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Research article

Biotechnology and Bioengineering (BB)  |  Cell Culture and Biomedical Engineering

Sensitive and Enzyme-Free Pseudomonas aeruginosa Detection and Isolation via DNAzyme Cascade Triggered DNA Tweezer

Furong Liu1†, Jingyuan Xu1†, and Lihua Yang2*

1Department of Urology, People’s Hospital of Chongqing Liang Jiang New Area, Chongqing, 401147, P.R. China
2Medical insurance pricing department, People’s Hospital Of Chongqing Liang Jiang New Area, Chongqing, 401147, P.R. China

Correspondence to:
Lihua Yang,      18523311807@163.com

†These authors contributed equally to this work.
Received: July 5, 2024; Revised: July 27, 2024; Accepted: July 28, 2024

J. Microbiol. Biotechnol. 2024; 34(9): 1919-1925

Published September 28, 2024 https://doi.org/10.4014/jmb.2407.07006

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

Effective isolation and sensitive detection of Pseudomonas aeruginosa (P. aeruginosa) is crucial for the early diagnosis and prognosis of various diseases, such as urinary tract infections. However, efficient isolation and simultaneous detection of P. aeruginosa remains a huge challenge. Herein, we depict a novel fluorescence assay for sensitive, enzyme-free detection of P. aeruginosa by integrating DNAzyme cascade-induced DNA tweezers and magnetic nanoparticles (MNPs)-based separation. The capture probe@MNPs is capable of accurately identifying target bacteria and transporting the bacteria signal to nucleic acid signals. Based on the DNAzyme cascade-induced DNA tweezers, the nucleic acid signals are extensively amplified, endowing the method with a high sensitivity and a low detection limit of 1 cfu/mL. In addition, the method also exhibits a wide detection of six orders of magnitudes. The proposed method could be extended to other bacteria detection by simply changing the aptamer sequence. Taking the merit of the high sensitivity, greatly minimized detection time (less than 1.5 h), enzyme-free characteristics, and stability, the proposed method could be potentially applied to diagnosing and preventing diseases caused by pathogenic bacteria.

Keywords

Pseudomonas aeruginosa, DNAzyme, DNA tweezer, urinary tract infections, magnetic nanoparticles

