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

An Alzheimer's disease, biochip technology, applied in measurement devices, material excitation analysis, fluorescence/phosphorescence, etc., can solve the problems of not reaching the picogram level, inaccurate results, low sensitivity, etc., and improve the emission intensity. , the effect of improving the detection signal and improving the sensitivity

Pending Publication Date: 2022-03-01
SHENZHEN UNIV +1
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AI-Extracted Technical Summary

Problems solved by technology

Among these research methods, the results of the colorimetric method are intuitively visible to the naked eye, but the sensitivity is low; although the sensitivity of the fluorescence method is slightly higher than that of the colorimetric method, it still cannot reach the picogram level, and it is susc...
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Method used

Described nano-gold biochip is a type of detection device based on fluorescence strength to determine the concentration of biomarkers to be detected, and the near-infrared fluorescent molecules it adopts can effectively avoid the background fluorescence signal of base material, improve signal noise ratio. The limitation of the current mainstream fluorescence detection is that the wavelength range is usually between 300nm and 700nm. In this range, samples and required consumables such as NC membrane, microwell plate, glass and other substrate materials have autofluorescence, and the detection background noise is large. . The near-infrared fluorescence used in the present invention has a wavelength range between 700nm and 800nm, which can effectively avoid the background fluorescence of the substrate material and improve the signal-to-noise ratio; and the fluorescence in this band has strong penetration, which can greatly reduce the fluorescence scattering effect , to improve detection sensitivity.
In summary, the present invention proposes a method for preparing a nano-gold biochip that detects Alzheimer's disease markers, gold nanoparticles are deposited on the chip substrate, so that the surface of the chip is distributed with many non- Continuous gold nanoparticles with uniform spacing, and the size of the gold nanoparticles is controlled at 20-50nm, and the spacing is controlled at 5-50nm. A gold film visible to the naked eye is formed on the substrate to construct a nano-gold chip. Then, the capture antibody for detecting Alzheimer's disease markers is fixed on the nano-gold chip by printing to form a capture layer to obtain a nano-gold biochip. Among them, the gap between the gold nanoparticles leads to a local electric field, which increases the emission intensity of the fluorescent molecules on the gold nanoparticles, and the surface plasmons of the gold nanoparticles resonate with the fluorescent molecules to achieve fluorescence amplification and improve the detection signal. In addition...
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Abstract

The invention provides a nanogold biochip for detecting an Alzheimer's disease marker and a preparation method and application of the nanogold biochip. Gold nanoparticles are deposited on a chip substrate, so that a plurality of discontinuous gold nanoparticles with uniform intervals are distributed on the surface of the chip; and fixing a capture antibody for detecting the Alzheimer's disease marker on the chip through printing to form a capture layer, thereby obtaining the nanogold biochip. A local electric field is generated due to gaps of the gold nanoparticles, so that the emission intensity of the coupled fluorescent molecules is improved, surface plasmas and the fluorescent molecules resonate, and fluorescence detection signal amplification is realized. The gold nanoparticles can also increase the specific surface area and increase binding sites for capturing the antibody. The near-infrared fluorescent molecules are adopted, so that background fluorescent signals of the substrate can be effectively avoided, and the signal-to-noise ratio is increased. Finally, background noise is reduced by detecting near-infrared fluorescence, a fluorescence output signal is enhanced by the gold nanoparticles, and the sensitivity of Alzheimer's disease biomarker detection is improved.

Application Domain

Technology Topic

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

Examples

  • Experimental program(2)

Example Embodiment

[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.

Example Embodiment

[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.
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PUM

PropertyMeasurementUnit
Diameter300.0 ~ 500.0µm
tensileMPa
Particle sizePa
strength10

Description & Claims & Application Information

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