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  • br Sensitivity test and detection limit br

    2022-05-04


    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 NCT-501 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
    Fig. 6. PCA3 detection from urine of healthy controls. (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 M1-M5, PCR products from urine of healthy subjects; 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/A640 ratio determined by UV–vis spec-trophotometer. Each bar represents the mean ± SD obtained from three experiments. The statistical analysis was evaluated by t-test. *P-value < .05 compared with NTC.
    of subjects by RT-PCR. The quality of the cDNA samples was in-vestigated by GAPDH amplification. By gel electrophoresis, PCA3 was detected only in PCa samples, while no band was observed in samples from other groups (Figs. 5A, 6A, 7A). Of note, GAPDH products were amplified and detected from all samples, reflecting the cDNA integrity (Figs. 5A, 6A, 7A). Thiolated PCR products were added into the AuNP solutions. As expected, no observable color change was detected when thiolated PCR products from PCa patients were tested (Fig. 5B). How-ever, an obvious color change was visualized when samples from both BPH patients and healthy subjects were tested as well as the NTCs (Figs. 6B, 7B). The A520/A640 ratio of each sample was calculated and graphs were plotted as shown in Figs. 5C, 6C, 7C. Notably, the average A520/A640 ratio from all positive samples was significantly greater than that of negative samples (P-value < .05) and higher than the cutoff point of 1.5.
    4. Discussion
    PCA3, a long non-coding RNA, is found to be strongly overexpressed in PCa tissue compared to normal prostate tissue [35]. Moreover, pre-vious studies indicate that the expression of PCA3 in urine of PCa pa-tients is high, while its expression is low or undetectable in BPH and normal subjects [36–38]. This characteristic of PCA3 makes it a pro-mising PCa biomarker [39]. Over the past decade, examination of PCA3 gene expression by various approaches was extensively studied [16,40]. 
    In this process, the Progensa PCA3 commercial assay (Hologic Gen-Probe, Marlborough, MA, USA) was developed. However, this assay is not ideal for routine clinical screening due to its high cost and need for specialized instrument.
    In the present study, the AuNP-based colorimetric assay for de-termination of PCA3 in urine was established. This assay used a strategy of combining RT-PCR and AuNP-based colorimetric methods. Unmodified AuNPs and a thiol-labeled primer were used to generate a visual detection readout. To evaluate the PCR efficiency of thiol-labeled and unlabeled primers, PCR reactions were performed using both pri-mers. The results revealed that thiol-labeled primer did not disturb the PCR reaction as compared to the unlabeled primer. This is consistent with the results of previous studies demonstrating that no reduction in band intensity of PCR products when a primer was thiolated [33,41]. Notably, thiol-labeled products prevented salt-induced AuNP aggrega-tion, while neither unlabeled PCR products nor NTC did so (Fig. 2B). Thiolated PCR products bind to unmodified AuNPs as a result of the strong interaction between thiol groups and the gold surface. The DNA grafted on AuNP surfaces forms a thick barrier which protects each particle from coming close to another [42]. These properties cause an increase in steric repulsion between neighboring DNA-bound AuNPs, which results in a resistance to color change due to salt-induced na-noparticle aggregation. On the other hand, AuNPs which lack thiolated PCR products undergo an abrupt and easily observed color change caused by particle aggregation upon the addition of salt solution [30].