Graphical Abstract


References

  1. Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. 2015. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat. Rev. Microbiol. 13: 269-284.
  2. Tullus K, Shaikh N. 2020. Urinary tract infections in children. Lancet 395: 1659-1668.
  3. Mittal R, Aggarwal S, Sharma S, Chhibber S, Harjai K. 2009. Urinary tract infections caused by Pseudomonas aeruginosa: a minireview. J. Infect. Public Health 2: 101-111.
  4. Newman JN, Floyd RV, Fothergill JL. 2022. Invasion and diversity in Pseudomonas aeruginosa urinary tract infections. J. Med. Microbiol. 71: 001458.
  5. Penaranda GE, Suasnabar DF, Foia E, Finello M, Ellena Leon MF, Panchuk A, et al. 2020. Urinary tract infections in hospitalized patients. Rev. Fac. Cien. Med. Univ. Nac. Cordoba. 77: 265-271.
  6. Song W, Zhang C, Lin H, Zhang T, Liu H, Huang X. 2023. Portable rotary PCR system for real-time detection of Pseudomonas aeruginosa in milk. Lab. Chip. 23: 4592-4599.
  7. Wang C, Ye Q, Jiang A, Zhang J, Shang Y, Li F, et al. 2022. Pseudomonas aeruginosa detection using conventional PCR and quantitative real-time PCR based on species-specific novel gene targets identified by pangenome analysis. Front. Microbiol. 13: 820431.
  8. Kim JK, Yun H, Yeom CH, Kim EJ, Kim W, Lee CS, et al. 2022. Flow cytometry-based rapid detection of Staphylococcus aureus and Pseudomonas aeruginosa using fluorescent antibodies. RSC Adv. 12: 34660-34669.
  9. Khatami SH, Karami S, Siahkouhi HR, Taheri-Anganeh M, Fathi J, Aghazadeh Ghadim MB, et al. 2022. Aptamer-based biosensors for Pseudomonas aeruginosa detection. Mol. Cell. Probes 66: 101865.
  10. Hussain M, Liu X, Tang S, Zou J, Wang Z, Ali Z, et al. 2022. Rapid detection of Pseudomonas aeruginosa based on lab-on-a-chip platform using immunomagnetic separation, light scattering, and machine learning. Anal. Chim. Acta 1189: 339223.
  11. Huang S, Wang X, Chen X, Liu X, Xu Q, Zhang L, et al. 2023. Rapid and sensitive detection of Pseudomonas aeruginosa by isothermal amplification combined with Cas12a-mediated detection. Sci. Rep. 13: 19199.
  12. Shen Y, Jia F, He Y, Fu Y, Fang W, Wang J, et al. 2022. A CRISPR-Cas12a-powered magnetic relaxation switching biosensor for the sensitive detection of Salmonella. Biosens. Bioelectron. 213: 114437.
  13. Rezaei B, Yari P, Sanders SM, Wang H, Chugh VK, Liang S, et al. 2024. Magnetic nanoparticles: A review on synthesis, characterization, functionalization, and biomedical applications. Small 20: e2304848.
  14. Zhao X, Luo C, Mei Q, Zhang H, Zhang W, Su D, et al. 2020. Aptamer-cholesterol-mediated proximity ligation assay for accurate identification of exosomes. Anal. Chem. 92: 5411-5418.
  15. Zhao X, Zhang W, Qiu X, Mei Q, Luo Y, Fu W. 2020. Rapid and sensitive exosome detection with CRISPR/Cas12a. Anal. Bioanal. Chem. 412: 601-609.
  16. Bai Y, Roncancio D, Suo Y, Shao Y, Zhang D, Zhou C. 2019. A method based on amino-modified magnetic nanoparticles to extract DNA for PCR-based analysis. Colloids Surf. B. Biointerfaces 179: 87-93.
  17. Sanchez Martin D, Oropesa-Nunez R, Zardan Gomez de la Torre T. 2023. Rolling circle amplification on a bead: Improving the detection time for a magnetic bioassay. ACS Omega 8: 4391-4397.
  18. Ivanov AV, Safenkova IV, Zherdev AV, Dzantiev BB. 2019. Recombinase polymerase amplification combined with a magnetic nanoparticle-based immunoassay for fluorometric determination of troponin T. Mikrochim. Acta 186: 549.
  19. Khan S, Burciu B, Filipe CDM, Li Y, Dellinger K, Didar TF. 2021. DNAzyme-based biosensors: Immobilization strategies, applications, and future prospective. ACS Nano 15: 13943-13969.
  20. Nie N, Tang W, Ding X, Guo X, Chen Y. 2022. DNAzyme based dual signal amplification strategy for ultrasensitive myocardial ischemia related MiRNA detection. Anal. Biochem. 640: 114543.
  21. Yang H, Weng B, Liu S, Kang N, Ran J, Deng Z, et al. 2022. Acid-improved DNAzyme-based chemiluminescence miRNA assay coupled with enzyme-free concatenated DNA circuit. Biosens. Bioelectron. 204: 114060.
  22. Chen H, Sun X, Cai R, Tian Y, Zhou N. 2019. Switchable DNA tweezer and G-quadruplex nanostructures for ultrasensitive voltammetric determination of the K-ras gene fragment. Mikrochim. Acta 186: 843.
  23. Yun W, Zhu H, Wu H, Zhuo L, Wang R, Ha X, et al. 2021. A "turn-on" and proximity ligation assay dependent DNA tweezer for onestep amplified fluorescent detection of DNA. Spectrochim. Acta A Mol. Biomol. Spectrosc. 249: 119292.
  24. Zhang R, Chen R, Ma Y, Liang J, Ren S, Gao Z. 2023. Application of DNA Nanotweezers in biosensing: Nanoarchitectonics and advanced challenges. Biosens. Bioelectron. 237: 115445.

