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Research article
Sensitive and Enzyme-Free Pseudomonas aeruginosa Detection and Isolation via DNAzyme Cascade Triggered DNA Tweezer
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
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
Keywords
Graphical Abstract

Introduction
Urinary tract infections (UTIs) are prevalent bacterial illnesses commonly acquired in communities and hospitals [1, 2].
The magnetic nanoparticles (MNPs)-based separation technique has garnered significant interest in biological analysis as a sample preparation method due to the easily manipulable adsorption properties of MNPs, which may be controlled using a magnet [12, 13]. Thus, various functional MNPs-based separation techniques have been employed as sample preparation methods to effectively enrich targets and eliminate interfering substances in complex samples, thereby significantly improving the anti-interference capability [14, 15]. However, the MNPs-based methods are insufficiently sensitive to detect low concentrations of pathogenic bacteria. Recently, a diverse array of MNPs-based methods has been proposed by integrating with isothermal signal amplification strategies, including polymerase chain reaction (PCR) [16], rolling circle amplification (RCA) [17], and recombinase polymerase amplification [18]. For example, sensitive bacteria detection methods have been proposed by integrating MNPs-based genomic DNA isolation and PCR for signal amplification. However, the signal amplification by these isothermal signal amplification strategies requires multiple enzymes and rigorous experimental conditions. In particular, the RCA-based methods necessitate the T4 DNA ligase and DNA polymerase, whereas the PCR-based methods necessitate the polymerase and thermal cycle.
DNAzyme is a catalytic nucleic acid capable of catalyzing various chemical processes [19]. Due to its superior stability and easy-to-produce capability, DNAzyme has been extensively used in biosensing, logical computing, and nanomachine engineering [20, 21]. However, few reports exist on constructing a DNAzyme cascade for
DNA tweezers are nano-devices constructed by Watson-Crick base pairing among several DNA sequences. They possess several distinct advantages, such as a predictable structure, controllable construction, excellent biocompatibility, and easy modification. DNA tweezers have been extensively utilized in biosensing, logic processing, and medication delivery [22]. The DNA tweezers can transition between open and closed states in response to many external stimuli, including nucleic acids, proteins, enzymes, metal ions, and pH levels [23, 24].
This study proposes an innovative technique for rapid, sensitive, enzyme-free, and accurate detection of
Experimental Section
Materials and Chemicals
The oligonucleotides (Table S1) utilized in this study were procured from Sangon Biotechnology Co., Ltd.(China). Phosphate buffer saline (PBS) and sodium chloride were purchased from Shanghai Sangon Biotechnology Co., Ltd. (China).
Preparation of the Capture probe@MNPs
The carboxyl-modified MNPs were initially synthesized using a solvothermal reduction method. The aptamer@MNPs were subsequently prepared. The carboxyl-modified MNPs (15 mg) were dispersed using sonication in 500 μl of Tris-HCl (20 mM, pH 5.5). Next, 250 μl of EDC (50 mM) and 250 μl of NHS (50 mM) were added to the suspension and heated at 37°C with continuous stirring for 30 min to activate the carboxyl groups of MNPs. Subsequently, the suspension was incubated at 3°C for 30 min with 0.8 nM of amino-modified aptamer. The unfixed moieties were subsequently removed through magnetic separation. The resultants were washed three times with Tris-HCl buffer (20 mM, pH 7.4). Lastly, the aptamer-MNPs were blocked by 1 mg/ml BSA in Tris-HCl buffer (20 mM, pH 7.4). The aptamer@MNPs were resuspended in 1 ml of Tris-HCl buffer (25 mM, pH 8.5) containing 0.3 M NaCl. Subsequently, the suspension was incubated at 37°C in the dark for 40 min, and 0.8 nM of cDNA was added. Lastly, the MNPs modified by the hybrid of aptamer and cDNA (aptamer/cDNA@MNPs) were washed with Tris-HCl buffer (20 mM, pH 7.4) to remove the unfixed cDNA. The fluorescent probe@MNPs was employed to detect
Detection Procedures
The capture probe@MNPs were suspended in 50 μl of Tris-HCl buffer (20 mM, pH 7.4). Next, 30 μl of
Results and Discussion
The Working Principle of the Proposed Method for P. aeruginosa Detection
Fig. 1 illustrates the schematic illustration of a DNA tweezer dependent on aptamer-modified MNPs for the sensitive and enzyme-free detection of
-
Fig. 1. The working mechanism of the method for sensitive and enzyme-free bacteria detection.
