A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a

English version

Autoři: Florian Katzmeier ^aff001; Lukas Aufinger ^aff001; Aurore Dupin ^aff001; Jorge Quintero ^aff002; Matthias Lenz ^aff001; Ludwig Bauer ^aff001; Sven Klumpe ^aff001; Dawafuti Sherpa ^aff002; Benedikt Dürr ^aff002; Maximilian Honemann ^aff001; Igor Styazhkin ^aff001; Friedrich C. Simmel ^aff001; Michael Heymann ^aff003
Působiště autorů: Physics Department and ZNN, Technical University of Munich, Garching, Germany ^aff001; Department of Biology, Ludwig-Maximilians-Universität Munich, Martinsried, Germany ^aff002; Intelligent Biointegrative Systems Group, Institute for Biomaterials and Biomolecular Systems, University Stuttgart, Germany ^aff003
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0220091

Souhrn

Point-of-care testing (POCT) in low-resource settings requires tools that can operate independently of typical laboratory infrastructure. Due to its favorable signal-to-background ratio, a wide variety of biomedical tests utilize fluorescence as a readout. However, fluorescence techniques often require expensive or complex instrumentation and can be difficult to adapt for POCT. To address this issue, we developed a pocket-sized fluorescence detector costing less than $15 that is easy to manufacture and can operate in low-resource settings. It is built from standard electronic components, including an LED and a light dependent resistor, filter foils and 3D printed parts, and reliably reaches a lower limit of detection (LOD) of ≈ 6.8 nM fluorescein, which is sufficient to follow typical biochemical reactions used in POCT applications. All assays are conducted on filter paper, which allows for a flat detector architecture to improve signal collection. We validate the device by quantifying in vitro RNA transcription and also demonstrate sequence-specific detection of target RNAs with an LOD of 3.7 nM using a Cas13a-based fluorescence assay. Cas13a is an RNA-guided, RNA-targeting CRISPR effector with promiscuous RNase activity upon recognition of its RNA target. Cas13a sensing is highly specific and adaptable and in combination with our detector represents a promising approach for nucleic acid POCT. Furthermore, our open-source device may be used in educational settings, through providing low cost instrumentation for quantitative assays or as a platform to integrate hardware, software and biochemistry concepts in the future.

Klíčová slova:

Artificial light – Colorimetric assays – Filter paper – Fluorescence – Nucleic acids – Ribonucleases – Signal filtering – Signal processing

Zdroje

1. Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, et al. Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing. Clin. Microbiol. Rev. 2006; 19:93. doi: 10.1128/CMR.19.1.165-256.2006

2. Wang S, Lifson MA, Inci F, Liang LG, Sheng YF, Demirci U. Advances in addressing technical challenges of point-of-care diagnostics in resource-limited settings. Expert Rev. Mol. Diagn. 2016; 16(4):449–459. doi: 10.1586/14737159.2016.1142877 26777725

3. Sia SK, Linder V, Parviz BA, Siegel A, Whitesides GM. An Integrated Approach to a Portable and Low-Cost Immunoassay for Resource-Poor Settings. Angew. Chem. Int. Ed. 2004; 43(4):498–502. doi: 10.1002/anie.200353016

4. Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. 2018; 360(6387):439–444. doi: 10.1126/science.aaq0179 29449508

5. Sharma S, Zapatero-Rodríguez J, Estrela P, O’Kennedy R. Point-of-Care Diagnostics in Low Resource Settings: Present Status and Future Role of Microfluidics. Biosensors. 2015; 5(3):577–601. doi: 10.3390/bios5030577 26287254

6. Vashist SK, Luppa PB, Yeo LY, Ozcan A, Luong JHT. Emerging Technologies for Next-Generation Point-of-Care Testing. Trends Biotechnol. 2015; 33(11):692–705. doi: 10.1016/j.tibtech.2015.09.001 26463722

7. Yang FB, Pan JZ, Zhang T, Fang Q. A low-cost light-emitting diode induced fluorescence detector for capillary electrophoresis based on an orthogonal optical arrangement. Talanta. 2009; 78(3):1155–1158. doi: 10.1016/j.talanta.2009.01.033 19269486

8. Wu J, Liu X, Wang L, Dong L, Pu Q. An economical fluorescence detector for lab-on-a-chip devices with a light emitting photodiode and a low-cost avalanche photodiode. Analyst. 2012; 137(2):519–525. doi: 10.1039/c1an15867h 22129542

9. Pais A, Banerjee A, Klotzkin D, Papautsky I. High-sensitivity, disposable lab-on-a-chip with thin-film organic electronics for fluorescence detection. Lab Chip. 2008; 8(5):794. doi: 10.1039/b715143h 18432351

10. Novak L, Neuzil P, Pipper J, Zhang Y, Lee S. An integrated fluorescence detection system for lab-on-a-chip applications. Lab Chip. 2007; 7(1):27–29. doi: 10.1039/b611745g 17180202

11. Obahiagbon U, Smith JT, Zhu M, Katchman BA, Arafa H, Anderson KS, et al. A compact, low-cost, quantitative and multiplexed fluorescence detection platform for point-of-care applications. Biosens. Bioelectron. 2018; 117:153–160. doi: 10.1016/j.bios.2018.04.002 29894852