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Article

Research article

J. Microbiol. Biotechnol. 2024; 34(9): 1919-1925

Published online September 28, 2024 https://doi.org/10.4014/jmb.2407.07006

Copyright © The Korean Society for Microbiology and Biotechnology.

Sensitive and Enzyme-Free Pseudomonas aeruginosa Detection and Isolation via DNAzyme Cascade Triggered DNA Tweezer

Furong Liu1†, Jingyuan Xu1†, and Lihua Yang2*

1Department of Urology, People’s Hospital of Chongqing Liang Jiang New Area, Chongqing, 401147, P.R. China
2Medical insurance pricing department, People’s Hospital Of Chongqing Liang Jiang New Area, Chongqing, 401147, P.R. China

Correspondence to:Lihua Yang,      18523311807@163.com

†These authors contributed equally to this work.

Received: July 5, 2024; Revised: July 27, 2024; Accepted: July 28, 2024

Abstract

Effective isolation and sensitive detection of Pseudomonas aeruginosa (P. aeruginosa) is crucial for the early diagnosis and prognosis of various diseases, such as urinary tract infections. However, efficient isolation and simultaneous detection of P. aeruginosa remains a huge challenge. Herein, we depict a novel fluorescence assay for sensitive, enzyme-free detection of P. aeruginosa by integrating DNAzyme cascade-induced DNA tweezers and magnetic nanoparticles (MNPs)-based separation. The capture probe@MNPs is capable of accurately identifying target bacteria and transporting the bacteria signal to nucleic acid signals. Based on the DNAzyme cascade-induced DNA tweezers, the nucleic acid signals are extensively amplified, endowing the method with a high sensitivity and a low detection limit of 1 cfu/mL. In addition, the method also exhibits a wide detection of six orders of magnitudes. The proposed method could be extended to other bacteria detection by simply changing the aptamer sequence. Taking the merit of the high sensitivity, greatly minimized detection time (less than 1.5 h), enzyme-free characteristics, and stability, the proposed method could be potentially applied to diagnosing and preventing diseases caused by pathogenic bacteria.

Keywords: Pseudomonas aeruginosa, DNAzyme, DNA tweezer, urinary tract infections, magnetic nanoparticles