Feasibility Analysis
To confirm the fixation of the modified hybrid of aptamer and cDNA on the surface of MNPs, the end of the cDNA was tagged with carboxyfluorescein (FAM). The fluorescence intensity of MNPs, FAM-labeled cDNA (FAM-cDNA)/MNPs, and capture probe@MNPs were quantified in Tris-HCl solutions with a concentration of 20 mM. The MNPs were separated via magnetic separation, and the fluorescence intensities were evaluated following incubation with either aptamer or cDNA. As depicted in Fig. 2A, the fluorescence signal of MNPs is negative. Moreover, the fluorescence intensity of the capture probe@MNPs was significantly elevated, suggesting the successful immobilization of the aptamer/cDNA duplex (capture probe) onto the MNPs’ surface. In contrast, the FAM-cDNA/MNPs exhibited a minimal increase in fluorescence intensity, indicating that the aptamer plays a vital role in forming the capture probe@MNPs complex. The result in Fig. 2B showed the fluorescence intensity exhibits a positive correlation with the concentration of aptamers, demonstrating that aptamer mediated the fixation of cDNA on MNPs.
-
Fig. 2. Feasibility analysis of the proposed method. (A) Fluorescence spectrum of the FAM-labeled cDNA during the construction of the capture probe@MNPs. Line 1: MNPs; Line 2: MNPs + cDNA; Line 3: MNPs + cDNA + aptamer. (B) Fluorescence intensity of the capture probe@MNPs with different concentrations of aptamer. (C) Fluorescence intensity of the method when essential components existed or not.
Several samples with varying settings were created to examine the viability of the DNAzyme cascade-dependent DNA tweezer. Fig. 2C demonstrates that the blank sample exhibits low fluorescence intensity because the DNA tweezer is in a “close” state (sample 1). Sample 2, in the absence of the S probe, has a feeble fluorescence intensity, suggesting no occurrence of DNA cascade. This indicates that the DNA tweezer remains closed. Sample 3 also exhibits a low fluorescence intensity due to the lack of target bacteria. The sample 4 exhibits a relatively low fluorescent signal, excluding the L probe. The “8” sequence was liberated upon the inclusion of the L probe, leading to the activation of DNA tweezers and a significant increase in fluorescence intensity.
Parameters Optimization of DNAzyme Cascade-Dependent DNA Tweezers
The incubation temperature is a crucial factor that can affect the thermal stability of DNA tweezers and the hybridization efficiency between target DNA and capture DNA. Therefore, the reaction temperature was first optimized in this study. The fluorescent signal initially increases with the incubation temperature. Subsequently, it significantly reduces when the temperature exceeds 37°C (Fig. 3A). This phenomenon can be explained by the reduced effectiveness of DNA binding between the target and capture DNA at elevated reaction temperatures. Therefore, the incubation temperature was selected to be 37°C.
-
Fig. 3. Optimization of experimental parameters. Fluorescence intensities of the method with different experimental temperatures (A) DNA tweezer concentrations (B) incubation time (C) and concentration of S probe and L probe (D).
The concentration of DNA tweezers is the primary factor determining their fluorescent intensity. The concentration of DNA forceps has been optimized from 100 nM to 700 nM. The concentration of DNA tweezers significantly increases the fluorescent intensity. Additionally, the fluorescent signal remains consistent when the concentration of DNA tweezers is approximately 500 nM, suggesting that the DNA tweezers are nearly saturated at this concentration (Fig. 3B).
The temperature was maintained at 37°C during the investigation of the incubation time of capture probe@MNPs with bacteria. The results indicate that the fluorescence intensity increases as the incubation time increases from 0 to 40 min. However, the fluorescence intensity reaches a plateau after 40 min. This implies that the competitive substitution of cDNA by bacteria can be completed in 40 min. Consequently, the linearity of the procedure for bacteria is investigated for 40 min (Fig. 3C). The optimized concentrations of the S and L probes were 100 nM and 200 nM, respectively, as demonstrated in Fig. 3D.
Sensitivity and Specificity of the Method
The fluorescence intensity of the DNA tweezers in the supernatant was measured for quantitative analysis of
-
Fig. 4. Analytical performance of the method. (A) The method’s fluorescence spectrum when detecting different concentrations of
P. aeruginosa . (B) The correlation between the peak fluorescence intensity and the concentration ofP. aeruginosa . (C) The method’s fluorescence intensity when detecting different bacteria. (D) The calculatedP. aeruginosa concentration with the same batch and distinct batches of the method.
