Automated miRNA detection with one-step amplification on the analyzer
We measured different concentrations of synthetic miR-21-5p from 0.01 to 1000 pM in triplicate, and 0 pM in replicates of 5 via the one-step amplification assay for miR-21-5p using Converter DNA-21. The Converter DNA was designed to form a hairpin structure by the introduction of cover sequence as shown in Fig. 1. The hairpin structure accelerates DNA amplification by preventing signal DNA released into the solution from hybridizing to signal generation sequence of intact Converter DNA (see ESM Methods, ESM Results, ESM Fig. S1, Table S2). The assay results were obtained in 44 min after initial sample dispense with throughput of 66 tests per hour. The resulting dose-response curve of the miR-21-5p assay is shown in Fig. 3a. The coefficient of variations (CVs) of chemiluminescence intensity (CI) for the target at concentrations of 1 pM and higher were less than 6%. The CVs of CI even at low target concentrations both of 10 and 100 fM were 10%, and that of the background CI at 0 pM was 13%. The detection sensitivity was in the range from 10 to 100 fM. Similarly, we also measured miR-18a-5p and miR-500a-3p on the analyzer with Converter DNA-18 and Converter DNA-500, respectively (Fig. 3a). For clarity, each dose-response curve was separately shown in ESM Fig. S2A–C.
The one-step amplification assays for miR-18a-5p and miR-500a-3p, which were developed by changing the TBS of Converter DNA-21, exhibited comparable sensitivity as shown in the dose-response curves (Fig. 3a). In the present assay system, the target miRNA is easily altered only by changing the TBS of Converter DNA since miRNA is converted to the common signal DNA to be detected with the common Capture DNA probe and Chemiluminescence DNA probe. More than a hundred circulating miRNAs in blood have been so far identified as diagnostic, prognostic, or predictive biomarkers for different types of cancers [15]. The proposed assay allows the use of a common detection system and promotes the development of multiple miRNA assays.
Next, we investigated the cross reactivity of the one-step amplification assay for miR-21-5p to evaluate the sequence specificity. We selected fifteen human miRNAs which have sequences similar to that of miR-21-5p according to the miRNA database, miRBase [2, 40]. The numbers of bases identical to those in miR-21-5p sequence ranged from 8 to 14 (ESM Table S3). We measured the samples of each synthetic miRNA at the concentrations from 0 to 1000 pM. Concentrations of synthetic miRNAs which interfered with the assay of miR-21-5p were estimated by the dose-response curve of miR-21-5p. The concentration was defined as interfering miR-21-5p concentration (Fig. 3b). The cross reactivity was evaluated with the ratio of the interfering miR-21-5p concentration to each synthetic miRNA concentration. No cross reactivity was observed for fourteen miRNAs (miR-15a-5p, miR-30a-3p, miR-195-5p, miR-491-3p, miR-16-1-3p, miR-34a-5p, miR-200a-3p, miR-337-5p, miR-340-5p, miR-425-5p, miR-9-1-5p, miR-338-3p, miR-505-5p, miR-660-5p) (Fig. 3b). At the highest, 0.02% of cross reactivity was found in miR-590-5p at the concentration of 1000 pM, which has 13 bases identical to those in miR-21-5p sequence. These results indicated high sequence specificity of the proposed miR-21-5p assay.
Automated miR-21-5p detection with two-step amplification on the analyzer
The detection sensitivity of the one-step amplification assay for miR-21-5p in the range from 10 to 100 fM was not sufficient because the cutoff concentration of miR-21-5p in serum and plasma for cancer diagnosis is around 50 fM [13]. We extended the one-step amplification assay to the two-step amplification assay by layering linear amplification reactions [41]. We measured different concentrations of synthetic miR-21-5p from 0 to 1000 pM in replicates of 5 on the analyzer. The obtained dose-response curve is shown in Fig. 4a. The curve fitting of the dose-response curve with fitted equation and correlation coefficient is shown in ESM Fig. S3A. For determining the precise detection limit, we further measured different concentrations of synthetic miR-21-5p from 0 to 50 fM in the samples in replicates of 20 (Fig. 4b). The sample with 0-fM (blank) and 3-fM miR-21-5p showed the mean CI of 79 relative light unit (RLU) (standard deviation (SD) 15) and 322 RLU (SD 30), respectively. The 3 fM of miR-21-5p was distinguished from the blank sample (P (two-tailed unpaired t test) < 10−21). The two-step amplification assay achieved the detection sensitivity value of 3 fM for miR-21-5p, significantly lower than the cutoff concentrations in serum around 50 fM [13]. This highly sensitive detection is attributed not only to the introduction of cover sequence and layering linear amplification reactions, but also to the application of chemiluminescence reaction with Capture DNA probe and Chemiluminescence DNA probe emulating the common sandwich assay in immunoassay. This reaction design is suitable for the fully automated immunoassay analyzer.
