博文
SERS-RCA biosensor for profiling dual miRNAs
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aKey Laboratory of Hainan Trauma and Disaster Rescue, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou 571199, China
bEngineering Research Center for Hainan Bio-Smart Materials and Bio-Medical Devices, College of Emergency and Trauma, Hainan Medical University, Haikou 571199, China
cCollege of Pharmacy, Hainan Medical University, Haikou 571199, China
Received 4 August 2023, Revised 26 December 2023, Accepted 21 January 2024, Available online 24 January 2024, Version of Record 25 January 2024.
Chemical Engineering Journal
Volume 482, 15 February 2024, 149012
https://doi.org/10.1016/j.cej.2024.149012
Highlights•A SERS-RCA microdroplet biosensor for precise IPF diagnosis via padlock probes.
•Integrated RCA chip enhances repeatability in SERS detection.
•SERS-RCA offers ultra-low miRNA detection limits with broad dynamic ranges.
•Dual miRNA-21 and miRNA-155 detection boosts IPF diagnosis accuracy (AUC: 0.884).
•SERS-RCA sensors effectively assess IPF risk for treatment monitoring.
Accurate detection of low-abundance, highly homologous miRNAs is crucial for the precise diagnosis and monitoring of idiopathic pulmonary fibrosis (IPF). In this study, a fully integrated microdroplet analysis platform based on rolling circle amplification (RCA) technology and surface-enhanced Raman spectroscopy (SERS) has been developed to precisely detect miRNA-21 and miRNA-155 in the serum of IPF patients. By utilizing the RCA strategy, the constructed sensor is able to realize single nucleotide variation detection with prominent specificity. The integration of microfluidics provides satisfactory directions to the reproducibility issue in SERS, thereby improving detecting sensitivity. With this sophisticated biosensing platform, low detection limits of 0.398 fM and 0.215 fM have been achieved for miRNA-21 and miRNA-155, respectively. Moreover, the combined determination of these two miRNAs demonstrates a more substantial diagnostic potential, with an AUC value of 0.884, higher than their evaluations. When used in conjunction with high-resolution chest computed tomography, this innovative SERS-RCA microfluidic biosensor can serve as an auxiliary diagnostic tool for IPF risk assessment, particularly for real-time monitoring of miRNA levels in patients undergoing chemotherapy. This sensing technique holds promise to extend into versatile platforms for diverse miRNA detection in the rapid and accurate diagnosis of various diseases beyond IPF.
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung disease with a mortality rate higher than most cancers, leading it to be referred to as a “cancer-like disease”[1]. Due to the lack of typical symptoms in the early stages, most patients are diagnosed when the disease has progressed to the middle and late stages, resulting in irreversible lung damage, prolonged suffering, and poor prognosis [2]. Currently, the diagnosis of pulmonary fibrosis largely relies on invasive procedures, with tissue biopsy confirming the presence of abnormal extracellular matrix (ECM) deposition and destruction of tissue architecture [3]. However, histologic assessment of pulmonary fibrosis is often semi-quantitative, and subjective, potentially causing harm to patients and increasing the risk of complications [4]. Recent studies have shown that certain microRNAs (miRNAs) are closely associated with the development and progression of IPF [5], [6]. Further research has revealed that miRNA-21 and miRNA-155, as IPF-related miRNAs, are significantly upregulated in both IPF model mice and patient serum, promote epithelial-mesenchymal transition (EMT) through TGF-β signalling, and positively correlate with IPF progression [7], [8], [9]. Given the characteristics of these two miRNAs in the IPF process, the combined detection of miRNA-21 and miRNA-155 may not only improve the diagnostic efficacy and prognostic assessment of IPF but also assist clinicians in devising more effective treatment strategies. However, due to the low abundance and high sequence homology of miRNAs in IPF, the accurate detection of these miRNAs remains challenging. Therefore, the rapid, sensitive, and selective detection of miRNA-21 and miRNA-155 expression levels is crucial for accurate diagnosis and disease monitoring in IPF.
