Multiplexed analyses of test samples
A technology for testing samples and samples, which is applied in the field of detecting target molecules in samples, and can solve problems such as inaccurate detection of target molecules
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Embodiment 1
[0149] Example 1. Photoaptamer analysis using hybridization capture via surface-immobilized probes Measurement buffer and plasma serial dilutions of VEGF for detection and quantification
[0150] This example illustrates how Figure 2A , 2B and the analysis steps shown in 2C for a single photoaptamer and its target protein. The analysis was performed with two different test samples, buffer and plasma.
[0151] A. Preparation of oligonucleotides: A DNA oligonucleotide tag included at the 3' end of the photoptamer was used here. In this example, the marker is the 3' fixed region used in the SELEX method. VEGF photoaptamer 509-80 was synthesized using standard methods for solid-phase phosphoramidite DNA synthesis on a conventional synthesizer. The 5-BrdU containing photoptamer was cleaved and deprotected under milder conditions than DNA synthesis standards (tert-butylamine:methanol:water=1:1:2, 70°C, 5 hours), filtered and evaporated to dryness. Aptamers were purified by eth...
Embodiment 2
[0155] Example 2. Detection and Quantification of Multiple Target Proteins in Buffer by Photoaptamer Analysis Using Surface-immobilized Probes for Hybridization Capture
[0156] This example illustrates how Figure 2A , 2B and 2C for the analysis steps of the multiplexed format with 10 photoaptamers and their target proteins in buffer.
[0157] A. Preparation of Tagged Photoaptamers: Unique Oligonucleotide Tags Assigned to AffymetrixGeneChip A set of gene expression probes obtained from the Test3 Array reverse complement sequence for each photoaptamer. Labeled photoaptamers were prepared as described in Example 1, except that an amine group was added to the 5' end of the photoaptamer. Prior to use, the aptamer solution was heated at 95°C for 3 minutes and then cooled to 37°C at a rate of 0.1°C per second under control.
[0158] B. Immobilization of Capture Probes to Amine Reactive Surfaces: Reverse Complementary Capture Labels are immobilized as described in Example 1, ex...
Embodiment 3
[0161] Example 3. Detection and quantification of multiple target proteins in serum by photoaptamer analysis using hybridization-captured probes immobilized on the surface
[0162] This example demonstrates that if Figure 2A , 2B Analysis of multiplexed forms of serum samples measured with 57 photoptamers shown in 2C and 2C.
[0163] A. Preparation of labeled photoaptamers: The labeled photoaptamers were prepared as described in Example 2.
[0164] B. Immobilization of capture probes on amine-reactive surfaces: Immobilization of reverse complementary capture labels as described in Example 2.
[0165] The 57 photoptamers were divided into 2 groups for diversification, 1 group of 27 photoptamers for low-abundance targets in human serum or plasma, and a group of 30 photoptamers for high-abundance targets. 2 dilutions were prepared from 14 serum samples and mixed with 2 sets of aptamers so that the final concentration of each aptamer was 1 nM, and 10% serum, 0.9xSB1, 0.1% Twee...
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