br Fig PCA detection from urine
Fig. 5. PCA3 detection from urine of PCa patients. (A) PCR products of PCA3 (upper gel) and GAPDH (lower gel) analyzed by 1.5% agarose gel electrophoresis. Amplicon sizes of PCA3 and GAPDH were 838 bp and 106 bp, respectively. Lane M, 100 bp ladder; Lane PC, PCR products from LNCaP cells; Lanes P1-P5, PCR products from urine of PCa patients; Lane NTC, no-target control. (B) AuNP-based colorimetric assay. Color change was visualized by the naked eye. The order of the reactions is similar to gel electrophoresis. (C) Absorption spectra of mixtures of AuNP solutions and samples. A520/A650 ratio determined by UV–vis spectro-photometer. Each bar represents the mean ± SD obtained from three experiments. The statistical analysis was evaluated by t-test. *P-value < .05 compared with NTC.
3.2. Specificity test
The specificity of PCA3 Dorsomorphin was investigated by using cDNA templates extracted from various cell lines, i.e., LNCaP and VCaP as prostate cancer cell lines and RWPE-1 as a prostate epithelial cell line. The quality of the cDNA templates was examined by RT-PCR using specific primers for the GAPDH gene. As shown in Fig. 3A, 106 bp PCR products of GAPDH were amplified from the cDNA of all tested cell lines. This result indicated the good quality of the cDNA templates. Notably, 838 bp PCR products of PCA3 were detected from both LNCaP and VCaP cell lines, while not detected from RWPE-1. In the colori-metric assay, these PCR products were added to AuNP solutions. After adding salt, the AuNP solutions containing thiol-labeled PCR amplicons remained red, whereas color change was observed in the AuNP solu-tions containing non-amplified PCR products (Fig. 3B). The color could be distinguished by the naked eye. Spectral scanning of each tested solution is shown in Fig. 3C. Moreover, the A520/A640 ratio was used to compare positive (red) and negative (blue) results. The ratios of positive results were significantly greater than those of negative results (Fig. 3C).
3.3. Sensitivity test and detection limit
To verify the sensitivity of this method, diﬀerent concentrations of PCR products amplified from the LNCaP cell line were applied to the system. Color changes of AuNP solutions were observed by the naked eye and then scanned within the absorption spectra ranging from 400 to
700 nm. The same concentrations of PCR products were also subjected to gel electrophoresis. At < 62.5 ng of PCR products, no band appeared on the gel electrophoresis (Fig. 4A). Interestingly, the red color was obviously detected at 31.25 ng of PCR products. Moreover, the A520/ A640 ratio at this concentration was also statistically diﬀerent than that of the NTC. As a result, the detection limit of the assay was approxi-mately 31.25 ng (Fig. 4B). These results indicated that the proposed colorimetric assay was more sensitive than gel electrophoresis. The absorption spectra were shifted when decrease of PCR product con-centrations (Fig. 4C). Notably, the intensity of blue color increased with lower amounts of thiol-labeled amplified DNA. The degree of color change (A520/A640) was linearly dependent on the concentration of thiolated PCR products (R2 = 0.9798) as shown in Fig. 4D. These findings clearly indicated that the presence of thiol-labeled DNA caused an increase in the resistance to salt induced-aggregation of AuNPs.
3.4. PCA3 detection in urine samples using AuNP-based colorimetric assay
After establishing assay specificity and sensitivity, the AuNP-based colorimetric assay was utilized for detection of PCA3 in clinical sam-ples. Fifteen clinical urinary samples used in this study: 5 were from BPH patients, 5 were from biopsy-proven PCa patients and the re-maining 5 were from healthy controls. In order to verify our detection system, all samples were tested for PCA3 expression by qRT-PCR. The relative PCA3 expression level in urine from PCa patients was sig-nificantly higher than that from both healthy subjects and BPH patients (Table 2). Thiolated PCR products of PCA3 were amplified from cDNA