This method was employed to detect
The method’s reliability was evaluated by measuring the parallel samples with the same batch and distinct batches. The method’s reproducibility is demonstrated by the fact that the intra-assay (same batch) and inter-assay (different batches) relative standard deviations (RSDs,
Detection of P. aeruginosa in Constructed Human Serum Sample
The performance of this DNA tweezer, which relies on MNPs, was assessed by detecting
-
Fig. 5. Calculated concentration of
P. aeruginosa by the method and the colony count method.
Conclusion
Concisely, we have created a highly responsive and enzyme-independent method for detecting
Supplemental Materials
Author Contributions
Lihua Yang is responsible for study conception and design; Furong Liu and Jingyuan Xu, as the co-first authors, are responsible for acquisition of data, data analysis and drafting the manuscript.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
References
- 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. - Tullus K, Shaikh N. 2020. Urinary tract infections in children.
Lancet 395 : 1659-1668. - 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. - Newman JN, Floyd RV, Fothergill JL. 2022. Invasion and diversity in
Pseudomonas aeruginosa urinary tract infections.J. Med. Microbiol. 71 : 001458. - 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. - 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. - 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. - Kim JK, Yun H, Yeom CH, Kim EJ, Kim W, Lee CS,
et al . 2022. Flow cytometry-based rapid detection ofStaphylococcus aureus andPseudomonas aeruginosa using fluorescent antibodies.RSC Adv. 12 : 34660-34669. - Khatami SH, Karami S, Siahkouhi HR, Taheri-Anganeh M, Fathi J, Aghazadeh Ghadim MB,
et al . 2022. Aptamer-based biosensors forPseudomonas aeruginosa detection.Mol. Cell. Probes 66 : 101865. - Hussain M, Liu X, Tang S, Zou J, Wang Z, Ali Z,
et al . 2022. Rapid detection ofPseudomonas aeruginosa based on lab-on-a-chip platform using immunomagnetic separation, light scattering, and machine learning.Anal. Chim. Acta 1189 : 339223. - Huang S, Wang X, Chen X, Liu X, Xu Q, Zhang L,
et al . 2023. Rapid and sensitive detection ofPseudomonas aeruginosa by isothermal amplification combined with Cas12a-mediated detection.Sci. Rep. 13 : 19199. - 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 ofSalmonella .Biosens. Bioelectron. 213 : 114437. - 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. - 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. - 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. - 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. - 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. - 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. - 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. - 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. - 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. - 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. - 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. - 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.
Related articles in JMB

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.
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
Introduction
Urinary tract infections (UTIs) are prevalent bacterial illnesses commonly acquired in communities and hospitals [1, 2].
The magnetic nanoparticles (MNPs)-based separation technique has garnered significant interest in biological analysis as a sample preparation method due to the easily manipulable adsorption properties of MNPs, which may be controlled using a magnet [12, 13]. Thus, various functional MNPs-based separation techniques have been employed as sample preparation methods to effectively enrich targets and eliminate interfering substances in complex samples, thereby significantly improving the anti-interference capability [14, 15]. However, the MNPs-based methods are insufficiently sensitive to detect low concentrations of pathogenic bacteria. Recently, a diverse array of MNPs-based methods has been proposed by integrating with isothermal signal amplification strategies, including polymerase chain reaction (PCR) [16], rolling circle amplification (RCA) [17], and recombinase polymerase amplification [18]. For example, sensitive bacteria detection methods have been proposed by integrating MNPs-based genomic DNA isolation and PCR for signal amplification. However, the signal amplification by these isothermal signal amplification strategies requires multiple enzymes and rigorous experimental conditions. In particular, the RCA-based methods necessitate the T4 DNA ligase and DNA polymerase, whereas the PCR-based methods necessitate the polymerase and thermal cycle.
DNAzyme is a catalytic nucleic acid capable of catalyzing various chemical processes [19]. Due to its superior stability and easy-to-produce capability, DNAzyme has been extensively used in biosensing, logical computing, and nanomachine engineering [20, 21]. However, few reports exist on constructing a DNAzyme cascade for
DNA tweezers are nano-devices constructed by Watson-Crick base pairing among several DNA sequences. They possess several distinct advantages, such as a predictable structure, controllable construction, excellent biocompatibility, and easy modification. DNA tweezers have been extensively utilized in biosensing, logic processing, and medication delivery [22]. The DNA tweezers can transition between open and closed states in response to many external stimuli, including nucleic acids, proteins, enzymes, metal ions, and pH levels [23, 24].
