Method for preparing composite nano microsphere for enriching lung cancer cells
A technology of lung cancer cells and complex proteins, which is applied in the fields of polymer chemistry and cell biomedicine, can solve the problems of high false negative rate of lung cancer exfoliated cells and complex cell components, and achieve increased detection sample size, reduced costs, improved reliability and The effect of accuracy
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Embodiment 1
[0034] Example 1 Fe 3 o 4 Preparation of nanoparticles
[0035]1) form ferric nitrate and ferrous nitrate in ethanol with the molar mass ratio of 4:1 to form a uniformly dispersed ethanol solution, wherein the concentration of iron ions is 0.5 to 1.0 mol / L; Epichlorohydrin whose mass is 10 times that of iron ions forms a sol, and forms a gel after standing; the prepared gel is aged and washed with ethanol and dried; followed by annealing at 400°C to obtain Fe 3 o 4 .
[0036] 2) After the reaction finishes, the Fe 3 o 4 Wash with deionized water and ethanol alternately until the pH value is 7. Finally, dry it under vacuum to get Fe 3 o 4 magnetic nanoparticles.
[0037] 3) Take 5g of Fe prepared above 3 o 4 Magnetic nanoparticles, placed in 20ml of deionized water, ultrasonic (1000kHz) dispersed for 30min and then centrifuged (or magnetically) separated to obtain nano-Fe 3 o 4 Precipitation A.
[0038] 4) Reset 5g of Precipitate A in 10ml of PBS buffer solution, ...
Embodiment 2
[0042] Example 2 Preparation of Composite Nanomagnetic Particles
[0043] 1. Take PEI-modified Fe 3 o 4 Nanoparticles 100mg were suspended in 10ml 0.01M pH7.2TBS to obtain a nanoparticle solution, in which PEI-modified Fe 3 o 4 The concentration of nanoparticles is about 3 × 10 13 pieces / ml.
[0044] 2. Preparation of complex proteins: Dissolve protein A (SPA) and protein B (rabbit anti-human CD44 and CK7) with 3ml 0.01M pH7.2TBS (both purchased from Yusen Biotechnology Co., Ltd.), mix well, and react at 4°C For 30 minutes, the protein A concentration was 10 mg / ml, and the protein B concentration was 1 mg / ml.
[0045] 3. Take 0.5ml of the above-mentioned AB composite protein solution and mix evenly with 10ml of the above-mentioned nanoparticle solution, and leave it at room temperature for 60 minutes to obtain a composite nanomagnetic particle solution.
Embodiment 3
[0046] Pathological detection of lung cancer cells in the sputum of embodiment 3
[0047] Sputum samples from 850 patients with lung cancer were randomly divided into two groups. Patients need to leave morning sputum for 3 consecutive days and send it to the hospital within 2 hours, otherwise, the sputum will deteriorate and the cells will autolyze, making it impossible to diagnose. Collect 5ml of the patient's sputum into a sputum box, add 0.5ml of alkaline lysate (containing 1% proteinase K, 10% NaOH in water) and mix well, keep at room temperature for 10min to fully liquefy the sputum.
[0048] The experimental group was subjected to lung cancer cell enrichment and liquid-based cytopathological detection. Add 1 / 10 volume of the composite nano-magnetic particle solution prepared in Example 2 to the above liquefied sputum, mix well, and let stand for 15 minutes. Centrifuge at 500 rpm for 5 minutes, take the precipitate and suspend it in preservation solution (10% aqueous me...
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