Surface-enhanced Raman Spectroscopy Of Serum Albumin In Liver Cancer Patient

In this study,we used hydroxyapatite(HAp)to adsorb-exfoliate albumin from serum for SERS analysis,with the aim to develop a novel method for noninvasive and label-free cancer screening.The major results are as follows:(1)The HAp particles were prepared by the hydrothermal method with uniformly microsphere and the average diameter of the particles was approximately 4 to 5 μm.Based on HAp nanoparticle adsorption and in vitro release of BSA,and combined with SERS detection method.Moreover,HAp method can achieve quantitative analysis of protein,demonstrating that the proteins are mainly bound to the surface of HAp by physical adsorption(electrostatic attraction),which has no obvious effect on the structure of proteins.Moreover,high quality and reproducible SERS spectra can be obtained during SERS measurement,which shows that HAp has high reliability for protein adsorption.More importantly,HAp method can be achieved quantitative analysis of proteins,and can be easily extracted proteins from serum by the adsorption-exfoliation ability of HAp.And then,the cellulose acetate membrane electrophoresis was further demonstrated that the HAp method mainly adsorbed albumin from serum.The normal range of serum albumin in adults is about 3.5 to 5.0 g/d L,and previous study has demonstrated that low serum albumin level(≤3.5 g/d L)is a poor prognostic factor for the survival of patients with liver cancer.The quantitative analysis can be achieved by our HAp method with a good linear relationship from 1 to 10 g/d L.Moreover,the detection range of HAp method(from 1 g/d L to 10 g/d L)includes the normal range of serum albumin(3.5–5.0 g/d L).Especially,the lower limit detection of our method(1 g/d L)was less than the albumin prognostic factor for liver cancer(≤ 3.5 g/d L),suggesting the promising potential of this method for clinical cancer screening.(2)Based on the adsorption-exfoliation ability of HAp,albumin was extracted from serum,and then mixed with Ag colloids to perform SERS analysis for liver cancer and breast cancer screening.The feasibility of this method for cancer screening has been demonstrated by analyzing serum protein samples from liver cancer patients(n=58),breast cancer patients(n=30)and healthy volunteers(n=60).A discrimination threshold of 0.5 was set for accurate discriminate liver cancer versus healthy,breast cancer versus healthy,and liver cancer versus breast cancer,yielding the diagnostic sensitivities of95.1%,93.3%和 87.32%,respectively.The corresponding diagnostic specificities for each combination were 100.0%,92.5%和 90.2%,respectively.The AUC values for each combination classification were 0.980 、 0.946 和 0.911.The result demonstrates that PCA-LDA-based serum albumin SERS technique has potential capability for liver cancer and breast cancer detection.The RBF kernel SVM diagnostic model based on the PLS components distinguished protein SERS spectra of liver cancer,breast cancer and healthy subjects simultaneously with an accuracy rate of 88%.Moreover,a diagnostic accuracy of93.75% was achieved by PLS-SVM in the unknown testing set.These results suggest great potential for this new method for noninvasive and label-free cancer detection and screening.In future,we will continue to collect and detect more cancer samples of different pathological stages and different types to verify the reliability of the method.(3)Based on the adsorption ability of HAp,albumin was extracted from serum by adjusting the concentration of phosphate particles in the solution without damaging the HAp particles.We further explored the relationship between albumin and liver cancer of different tumor stages,and then combined with SERS spectroscopy technology to achieve label-free,non-invasive and more accurate detection of liver cancer at different stages.Moreover,it should be noted that the body fluids are complex system that includes all the electrolytes,antibodies,antigens,hormones,and also exogenous substances(e.g.drugs,bacteria,viruses,etc.),and SERS analysis of these body fluids are easily subject to the interference of these substances.Notably,our new method can eliminate the interference of other exogenous substances in serum samples and albumin was extracted from serum.Based on the PCA-LDA algorithm,the SERS spectra of early(T1)stage LC and advanced(T2-T4)stage LC can be well distinguished from normal subjects with accuracies of 88.33% and 94.92%,respectively.Excitingly,compared with other protein purification techniques(such as salting-out precipitation or HPLC),the HAp-preparation procedure is easy,and this preparation method is characterized by a high success rate.The reproducibility is greatly important for clinical sample detection.