This study proposes an innovative technique for rapid, sensitive, enzyme-free, and accurate detection of
Experimental Section
Materials and Chemicals
The oligonucleotides (Table S1) utilized in this study were procured from Sangon Biotechnology Co., Ltd.(China). Phosphate buffer saline (PBS) and sodium chloride were purchased from Shanghai Sangon Biotechnology Co., Ltd. (China).
Preparation of the Capture probe@MNPs
The carboxyl-modified MNPs were initially synthesized using a solvothermal reduction method. The aptamer@MNPs were subsequently prepared. The carboxyl-modified MNPs (15 mg) were dispersed using sonication in 500 μl of Tris-HCl (20 mM, pH 5.5). Next, 250 μl of EDC (50 mM) and 250 μl of NHS (50 mM) were added to the suspension and heated at 37°C with continuous stirring for 30 min to activate the carboxyl groups of MNPs. Subsequently, the suspension was incubated at 3°C for 30 min with 0.8 nM of amino-modified aptamer. The unfixed moieties were subsequently removed through magnetic separation. The resultants were washed three times with Tris-HCl buffer (20 mM, pH 7.4). Lastly, the aptamer-MNPs were blocked by 1 mg/ml BSA in Tris-HCl buffer (20 mM, pH 7.4). The aptamer@MNPs were resuspended in 1 ml of Tris-HCl buffer (25 mM, pH 8.5) containing 0.3 M NaCl. Subsequently, the suspension was incubated at 37°C in the dark for 40 min, and 0.8 nM of cDNA was added. Lastly, the MNPs modified by the hybrid of aptamer and cDNA (aptamer/cDNA@MNPs) were washed with Tris-HCl buffer (20 mM, pH 7.4) to remove the unfixed cDNA. The fluorescent probe@MNPs was employed to detect
Detection Procedures
The capture probe@MNPs were suspended in 50 μl of Tris-HCl buffer (20 mM, pH 7.4). Next, 30 μl of
Results and Discussion
The Working Principle of the Proposed Method for P. aeruginosa Detection
Fig. 1 illustrates the schematic illustration of a DNA tweezer dependent on aptamer-modified MNPs for the sensitive and enzyme-free detection of
-
Figure 1. The working mechanism of the method for sensitive and enzyme-free bacteria detection.
Feasibility Analysis
To confirm the fixation of the modified hybrid of aptamer and cDNA on the surface of MNPs, the end of the cDNA was tagged with carboxyfluorescein (FAM). The fluorescence intensity of MNPs, FAM-labeled cDNA (FAM-cDNA)/MNPs, and capture probe@MNPs were quantified in Tris-HCl solutions with a concentration of 20 mM. The MNPs were separated via magnetic separation, and the fluorescence intensities were evaluated following incubation with either aptamer or cDNA. As depicted in Fig. 2A, the fluorescence signal of MNPs is negative. Moreover, the fluorescence intensity of the capture probe@MNPs was significantly elevated, suggesting the successful immobilization of the aptamer/cDNA duplex (capture probe) onto the MNPs’ surface. In contrast, the FAM-cDNA/MNPs exhibited a minimal increase in fluorescence intensity, indicating that the aptamer plays a vital role in forming the capture probe@MNPs complex. The result in Fig. 2B showed the fluorescence intensity exhibits a positive correlation with the concentration of aptamers, demonstrating that aptamer mediated the fixation of cDNA on MNPs.
-
Figure 2. Feasibility analysis of the proposed method. (A) Fluorescence spectrum of the FAM-labeled cDNA during the construction of the capture probe@MNPs. Line 1: MNPs; Line 2: MNPs + cDNA; Line 3: MNPs + cDNA + aptamer. (B) Fluorescence intensity of the capture probe@MNPs with different concentrations of aptamer. (C) Fluorescence intensity of the method when essential components existed or not.
Several samples with varying settings were created to examine the viability of the DNAzyme cascade-dependent DNA tweezer. Fig. 2C demonstrates that the blank sample exhibits low fluorescence intensity because the DNA tweezer is in a “close” state (sample 1). Sample 2, in the absence of the S probe, has a feeble fluorescence intensity, suggesting no occurrence of DNA cascade. This indicates that the DNA tweezer remains closed. Sample 3 also exhibits a low fluorescence intensity due to the lack of target bacteria. The sample 4 exhibits a relatively low fluorescent signal, excluding the L probe. The “8” sequence was liberated upon the inclusion of the L probe, leading to the activation of DNA tweezers and a significant increase in fluorescence intensity.
