Access the full text.
Sign up today, get DeepDyve free for 14 days.
For the clinical diagnosis of diseases and for basic biological research, it is crucial to develop a trustworthy and efficient method for detecting small extracellular vesicles (sEVs) in multiple experimental conditions. Here, we create a colorimetric assay that enables sensitive and precise sEVs identification without the need for pricey equipment. In this assay, the exonuclease III (Exo III)-assisted signal recycle is activated by the released single-strand DNA (ssDNA) from SMBs (streptavidin magnetic beads)-aptamer-ssDNA complex after identification of sEVs. By integrating with the strand displacement amplification (SDA) process, a significant amount of double-strand DNA products with G-rich tails is produced. The G-rich tails fold to G-quadruplex under the assistance of hemin to catalyze the oxidation of TMB, yielding a color change. The approach offers a broad detection range of 5 orders of magnitudes based on the signal recycles and SDA. In addition, single-stranded DNA binding protein (SSB) is exploited in this method to minimize the background signal from non-specific digestion of Exo-III, making the method a robust tool for sEVs detection and disease diagnosis. Keywords Exonuclease III (Exo III), Single-stranded DNA binding protein (SSB), Strand displacement amplification (SDA), DNAzyme, Pancreatitis targets because they can transport proteins, nucleic Introduction acids, and other biological components to close or dis- Cell culture supernatants and biofluids contain small tant cells (Garcia-Martin et al. 2022; Zhao et al. 2022a; extracellular vesicles (sEVs), a type of phospholipid- Niel et al. 2022). With pancreatitis and pancreatic can- enclosed vesicle generated by parent cells (Malys et al. cer, which belong to the deadliest malignancy among 2021; Moller and Lobb 2020; Urabe et al. 2020). sEVs pancreatic diseases, as example, the aberrant expression are of relevance as disease indicators and therapeutic of sEVs is closely associated with cancer development (Armacki et al. 2020; Waldenmaier et al. 2021; Liu et al. *Correspondence: 2021). Thus, the creation of a technique that can iden - Feng Yu tify low abundance sEVs is important for the screening firstname.lastname@example.org Heng Cheng of cancers and helps to manage disease. In recent years, email@example.com much efforts have been made to establish sensitive and College of Chongqing Medical and Pharmaceutical College, reliable approaches for the sEVs detection. Among them, Chongqing 401331, China Department of Gastroenterology, Chenjiaqiao Hospital of Shapingba nanoparticle tracking analysis (NTA) has been employed District Affiliated to Chongqing Medical and Pharmaceutical College, extensively in clinical and experimental settings due to its Chongqing 401331, China superior capability in detecting trace amount of sEVs and Laboratory Medicine, Chenjiaqiao Hospital of Shapingba District Affiliated to Chongqing Medical and Pharmaceutical College, characterizing the size distributions (Yahata et al. 2021; Chongqing 401331, China Zhao et al. 2020a, 2022b). However, the NTA method © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Ning et al. Journal of Analytical Science and Technology (2023) 14:5 Page 2 of 7 requires expensive equipment, complicated preparation, contains a G-rich sequence that can be fold to G-quad- and laborious calculations, hampering the further appli- ruplex under the assistance of hemin to catalyze the cation in non-laboratory sceneries (Zhao et al. 2020b, oxidation of TMB, yielding a color change. In addition, 2020c). the single-stranded DNA binding protein (SSB), which Colorimetric assays have attracted considerable atten- can bind to single-stranded DNA and prevent ssDNA tion recently because they allow for simple processing from being repaired to form double-stranded DNA or and direct readout of detection result with cheap and degraded by nucleases, is exploited in this approach portable devices (Xie et al. 2012; Marin et al. 2015; Yeas- to minimize the background signal from non-specific min et al. 2020). In colorimetric assays, the presence of digestion of Exo-III, thus endowing the method a high analytes in the sensing system lead to the color change specificity. that can be observed by naked eye and measured through optical instruments. For example, Yang et al. (2020) pro- Experimental section posed a colorimetric bacteria detection approach based Materials and reagents on the Exonuclease III (Exo-III) assisted stochastic DNA The