Scheme 1. A) principle of the SERS-RCA-microfluidic biosensor for detecting ipf-related mirna. b) design of the microfluidic chip. i: Target miRNA recognition and ligation. ii: RCA initiation, where streptavidin-coated MBs&zipDNA, DNA polymerase, DTT, dNTP, and buffer are introduced. iii: Au NPs coupling area, where Au NPs&MGITC&pDNA are introduced. iv: SERS signal detection area, where a magnet is placed to capture MB-RCA-Au NPs complexes and separate the supernatant. c) Design of the padlock probe and functional descriptions of the D1, D2, P, and zip regions. d) i: Target recognition. ii) RCA initiation. iii-iv) SERS signal output, where RCA products are connected to Au NPs in iii, and MB-RCA-Au NPs complexes are magnetically captured and subjected to SERS detection in iv.
Fig. 1. Design and Construction of functional Au NPs-DNA conjugates, MBs and SERS-RCA complexes. a) Scheme of linking thiolated DNA to Au NPs utilizing a microwave-assisted method during the heating-dry process under the protection of N2. b) Zeta potential analysis and c) DLS characterization of Au NPs, Au NPs&MGITC, and Au NPs&MGITC&DNA, respectively. d) Quantitative analysis of the number of DNA bound on Au NPs and MBs, respectively. e) Raman intensity at 1614 cm-1 of SERS-RCA complexes triggered by the padlock probes with (5′-folding, middle-folding, and 3′ folding) and without secondary structure in the detection of miRNA-21 and miRNA-155, respectively. f) The optimized sequences of miRNA-21 and miRNA-155 with no secondary structure. g) Electrophoretic identification of the circularization products upon target recognition and ligation in the presence of an unlocked padlock probe, target miRNA-21, and T4 ligase (left). Electrophoresis analysis of different ligation reaction times from 10 to 60 min (right). h) Identification of the RCA reaction's amplification products by electrophoresis in the existence of a padlock probe for miRNA-21, zipDNA, and phi29 DNA polymerase (left). Electrophoresis analysis of different RCA reaction times from 0 to 60 min (right). i) Raman intensity at 1614 cm-1 of SERS-RCA complexes with different ligation times (10 min, 30 min, 60 min) and different amplification times (0 min, 10 min, 30 min, 60 min), respectively. j) TEM characterization of the constructed MB-SERS-RCA complexes, it consists of MB (Orange dotted line), Au NPs (Red dotted line) and RCA products (Blue arrow). Data are presented as means ± SEM, n = 3, NS = not significant, *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Comprehensive representation and functionality of the microfluidic system. a) Composite image featuring a physical realization of the microfluidic chip alongside a layered schematic diagram comprising a 3D-printed nylon substrate, a ceramic heating layer, a glass slide, and a PDMS layer, together forming the structural integrity of the device. b) Graphical illustration of the microdroplet sensor purposed for targeted miRNA detection. The chip encompasses five specialized compartments and three thermally controlled regions, each possessing unique attributes: i) Zone for droplet formation and combination to enable ambient-temperature ligation. ii) Sector for integrated component interaction facilitating RCA reaction in a dedicated 45 °C region. iii) Area for component assembly and the subsequent initial unfolding of RCA products in a 95 °C region, in conjunction with the complementary pairing of RCA products and SERS probe within a 65 °C environment. The 95 °C region also serves as the locus for enzyme denaturation and inactivation, thus curtailing relevant reactions. iv) Section for magnetic bar-mediated isolation of MB-SERS-RCA complexes, and v) Space designated for SERS analysis of the supernatant. c) Process schematic displaying the entirety of the procedure within a streamlined channel. Key parameters of each region, including the addition of reactants, associated reaction temperatures, the passage of droplets through varying temperature zones, and regional functionalities, are explicitly marked. d) Microscopic captures of distinct phases within the microfluidic device, showcasing i) the origin of droplets, ii) the primary stage of droplet fusion, iii) the secondary stage of droplet fusion, and iv) the magnetic partition of droplets within the respective compartments.
