MicroRNA (miRNA) has emerged as a promising class of non-invasive and sensitive biomarkers for cancer diagnosis due to its close association with tumorigenesis. However, the accurate detection of miRNA remains challenging owing to its short sequence, high sequence homology among family members, and low abundance in biological samples. To address the demands of early cancer diagnosis and clinical prognosis monitoring, this study designed and fabricated a colorimetric microfluidic paper-based analytical device (µPAD) for the quantitative detection of miRNA-21. Leveraging the inherent porous structure and ease of functionalization of paper-based materials, along with the specific binding of streptavidin and biotin, this chip integrates microfluidic technology with the peroxidase-mimicking activity of gold-platinum core-shell nanoparticles (Au@Pt NPs) to achieve rapid and sensitive detection of miRNA-21. In the presence of the target miRNA-21, it preferentially hybridizes with the complementary DNA (cDNA), competitively releasing the pre-hybridized Au@Pt NPs-cDNA conjugate. This conjugate then migrates via capillary action to the detection zone, where the Au@Pt NPs catalyze the oxidation of the colorless 3,3’,5,5’-tetramethylbenzidine, producing a deep blue product, thereby converting the biological recognition event into a detectable optical signal. The miRNA-21 concentration can be conveniently quantified by capturing the image with a smartphone and analyzing the grayscale intensity using the freely available Image J software. Under optimized conditions, the proposed µPAD demonstrated a linear detection range from 1 to 2000 nmol/L with a limit of detection of 0.25 nmol/L. The method also showed good specificity and was successfully applied to the detection of miRNA-21 spiked in human serum samples. This work presents a rapid, simple, and cost-effective paper-based platform with considerable potential for point-of-care testing in early cancer diagnosis.
| [1] |
Ala U. Competing endogenous RNAs, non-coding RNAs and diseases: an intertwined story. Cells, 2020, 9(7): 1574
|
| [2] |
Avanu AE, Ciubotariu AM, Dodi G. Can nano yield big insights? Oligonucleotide-based biosensors in early diagnosis of gastric cancer. Chemosensors, 2024, 12(3): 44
|
| [3] |
Cai BJ, Guo S, Li Y. MoS2-based sensor for the detection of MiRNA in serum samples related to breast cancer. Anal Methods, 2018, 10: 230-6
|
| [4] |
Cao T, Wang Y, Yu X. Co-allosteric hairpin probe for detecting MicroRNA with high specificity. RSC Adv, 2025, 15(43): 35921-9
|
| [5] |
Cardinali B, Tasso R, Piccioli P, et al. . Circulating MiRNAs in breast cancer diagnosis and prognosis. Cancers, 2022, 14(9): 2317
|
| [6] |
Chi H, Cui X, Lu Y, et al. . Colorimetric determination of cysteine based on Au@ Pt nanoparticles as oxidase mimetics with enhanced selectivity. Microchim Acta, 2022, 189(1): 13
|
| [7] |
Chi Y, Cheng H, Sun DW, et al. Food nanozymology: nanozyme engineering toward future food. Adv Mater. 2025;2505552. https://doi.org/10.1002/adma.202505552.
|
| [8] |
Das D, Singh T, Ahmed I, et al. . Effects of relative humidity and paper geometry on the imbibition dynamics and reactions in lateral flow assays. Langmuir, 2022, 38(32): 9863-73
|
| [9] |
Fatica A, Bozzoni I. Long non-coding rnas: new players in cell differentiation and development. Nat Rev Genet, 2014, 15(1): 7-21
|
| [10] |
Gao ZQ, Ye HH, Tang DY, et al. . Platinum-decorated gold nanoparticles with dual functionalities for ultrasensitive colorimetric in vitro diagnostics. Nano Lett, 2017, 17(9): 5572-9
|
| [11] |
Gomez-Gijon S, Ortiz‐Gómez I, Rivadeneyra A. Paper‐based electronics: toward sustainable electronics. Adv Sustain Syst, 2025, 9(1): 2400486
|
| [12] |
Jiang W, Chen Z, Lu J, et al. . Ultrasensitive visual detection of miRNA-143 using a CRISPR/Cas12a-based platform coupled with hyperbranched rolling circle amplification. Talanta, 2023, 251: 123784
|
| [13] |
Kizhepat S, Rasal AS, Chang JY, et al. . Development of two-dimensional functional nanomaterials for biosensor applications: opportunities, challenges, and future prospects. Nanomaterials, 2023, 13(9): 1520
|
| [14] |
Kumar L, Mohan L, Anand R, et al. . Chlorella minutissima-assisted silver nanoparticles synthesis and evaluation of its antibacterial activity. Syst Microbiol Biomanufacturing, 2024, 4(1): 230-9
|
| [15] |
Kutluk H, Bruch R, Urban GA, et al. . Impact of assay format on MiRNA sensing: electrochemical microfluidic biosensor for MiRNA-197 detection. Biosens Bioelectron, 2020, 148: 111824
|
| [16] |
Li S, Zhang Y, Wang Q et al. Nanozyme-enabled analytical chemistry. Analytical Chem. 2021; 94(1): 312–323. https://orcid.org/0000-0003-0870-7142.