12. Dandin M, Abshire P, Smela E. Optical filtering technologies for integrated fluorescence sensors. Lab Chip. 2007; 7(8):955. doi: 10.1039/b704008c 17653336

13. Martinez AW, Phillips ST, Whitesides GM, Carrilho E. Diagnostics for the Developing World: Microfluidic Paper-Based Analytical Devices. Anal. Chem. 2010; 82(1):3–10. doi: 10.1021/ac9013989 20000334

14. Pardee K. Perspective: Solidifying the impact of cell-free synthetic biology through lyophilization. Biochem. Eng. J. 2018; 138:91–97. doi: 10.1016/j.bej.2018.07.008 30740032

15. Yetisen AK, Akram MS, Lowe CR. Paper-based microfluidic point-of-care diagnostic devices. Lab Chip. 2013; 13(12):2210. doi: 10.1039/c3lc50169h 23652632

16. Pardee K, Green A, Ferrante T, Cameron DE, DaleyKeyser A, Yin P, et al. Paper-Based Synthetic Gene Networks. Cell. 2014; 159(4):940–954. doi: 10.1016/j.cell.2014.10.004 25417167

17. Green A, Silver P, Collins J, Yin P. Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Cell. 2014; 159(4):925–939. doi: 10.1016/j.cell.2014.10.002 25417166

18. Pardee K, Green A, Takahashi M, Braff D, Lambert G, Lee J, et al. Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell. 2016; 165(5):1255–1266. doi: 10.1016/j.cell.2016.04.059 27160350

19. Piepenburg O, Williams CH, Stemple DL, Armes NA. DNA Detection Using Recombination Proteins. PLoS Biol. 2006; 4(7):e204. doi: 10.1371/journal.pbio.0040204 16756388

20. Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017; 356(6336):438–442. doi: 10.1126/science.aam9321 28408723

21. Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DBT, et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science. 2016; 353(6299):aaf5573. doi: 10.1126/science.aaf5573 27256883

22. East-Seletsky A, O’Connell MR, Burstein D, Knott GJ, Doudna JA. RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes. Mol. Cell. 2017; 66(3):373–383.e3. doi: 10.1016/j.molcel.2017.04.008 28475872

23. Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 2018; 360(6387):436–439. doi: 10.1126/science.aar6245 29449511

24. Li SY, Cheng QX, Wang JM, Li XY, Zhang ZL, Gao S, et al. CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov. 2018; 4(1):20. doi: 10.1038/s41421-018-0028-z 29707234

25. Myhrvold C, Freije CA, Gootenberg JS, Abudayyeh OO, Metsky HC, Durbin AF, et al. Field-deployable viral diagnostics using CRISPR-Cas13. Science. 2018; 360(6387):444–448. doi: 10.1126/science.aas8836 29700266

26. Paige JS, Wu KY, Jaffrey SR. RNA mimics of green fluorescent protein. Science. 2011; 333(6042):642–646. doi: 10.1126/science.1207339 21798953

27. Autour A, Westhof E, Ryckelynck M. iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications. Nucleic Acids Res. 2016; 44(6):2491–2500. doi: 10.1093/nar/gkw083 26932363

28. Reisch M. Elektronische Bauelemente: Funktion, Grundschaltungen, Modellierung mit SPICE. Springer-Verlag; 2013.

29. Datasheet ATmega48P/88P/168P/328P; 2009. Available from: https://www.sparkfun.com/datasheets/Components/SMD/ATMega328.pdf.

30. McNaught AD, Wilkinson A. IUPAC. Compendium of Chemical Terminology, 2nd ed. (the “Gold Book”). Blackwell Scientific Publications, Oxford; 1997. Available from: https://doi.org/10.1351/goldbook.L03540.

31. Tambe A, East-Seletsky A, Knott GJ, Doudna JA, O’Connell MR. RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a. Cell Rep. 2018; 24(4):1025–1036. doi: 10.1016/j.celrep.2018.06.105 30044970

32. Oesinghaus L, Simmel FC. Switching the activity of Cas12a using guide RNA strand displacement circuits. Nat. Commun. 2019; 10(1):2092. doi: 10.1038/s41467-019-09953-w 31064995

33. Stark JC, Huang A, Nguyen PQ, Dubner RS, Hsu KJ, Ferrante TC, et al. BioBits™ Bright: A fluorescent synthetic biology education kit. Sci. Adv. 2018; 4(8):eaat5107. doi: 10.1126/sciadv.aat5107 30083609

34. Stark JC, Huang A, Hsu KJ, Dubner RS, Forbrook J, Marshalla S, et al. BioBits™ Health: Classroom activities exploring engineering, biology, and human health with fluorescent readouts. ACS Synth. Biol. 2019; 8(5):1001–1009. doi: 10.1021/acssynbio.8b00381 30925042

A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a

Souhrn

Klíčová slova:

Zdroje

PLOS One

Cesta pacienta nejen s SMA do nervosvalového centra

Svět praktické medicíny 2/2025 (znalostní test z časopisu)

Eozinofilní zánět a remodelace

Svět praktické medicíny 1/2025 (znalostní test z časopisu)

Hypertrofická kardiomyopatie: Moderní přístupy v diagnostice a léčbě