Fig. 3. Specificity, reproducibility, and stability of the SERS-RCA microdroplet sensor for miRNA detection. a) Selectivity of the SERS-RCA microfluidic platform for different targets: a blank, one-base mismatched primer to miRNA-21, random primer, miRNA-155, and miRNA-21 for the detection of miRNA-21; blank, one-base mismatched primer to miRNA-155, random primer, miRNA-155, and miRNA-21 for the detection of miRNA-155. NS: no statistical significance, *p < 0.05, **p < 0.01, ***p < 0.001. b) Comparison of the Raman intensity at 1614 cm-1. (b) Reproducibility analysis within the microfluidic chip: comparison of relative standard deviation (RSD) of Raman intensity at 1614 cm-1 for measurements performed off-chip (miRNA-21: 13.59 %, miRNA-155: 15.26 %) and on-chip (miRNA-21: 3.69 %, miRNA-155: 6.56 %). (c) Stability assessment of SERS probes by comparing Raman intensities at 1614 cm-1 between probes stored at 4 °C for 30 days and freshly synthesized probes.
Fig. 4. SERS spectra of a) miRNA-21 and b) miRNA-155 with different concentrations in the SERS-RCA microfluidic system. The concentration ranges of miRNA-21 and miRNA-155 were 1 fM – 10 nM and 1 fM – 10 nM, respectively. For quantitative assessments of miRNA-21 and miRNA-155, variations in the Raman intensity at 1614 cm-1 were selected.Calibration curves for b) miRNA-21 and d) miRNA-155, respectively, were obtained by plotting the corresponding SERS intensity against the concentration. Error bars represented the standard deviation (SD) of five replicates. Limits of detection (LOD) were determined as 0.29 fM for miRNA-21 and 0.37 fM for miRNA-155. e) Comparison of LODs and detection ranges between the proposed SERS-RCA droplet sensor and qRT-PCR. The red region represented the result area where PCR data was not obtained. Red dots represented SERS sensor results, and blue dots represented RT-PCR results. f) Bland-Altman analysis of the correlation between the two detection methods, showing the 95 % consistency bias and agreement limits. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 1. Recovery and relative standard deviation of SERS-RCA microdroplet sensor.
| No. | Added (mol/L) | Detected (mol/L) | Recovery (%) | RSD(%) | ||||
|---|---|---|---|---|---|---|---|---|
| miRNA-21 | miRNA-155 | miRNA-21 | miRNA-155 | miRNA-21 | miRNA-155 | miRNA-21 | miRNA-155 | |
| 1 | 1 × 10-8 | 1 × 10-10 | 1.03 × 10-8 | 1.1 × 10-10 | 103.3 | 110.8 | 6.3 | 1.5 |
| 2 | 1 × 10-9 | 1 × 10-11 | 9.78 × 10-10 | 1.04 × 10-11 | 97.8 | 104.4 | 3.5 | 7.3 |
| 3 | 1 × 10-10 | 1 × 10-8 | 1.05 × 10-10 | 1.02 × 10-8 | 105.1 | 102.1 | 2.1 | 5.2 |
| 4 | 1 × 10-12 | 1 × 10-14 | 1.1 × 10-12 | 9.7 × 10-15 | 110.3 | 97.4 | 5.7 | 8.0 |
| 5 | 1 × 10-14 | 1 × 10-12 | 1.04 × 10-14 | 1.07 × 10-12 | 104.5 | 107.3 | 4.8 | |
Table 2. Comparison of the SERS-based platform with other biosensors for miRNA-21/miRNA-155 detection.
