Surface-enhanced Raman spectroscopic analysis of centrifugally filtered HBV serum samples.

BACKGROUND: Raman spectroscopy is an effective tool for detecting and discriminating centrifugally filtered hepatitis B virus serum and centrifugally filtered control serum. OBJECTIVES: The purpose of current study is to separate high molecular weight fractions from low molecular weight fractions present hepatitis B serum to increase the disease diagnostic ability of surface enhanced Raman spectroscopy (SERS). METHODS: Clinically diagnosed centrifugally filtered serum samples of hepatitis B patients are subjected for surface enhanced Raman spectroscopy (SERS) in comparison with centrifugally filtered serum samples of healthy individuals by using silver nanoparticles (Ag-NPs) as SERS substrates. Some SERS spectral features are solely observed in centrifugally filtered serum samples of hepatitis B and some SERS spectral are solely observed in centrifugally filtered serum samples of healthy individuals. The diagnostic ability of SERS is further enhanced with different statistical techniques like principal component analysis (PCA), partial least square discriminant analysis (PLS-DA) and partial least square regression analysis (PLSR) have applied. RESULTS: The disease biomarkers of hepatitis B are more pronounced after their centrifugation as compared with uncentrifuged form. Statistical tools like principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) clearly differentiated centrifugally filtered serum samples of hepatitis B from centrifugally filtered serum samples of healthy individuals. Furthermore, partial least square regression analysis (PLSR) has been applied for predicting unknown viral load of centrifugally filtered serum sample of hepatitis B. CONCLUSION: SERS technique along with chemometric tools have successfully differentiated centrifugally filtered serum samples of hepatitis B from centrifugally filtered serum samples of healthy individuals. The centrifugal filtration process has increased the differentiation accuracy of PLS-DA in terms of percentage 98% and regression accuracy of PLSR regression analysis in terms of RMSEP (0.30 IU/mL) of this diagnostic method as compared with that of uncentrifuged method.

Surface-enhanced Raman spectral analysis for comparison of PCR products of hepatitis B and hepatitis C.

BACKGROUND: Surface-enhanced Raman spectroscopy is a reliable tool for identification and differentiation of two diseases showing similar symptoms, hepatitis B (HBV) and hepatitis C (HCV). OBJECTIVES: To develop a polymerase chain reaction technique (PCR) based SERS technique for differentiation of two human pathological conditions sharing the same symptoms using multivariate data analysis techniques e.g. principle component analysis (PCA) and partial least square discriminate analysis (PLS-DA). METHODS: PCR products of HBV and HCV were differentiated by SERS using silver nanoparticles (AgNPs) as a SERS substrate. For this analysis, PCR products of both the diseases with predetermined viral loads were collected and analyzed under SERS instrument and unique SERS spectra of HBV and HCV was compared showing many differences at various points. Diseased classes of HBV and HCV and their negative control classes (viral load less than 1) were compared. PCR products of true healthy DNA and RNA were also compared, which were significantly separated. Moreover, SERS data was analyzed using multivariate data analysis techniques including principle component analysis (PCA) and partial least square discriminate analysis (PLS-DA) and differences were so prominent to observe. RESULTS: SERS spectral data of HBV and HCV showed clear differences and were significantly separated using PCA. Negative control samples of both disorders and their true healthy samples of DNA and RNA were separated according to 1st principle component. By analyzing data using partial least square discriminate analysis, differentiation of two disease classes was considered more valid with sensitivity, specificity and accuracy value of 96%, 94% and 98% respectively. Value of area under curve (AUROC) was 0.7527. CONCLUSION: SERS can be employed for identification and comparison of two human pathological conditions sharing the same symptomology.