid the fundamental fluorescent signal of the base material and improve the signal-to-noise ratio. Finally, the nano-gold biocontrol provided by the present invention, by detecting near-infrared fluorescence, reducing the background noise, increasing the antibody binding site and the gold nanoparticle enhances fluorescence output signal, and improves the sensitivity of the Alzheimer's biological marker detection, realization Super sensitive detection of the AD marker.