oligonucleotides used in this colorimetric assay is dual-walker for ultrafast target detection. Compared listed in Additional file 1: Table S1. The sequences were with the traditional approaches, the recently established synthesized and purified by Sangon Biotechnology colorimetric approaches possesses the advantages of Co. Ltd (Shanghai, China). Exonuclease III (Exo III) for low cost, fastness, and ease of working (Masumoto et al. sequence digestion and single-stranded DNA-binding 2022; Vogel et al. 2022). Therefore, the colorimetric assay protein (SSB) for reducing the background signal were can be potentially developed for sEVs detection. Despite obtained from New England Biolabs (Ipswich, MA, that considerable progresses have been made among the USA). Hydrogen peroxide (H O ) and deoxynucleotide 2 2 former established colorimetric assays, the drawback of solution mixture (dNTPs) were obtained from TaKaRa lacking enough sensitivity is inevitable in these colori- Biotech. Inc. (Dalian, China). The color signals of the metric approaches. Various signal amplification strategies approach was monitored by the UV–visible spectropho- have been integrated in construction of the colorimetric tometer (UV-2550, Shimadzu, Kyoto, Japan). assay to improve the sensitivity of DNA sequences detec- tion, such as rolling circle amplification (RCA) (Hamidi Preparations of the probes and Perreault 2019), exponential amplification reaction The synthesized activator probe was firstly diluted to (EXPAR) (Li et al. 2019), and strand displacement ampli- 10 μM by 1 × PBS buffer solution. The mixture was fication (SDA) (Gong et al. 2021). Among them, SDA heated to 90 °C for 10 min and was gradually cooled to method have attracted abundant attention due to its high room temperature. The assembled probes were stored at efficiency. However, the sensitivity and specificity of the 4 °C for the subsequent experiments. SDA based approach need further improvement due to the limitation of threshold value in strand displacement. Exo-III is sequence-independent enzyme that can digests Detection procedures of the established approach one strand of duplex DNA from a blunt end, 5′ overhang The Exo-III assisted signal recycles process was initiated or nick (Zhao et al. 2022b; Wang et al. 2021). Exo-III has by the addition of 2 μL sEVs (10 particles/μL), 2 μL MB been widely exploited in constructing signal recycles to probes (5 μM), 1 μL Exo-III enzyme (1 U/L), 2 μL H1 improve the sensitivity of current signal amplification probes (5 μM), 2 μL H2 probes (5 μM). The mixture was approaches. diluted to 20 μL in volume by mixing with 11 μL buffer Inspired by the unique feature of Exo-III enzyme, we solution containing Tris–HCl (10 mM), NaCl (50 mM), develop here a colorimetric approach for sensitive sEVs MgCl (10 mM), and 100 μg/mL BSA. The 20 μL solution detection by integrating the Exo-III assisted signal recy- was incubated at 37 °C for 60 min. Afterward, 200 μL of 2− cles with SDA. In this method, a SMBs (streptavidin ABTS (4 mM) and 1 μLH O (30%) were added to the 2 2 magnetic beads)-aptamer-ssDNA (single-strand DNA) mixture, and the mixture was incubated at room tem- complex is designed to specifically bind with target sEVs perature for 5 min. The absorbance of the mixture was and mediate subsequent signal amplification. In the sig - monitored by UV–visible spectrophotometer (UV-2550, nal amplification process, a hairpin structure probe is Shimadzu, Kyoto, Japan). designed and work as the activator of the amplification process through binding with released ssDNA sequences. Data analysis The hybridization between released ssDNA sequences All data in this paper are expressed as mean ± stand- and activator probe initiates the Exo-III assisted sig- ard deviations. Statistical significances between two nal recycles and SDA. The products after SDA process groups were calculated by SPSS 22.0 by using two-tailed N ing et al. Journal of Analytical Science and Technology (2023) 14:5 Page 3 of 7 Student’s t test. The threshold for significance was set as loop section of the MB probe to form active secondary P < 0.01. conformation. The active DNAzyme generates a nick in the loop section, leading to the release of the two single- Results and discussion strand DNA sequences (a and b). The b sequence, which The working mechanism of the established approach is a homologous sequence with the ssDNA sequences, for DNA sequences detection hybridizes with a next activator probe, inducing the sec- The working principle of the established colorimet - ond