| Technique | Target | LOD | Detection range | Detection time | Reference |
|---|---|---|---|---|---|
| Fluorescence | miRNA-21 | 0.35 fM | 1 fM – 1 pM | 280 min | [34] |
| Fluorescence | miRNA-21 | 55 fM | 1 pM – 10 nM | 130 min | [35] |
| SERS | miRNA-21 | 0.03 fM | 0.1 pM – 1 μM | 40 min | [36] |
| SERS | miRNA-21 | 3.5 fM | 10 fM – 0.1 μM | 120 min | [37] |
| Fluorescence | miRNA-155 | 0.18 nM | 1 nM – 0.1 μM | 135 min | [38] |
| Fluorescence | miRNA-155 | 11 pM | 50 pM – 10 nM | 95 min | [39] |
| SERS | miRNA-155 | 70.2 aM | 0.1 fM – 0.1 nM | 50 min | [40] |
| SERS | miRNA-155 | 0.22 fM | 1 fM – 10 nM | 120 min | [41] |
| qRT-PCR | miRNA-21 | 5.28 nM | 10 pM – 10 nM | 120 min | This study |
| miRNA-155 | 0.74 nM | 10 pM – 10 nM | |||
| SERS | miRNA-21 | 0.29 fM | 1 fM – 10 nM | 30 min | |
| miRNA-155 | 0.37 fM | 1 fM – 10 nM |
Fig. 5. (a) Expression levels of miRNA-21 and miRNA-155 in IPF patients and healthy individuals indicate significantly higher levels of these miRNAs in IPF patients (P < 0.01). (b) ROC curves and AUC values for miRNA-21 and miRNA-155, as well as their combination, demonstrate high diagnostic accuracy with AUC values of 0.863, 0.771, and 0.884, respectively. (c) Time-dependent variations in miRNA-155 and miRNA-21 concentrations for four IPF patients were categorized into three groups based on disease progression and chest CT results, with the green and red regions indicating concentrations below and above Youden's index cut-off points, respectively. (d) Chest CT images of the four patients corresponding to the different miRNA concentration changes shown in (c). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4. Conclusion
In conclusion, the proposed SERS-RCA microdroplet biosensor represents a promising multifunctional platform for the accurate diagnosis of IPF. The innovative integration of a fully integrated microfluidic chip with SERS-RCA technology offers remarkable sensitivity, specificity, a wide detection range, and ease of operation, surpassing the current gold standard of qRT-PCR and other similar detection methods. Compared with the traditional qRT-PCR technique, SERS-RCA is more cost-effective as it simplifies the process and minimizes the use of costly reagents. In addition, it also offers significant benefits in ease of operation by combining the advantages of microfluidic chips, enabling efficient implementation even in resource-limited environments. For current clinical POCT needs, the SERS-RCA microdroplet system faces challenges in detection throughput and equipment miniaturization. Excelling in automation and rapid response, it's limited to processing only 1 to 2 samples at a time, impacting efficiency in large hospitals, especially in fever clinics or emergency departments. The size of the SERS equipment and microdroplet pumps also hinders miniaturization, crucial for diverse clinical use. Future research will aim to increase throughput and develop more compact equipment, enhancing the system's diagnostic and risk assessment capabilities.
The clinical feasibility of the proposed method has been validated in serum samples from IPF patients and healthy individuals, demonstrating its potential for practical clinical applications. Furthermore, the analysis of ROC curves and AUC values emphasize the diagnostic potential of combined detection of miRNA-21 and miRNA-155, suggesting that they could serve as biomarkers for IPF diagnosis. Employing the SERS-RCA microdroplet biosensor technology for the simultaneous detection of target miRNAs significantly enhances the sensitivity and specificity for identifying IPF patients. By real-time monitoring of miRNA levels using SERS-RCA sensors, clinicians can conduct risk assessments of IPF patients and adjust treatment plans accordingly, serving as an assisted diagnostic tool to HRCT. These findings demonstrate the potential of SERS-RCA sensors as a supplementary diagnostic tool for IPF risk assessment, providing more accurate and comprehensive information for clinicians and improving patient outcomes. The SERS-RCA microdroplet biosensors demonstrate high specificity in miRNA detection, which is crucial for the early diagnosis of diseases such as acute renal injury. Additionally, their application is significant in the targeted detection of specific cancer biomarkers. These features establish the SERS-RCA platform as a reliable and versatile tool suitable for the rapid and accurate diagnosis of various diseases, highlighting its potential in diverse diagnostic scenarios.
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