In Fig. 4a, the CVs of CI for the target at concentrations from 0.01 to 1000 pM were less than 8%. The CV of CI even at low target concentration of 0.001 pM and that of the background CI at 0 pM were both 18%. In Fig. 4b, the CVs of CI for the target at concentrations of 3 fM were 9%, and those at five concentrations from 5 to 50 fM were less than 8%. These results indicated the high sensitivity and reproducibility of the assay.
Amplification rates as the ratios of amplified Signal DNA2 concentration to the target miR-21-5p concentration were estimated by using the calibration curve of Signal DNA2. The calibration curve of Signal DNA2 was generated in the measurement with serially diluted Signal DNA2 in 10 mM Tris-HCl, 0.01% BSA (pH 8.0) (ESM Fig. S3B, C). The mean amplification rate for the target concentrations from 0.1 to 10 pM was 103-fold. For comparison, we also estimated the amplification rate in the one-step amplification assay (ESM Fig. S4), based on the results shown in Fig. 3a. The obtained mean amplification rate for the target miR-21-5p at concentrations from 0.1 to 10 pM was 26-fold. The cascading of signal DNA generation achieved the higher amplification rate in the two-step amplification assay for miR-21-5p than that in the one-step amplification assay.
We also investigated whether precursor miR-21-5p (60 nt), which contains mature miR-21-5p sequence and forms a secondary structure, and mature miR-21-5p (22 nt) were distinguished in the two-step amplification assay. The samples of synthetic precursor miR-21-5p and mature miR-21-5p at the concentrations from 0 to 1000 pM were measured. The cross reactivity evaluated as the ratio of interfering mature miR-21-5p concentration to precursor miR-21-5p concentration was, at the highest, 0.01% at the concentration of 1000 pM (Fig. 4c). The result indicated that the miR-21-5p assay with Converter DNA-21 preferably detected mature miR-21-5p than precursor miR-21-5p.
Automated miR-200 family detection with two-step amplification on the analyzer
The miR-200 family consists of miR-200a, miR-200b, miR-200c, miR-141, and miR-429 which have highly homologous sequences (ESM Table S1B) [39]. We investigated the cross reactivity of the two-step amplification assays for miR-200a, miR-200b, and miR-200c by evaluating the ratio of interfering miRNA concentrations of each assay to each synthetic miRNA concentration. The samples of synthetic miRNAs, each containing miR-200a, miR-200b, miR-200c, miR-141, or miR-429 at the concentrations from 0 to 1000 pM, were measured in three respective assays with Converter DNAs having TBS for miR-200a, miR-200b, and miR-200c. In the assay for miR-200a, the cross reactivity values were not higher than 0.20, 0.01, 0.30, and 0.19% for miR-200b, miR-200c, miR-141, and miR-429, respectively (Fig. 5a). In the assay for miR-200b, the cross reactivity values were not higher than 0.06, 0.64, 0.02, and 0.01% for miR-200a, miR-200c, miR-141, and miR-429, respectively (Fig. 5b). In the assay for miR-200c, the cross reactivity values were 0.003, 0.44, 0.07, and 0.002% for miR-200a, miR-200b, miR-141, and miR-429, respectively (Fig. 5c). The similar miRNA sequences did not cause the false positive signal higher than 0.64%, showing high sequence specificity of the two-step amplification assays for miR-200 family detection.
These results indicated that our miRNA assays with two-step amplification on the analyzer have high reproducibility and sequence specificity and could be clinically useful in cancer detection.
Automated nucleic acid detection in human serum on the analyzer
We performed the two-step amplification assay in normal human serum containing the ssDNA on the analyzer. MiD-21-5p spiked in serum samples at different concentrations from 0.001 to 1000 pM and a blank at 0 pM were measured in replicates of 10 and 20, respectively, on the automated analyzer. The obtained dose-response curve is shown in Fig. 6a. The curve fitting of the dose-response curve with fitted equation and correlation coefficient is shown in ESM Fig. S5A.
To determine the sensitivity of the assay, we further measured different concentrations of miD-21-5p in serum samples from 1 to 100 fM in replicates of 10 and a blank at 0 fM in replicates of 20 (Fig. 6b). The mean CI calculated for the target miD-21-5p at the concentrations of 0 and 10 fM were 840 RLU (SD 60) and 1101 RLU (SD 100), respectively. The 10 fM of miD-21-5p in serum was distinguished from the blank serum sample (P (two-tailed unpaired t test) < 10−8). The CVs of CI in the measurement with the target miD-21-5p in serum at concentrations from 0 to 1000 pM were less than 8% except for 9% at 0.01 pM, which again represented the high reproducibility of the assay. The mean amplification rate for the target concentrations from 0.1 to 10 pM was 126-fold (see ESM Fig. S5B, C). These results indicated that the two-step amplification assay for ssDNA in normal human serum had high detection sensitivity and reproducibility.
The physiological temperature condition of constant 37 °C eliminated the need to purify the miRNA due to blood clotting caused by high-temperature conditions. Also, detergent addition, which is required for detaching miRNAs from the protein or breaking down exosomes in human serum [42], was implemented in the fully automated process.