Exosome detection method based on optical fiber evanescent wave fluorescence biosensor
A detection method, exosome technology, applied in fluorescence/phosphorescence, instruments, measuring devices, etc., can solve the problems that there are no reports on exosome detection, and achieve simple and fast detection operation, low requirements, and strong signal amplification Effect
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Embodiment 1
[0075] Example 1 Establishment of an exosome detection method based on optical fiber evanescent wave fluorescence biosensor
[0076] 1. Experimental materials
[0077] Freeze-dried exosome standard, sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, 3-aminopropyltriethoxysilane (APTS), citric acid, sodium citrate, dodecane Sodium disulfate (SDS), bovine serum albumin (BSA), glutaraldehyde, etc.
[0078] The nucleic acid sequences used in the experiment are as follows:
[0079]
[0080] Note: NH 2 It is a molecule modified on the nucleic acid sequence and covalently bound to the surface of the optical fiber;
[0081] (CH 2 ) 6 is the spacer molecule.
[0082] 2. Design principle of an exosome detection method based on optical fiber evanescent wave fluorescence biosensor
[0083] The double-labeled signal probe is attached to the surface of exosomes through the hydrophobic interaction between the cholesterol molecule and t...
Embodiment 2
[0088] Example 2 Detection condition optimization results
[0089] Mix different concentrations of double-labeled signaling probes with a concentration of 2.4×10 3 Particles / mL of exosomes were mixed, incubated at 37°C in the dark for 90 min, and passed into a fiber optic evanescent wave fluorescence detector to measure the fluorescence signal. It can be seen from the figure that when the concentration of the double-labeled signal probe is 100 nM, the ratio of the fluorescence signal intensity of the positive sample to the blank control sample is the highest ( Figure 4 A). Keeping the total concentration (400 nM) of exosome-specific aptamer sequence and short-chain nucleic acid sequence constant, the cones were modified with solutions containing different concentration ratios of exosome-specific aptamer sequence and short-chain nucleic acid sequence. After the optical fiber, put it into the sample pool of the optical fiber evanescent wave fluorescence detector in turn; mix...
Embodiment 3
[0090] The sensitivity determination of embodiment 3 detection method
[0091] According to the above optimization system, the exosome solution was diluted 10-fold, and the double-labeled signal probe with a concentration of 100 nM was mixed with different concentrations of exosomes. After incubation at 37°C in the dark for 60 min, pass through The fluorescence signal was measured in a fiber optic evanescent wave fluorescence detector. It can be seen from the figure that the fluorescence signal increases with the increase of exosome concentration ( Figure 5 A), the logarithm of the exosome concentration is the abscissa, the ratio of the fluorescence signal intensity of the positive sample to the fluorescence signal intensity of the blank control sample is the ordinate, and the standard curve is drawn: y = 0.1045x + 1.0153, R 2 = 0.9908 ( Figure 5 B), the linear range for exosome detection is 47.5 - 4.75 x 10 6 particles / mL, the detection limit was 7.66 particles / mL.
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