Parameters Optimization of DNAzyme Cascade-Dependent DNA Tweezers
The incubation temperature is a crucial factor that can affect the thermal stability of DNA tweezers and the hybridization efficiency between target DNA and capture DNA. Therefore, the reaction temperature was first optimized in this study. The fluorescent signal initially increases with the incubation temperature. Subsequently, it significantly reduces when the temperature exceeds 37°C (Fig. 3A). This phenomenon can be explained by the reduced effectiveness of DNA binding between the target and capture DNA at elevated reaction temperatures. Therefore, the incubation temperature was selected to be 37°C.
-
Figure 3. Optimization of experimental parameters. Fluorescence intensities of the method with different experimental temperatures (A) DNA tweezer concentrations (B) incubation time (C) and concentration of S probe and L probe (D).
The concentration of DNA tweezers is the primary factor determining their fluorescent intensity. The concentration of DNA forceps has been optimized from 100 nM to 700 nM. The concentration of DNA tweezers significantly increases the fluorescent intensity. Additionally, the fluorescent signal remains consistent when the concentration of DNA tweezers is approximately 500 nM, suggesting that the DNA tweezers are nearly saturated at this concentration (Fig. 3B).
The temperature was maintained at 37°C during the investigation of the incubation time of capture probe@MNPs with bacteria. The results indicate that the fluorescence intensity increases as the incubation time increases from 0 to 40 min. However, the fluorescence intensity reaches a plateau after 40 min. This implies that the competitive substitution of cDNA by bacteria can be completed in 40 min. Consequently, the linearity of the procedure for bacteria is investigated for 40 min (Fig. 3C). The optimized concentrations of the S and L probes were 100 nM and 200 nM, respectively, as demonstrated in Fig. 3D.
Sensitivity and Specificity of the Method
The fluorescence intensity of the DNA tweezers in the supernatant was measured for quantitative analysis of
-
Figure 4. Analytical performance of the method. (A) The method’s fluorescence spectrum when detecting different concentrations of
P. aeruginosa . (B) The correlation between the peak fluorescence intensity and the concentration ofP. aeruginosa . (C) The method’s fluorescence intensity when detecting different bacteria. (D) The calculatedP. aeruginosa concentration with the same batch and distinct batches of the method.
This method was employed to detect
The method’s reliability was evaluated by measuring the parallel samples with the same batch and distinct batches. The method’s reproducibility is demonstrated by the fact that the intra-assay (same batch) and inter-assay (different batches) relative standard deviations (RSDs,
Detection of P. aeruginosa in Constructed Human Serum Sample
The performance of this DNA tweezer, which relies on MNPs, was assessed by detecting
-
Figure 5. Calculated concentration of
P. aeruginosa by the method and the colony count method.
Conclusion
Concisely, we have created a highly responsive and enzyme-independent method for detecting
Supplemental Materials
Author Contributions
Lihua Yang is responsible for study conception and design; Furong Liu and Jingyuan Xu, as the co-first authors, are responsible for acquisition of data, data analysis and drafting the manuscript.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

Fig 5.

References
- 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. - Tullus K, Shaikh N. 2020. Urinary tract infections in children.
Lancet 395 : 1659-1668. - 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. - Newman JN, Floyd RV, Fothergill JL. 2022. Invasion and diversity in
Pseudomonas aeruginosa urinary tract infections.J. Med. Microbiol. 71 : 001458. - 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. - 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. - 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. - Kim JK, Yun H, Yeom CH, Kim EJ, Kim W, Lee CS,
et al . 2022. Flow cytometry-based rapid detection ofStaphylococcus aureus andPseudomonas aeruginosa using fluorescent antibodies.RSC Adv. 12 : 34660-34669. - Khatami SH, Karami S, Siahkouhi HR, Taheri-Anganeh M, Fathi J, Aghazadeh Ghadim MB,
et al . 2022. Aptamer-based biosensors forPseudomonas aeruginosa detection.Mol. Cell. Probes 66 : 101865. - Hussain M, Liu X, Tang S, Zou J, Wang Z, Ali Z,
et al . 2022. Rapid detection ofPseudomonas aeruginosa based on lab-on-a-chip platform using immunomagnetic separation, light scattering, and machine learning.Anal. Chim. Acta 1189 : 339223. - Huang S, Wang X, Chen X, Liu X, Xu Q, Zhang L,
et al . 2023. Rapid and sensitive detection ofPseudomonas aeruginosa by isothermal amplification combined with Cas12a-mediated detection.Sci. Rep. 13 : 19199. - 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 ofSalmonella .Biosens. Bioelectron. 213 : 114437. - 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. - 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. - 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. - 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. - 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. - 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. - 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. - 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. - 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. - 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. - 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. - 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.