|
| [17] |
Li CC, Hu J, Zou X, et al. . Construction of a structure-switchable toehold dumbbell probe for sensitive and label-free measurement of MicroRNA in cancer cells and tissues. Anal Chem, 2022, 94(3): 1882-9
|
| [18] |
Li S, Lei Z, Sun T. The role of MicroRNAs in neurodegenerative diseases: a review. Cell Biol Toxicol, 2023, 39(1): 53-83
|
| [19] |
Liao R, He K, Chen CY, et al. . Double-strand displacement biosensor and quencher-free fluorescence strategy for rapid detection of MicroRNA. Anal Methods, 2016, 88(8): 4254-8
|
| [20] |
Noviana E, Ozer T, Carrell CS, et al. . Microfluidic paper-based analytical devices: from design to applications. Chem Rev, 2021, 121(19): 11835-85
|
| [21] |
Ongaro AE, Ndlovu Z, Sollier E, et al. . Engineering a sustainable future for point-of-care diagnostics and single-use microfluidic devices. Lab Chip, 2022, 22(17): 3122-37
|
| [22] |
Ouyang WJ, Liu ZH, Zhang GF, et al. . Enzyme-free fluorescent biosensor for miRNA-21 detection based on MnO2 nanosheets and catalytic hairpin assembly amplification. Anal Methods, 2016, 8: 8492
|
| [23] |
Pal DB, Rathoure AK, Awasthi A, et al. . Microfluidic paper-based lab‐on‐a‐chip chemiluminescence sensing for healthcare and environmental applications: a review. Luminescence, 2025, 40(10): e70324
|
| [24] |
Pandey K, Pandey M, Kumar V, et al. . Bioprocessing 4.0 in biomanufacturing: paving the way for sustainable bioeconomy. Syst Microbiol Biomanufacturing, 2024, 4(2): 407-24
|
| [25] |
Panferov VG, Wang SH, Zhang WJ, et al. . Au@Ag, Au@Pd, Au@Pt and Au@Ir nanoparticles as colorimetric and peroxidase-like labels for lateral flow assays. ACS Appl Nano Mater, 2025, 8(36): 17754-67
|
| [26] |
Pastorino U, Boeri M, Sestini S, et al. . Baseline computed tomography screening and blood MicroRNA predict lung cancer risk and define adequate intervals in the biomild trial. Ann Oncol, 2022, 33(4): 395-405
|
| [27] |
Pham XH, Tran VK, Hahm E, et al. . Synthesis of gold-platinum core-Shell nanoparticles assembled on a silica template and their peroxidase nanozyme properties. Int J Mol Sci, 2022, 23(12): 6424
|
| [28] |
Rajwar D, Ammanath G, Cheema JA, et al. . Tailoring conformation-induced chromism of polythiophene copolymers for nucleic acid assay at resource limited settings. ACS Appl Mater Interfaces, 2016, 8(13): 8349-57
|
| [29] |
Raut JR, Schöttker B, Holleczek B, et al. . A MicroRNA panel compared to environmental and polygenic scores for colorectal cancer risk prediction. Nat Commun, 2021, 12(1): 4811
|
| [30] |
Smolarz B, Durczyński A, Romanowicz H, et al. . MiRNAs in cancer (review of literature). Int J Mol Sci, 2022, 23(5): 2805
|
| [31] |
Wang ZY, Sun MH, Zhang Q, et al. . Advances in point-of-care testing of MicroRNAs based on portable instruments and visual detection. Biosensors, 2023, 13(7): 747
|
| [32] |
Welch EC, Powell JM, Clevinger TB, et al. . Advances in biosensors and diagnostic technologies using nanostructures and nanomaterials. Adv Funct Mater, 2021, 31(44): 2104126
|
| [33] |
Zhang L, Huang H, Liao M, et al. . Ultrasensitive colorimetric immunoassay for aflatoxin B1 detection in Lotus seed powder based on enhanced catalysis of Au@ Pt with in situ deposition of PtNPs. Food Chem X, 2024, 24: 102030
|
| [34] |
Zhang Y, Wei G, Liu W, et al. . Nanozymes for nanohealthcare. Nat Rev Methods Primers, 2024, 4(1): 36
|
Funding
National Natural Science Foundation of China(62301234)
Wuxi Science and Technology Development Fund Project(K20221015)
RIGHTS & PERMISSIONS
Jiangnan University
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