References

  1. Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. 2015. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat. Rev. Microbiol. 13: 269-284.
  2. Tullus K, Shaikh N. 2020. Urinary tract infections in children. Lancet 395: 1659-1668.
  3. Mittal R, Aggarwal S, Sharma S, Chhibber S, Harjai K. 2009. Urinary tract infections caused by Pseudomonas aeruginosa: a minireview. J. Infect. Public Health 2: 101-111.
  4. Newman JN, Floyd RV, Fothergill JL. 2022. Invasion and diversity in Pseudomonas aeruginosa urinary tract infections. J. Med. Microbiol. 71: 001458.
  5. Penaranda GE, Suasnabar DF, Foia E, Finello M, Ellena Leon MF, Panchuk A, et al. 2020. Urinary tract infections in hospitalized patients. Rev. Fac. Cien. Med. Univ. Nac. Cordoba. 77: 265-271.
  6. Song W, Zhang C, Lin H, Zhang T, Liu H, Huang X. 2023. Portable rotary PCR system for real-time detection of Pseudomonas aeruginosa in milk. Lab. Chip. 23: 4592-4599.
  7. Wang C, Ye Q, Jiang A, Zhang J, Shang Y, Li F, et al. 2022. Pseudomonas aeruginosa detection using conventional PCR and quantitative real-time PCR based on species-specific novel gene targets identified by pangenome analysis. Front. Microbiol. 13: 820431.
  8. Kim JK, Yun H, Yeom CH, Kim EJ, Kim W, Lee CS, et al. 2022. Flow cytometry-based rapid detection of Staphylococcus aureus and Pseudomonas aeruginosa using fluorescent antibodies. RSC Adv. 12: 34660-34669.
  9. Khatami SH, Karami S, Siahkouhi HR, Taheri-Anganeh M, Fathi J, Aghazadeh Ghadim MB, et al. 2022. Aptamer-based biosensors for Pseudomonas aeruginosa detection. Mol. Cell. Probes 66: 101865.
  10. Hussain M, Liu X, Tang S, Zou J, Wang Z, Ali Z, et al. 2022. Rapid detection of Pseudomonas aeruginosa based on lab-on-a-chip platform using immunomagnetic separation, light scattering, and machine learning. Anal. Chim. Acta 1189: 339223.
  11. Huang S, Wang X, Chen X, Liu X, Xu Q, Zhang L, et al. 2023. Rapid and sensitive detection of Pseudomonas aeruginosa by isothermal amplification combined with Cas12a-mediated detection. Sci. Rep. 13: 19199.
  12. Shen Y, Jia F, He Y, Fu Y, Fang W, Wang J, et al. 2022. A CRISPR-Cas12a-powered magnetic relaxation switching biosensor for the sensitive detection of Salmonella. Biosens. Bioelectron. 213: 114437.
  13. Rezaei B, Yari P, Sanders SM, Wang H, Chugh VK, Liang S, et al. 2024. Magnetic nanoparticles: A review on synthesis, characterization, functionalization, and biomedical applications. Small 20: e2304848.
  14. Zhao X, Luo C, Mei Q, Zhang H, Zhang W, Su D, et al. 2020. Aptamer-cholesterol-mediated proximity ligation assay for accurate identification of exosomes. Anal. Chem. 92: 5411-5418.
  15. Zhao X, Zhang W, Qiu X, Mei Q, Luo Y, Fu W. 2020. Rapid and sensitive exosome detection with CRISPR/Cas12a. Anal. Bioanal. Chem. 412: 601-609.
  16. Bai Y, Roncancio D, Suo Y, Shao Y, Zhang D, Zhou C. 2019. A method based on amino-modified magnetic nanoparticles to extract DNA for PCR-based analysis. Colloids Surf. B. Biointerfaces 179: 87-93.
  17. Sanchez Martin D, Oropesa-Nunez R, Zardan Gomez de la Torre T. 2023. Rolling circle amplification on a bead: Improving the detection time for a magnetic bioassay. ACS Omega 8: 4391-4397.
  18. Ivanov AV, Safenkova IV, Zherdev AV, Dzantiev BB. 2019. Recombinase polymerase amplification combined with a magnetic nanoparticle-based immunoassay for fluorometric determination of troponin T. Mikrochim. Acta 186: 549.
  19. Khan S, Burciu B, Filipe CDM, Li Y, Dellinger K, Didar TF. 2021. DNAzyme-based biosensors: Immobilization strategies, applications, and future prospective. ACS Nano 15: 13943-13969.
  20. Nie N, Tang W, Ding X, Guo X, Chen Y. 2022. DNAzyme based dual signal amplification strategy for ultrasensitive myocardial ischemia related MiRNA detection. Anal. Biochem. 640: 114543.
  21. Yang H, Weng B, Liu S, Kang N, Ran J, Deng Z, et al. 2022. Acid-improved DNAzyme-based chemiluminescence miRNA assay coupled with enzyme-free concatenated DNA circuit. Biosens. Bioelectron. 204: 114060.
  22. Chen H, Sun X, Cai R, Tian Y, Zhou N. 2019. Switchable DNA tweezer and G-quadruplex nanostructures for ultrasensitive voltammetric determination of the K-ras gene fragment. Mikrochim. Acta 186: 843.
  23. Yun W, Zhu H, Wu H, Zhuo L, Wang R, Ha X, et al. 2021. A "turn-on" and proximity ligation assay dependent DNA tweezer for onestep amplified fluorescent detection of DNA. Spectrochim. Acta A Mol. Biomol. Spectrosc. 249: 119292.
  24. Zhang R, Chen R, Ma Y, Liang J, Ren S, Gao Z. 2023. Application of DNA Nanotweezers in biosensing: Nanoarchitectonics and advanced challenges. Biosens. Bioelectron. 237: 115445.