signal recycle; the a sequence binds with the H1 ric method is shown in Fig. 1. In this method, a SMBs- probe and gradually unfolds it to initiate the SDA pro- aptamer-ssDNA complex is designed, comprising CD63 cess among the H1 probes and H2 probes. Eventually, the aptamer, SMBs, and a ssDNA sequence that is partially G-rich section in the H1 probe is exposed and can fold complementary with CD63 aptamer. In the present of to active G quadruplex under the assistance of hemin to sEVs, the CD63 aptamer in the SMBs-aptamer-ssDNA catalyze the oxidation of TMB, yielding a color change. complex precisely binds with CD63 protein on the sur- face of sEVs, leading to the liberation of ssDNA. After Construction of SMBs‑aptamer‑ssDNA complex magnet based enrichment and isolation of the SMBs- and strategy to minimize the background signal aptamer-sEVs, the supernatant containing released of the established approach ssDNA initiated the signal amplification process. In the To verify the construction of SMBs-aptamer-ssDNA signal amplification process, a hairpin structure probe complex and its capability in identifying sEVs, a fluores - is designed with a tail which is complementary with cence assay was performed through labeling FAM and the ssDNA, and a DNAzyme section. In the present of corresponding quenching moiety (BHQ-1) on the ter- released ssDNA, it binds with the tail in the activator minal of CD63 aptamer and ssDNA, respectively. From probe, forming a blunt end in the probe. Under the assis- the result in Fig. 2A, the signal of FAM labeled CD63 tance of the Exo-III enzyme, the stem section is digested aptamer significantly decreased in the SMBs-aptamer- from the 3’ terminal, resulting the liberation of the DNA- ssDNA complex, which was 5.67% of that in FAM labeled zyme sequences and the ssDNA. The released ssDNA CD63 (2135.33 ± 78.5 a.u.), indicating that ssDNA was binds with a next activator probe to induce a signal hybridized with CD63 aptamer and the SMBs-aptamer- recycle, and the DNAzyme sequence can bind with the ssDNA complex was successfully constructed. Upon the Fig. 1 The working mechanism of the established approach for DNA targets detection Ning et al. Journal of Analytical Science and Technology (2023) 14:5 Page 4 of 7 Fig. 2 Construction of SMBs-aptamer-ssDNA complex. A Fluorescence spectrum of FAM labeled CD63 aptamer before and after assembly. B Real time monitoring of the fluorescence intensities with or without the addition of SSB addition of sEVs, the FAM signal recovered, implying the ssDNA disassociated from SMBs-aptamer-ssDNA complex. One of the biggest challenge for the Exo-III based approaches is high background signal derived from the ono-specific digestion of probes by Exo-III. Herein, we exploited the single-stranded DNA binding protein (SSB) which possesses a high affinity to the single-strand DNA sequences (ssDNA), to reduce the background signals. To demonstrate the efficiency of the SSB to minimize the background signal, a fluorescence assay was performed. In this fluorescence assay, the two terminals of the acti - vator probe were labeled with FAM and BHQ-1, respec- tively. If the activator probe was digested by the Exo-III, fluorescence signal of FAM would reappeared. As shown in Fig. 2B, the fluorescence intensity in the solution con - taining only activator probe and the synthesized ssDNA sequence (control group) was 5.65 a.u.. The intensities of the group containing Exo-III enzyme and activator probe gradually elevated with the time ranged from 0 to 60 min, and the intensity at 60 min was approximate 2.1 times higher than that in the control group. On the contrary, Fig. 3 Absorbance and the colorimetric assay when essential experimental components (target, MB, H1, and H2) existed or not the intensity of the group containing Exo-III enzyme, SSB and activator probe showed no obvious increment com- pared with the control group. the sEVs was absent, indicating the Exo-III alone can- not induce signal recycles and color generation. Simi- Feasibility of the colorimetric approach lar results were obtained when MB, H1 probe and H2 To test the feasibility of the established colorimetric probe absent in the sensing system, implying all the assay for sensitive sEVs detection, the color change and probes were essential for the feasibility of the colori- quantitative absorbance of the approach under differ - metric approach. When all essential experimental com- ent experimental conditions was monitored. From the ponents existed in this system, an obvious color change result in Fig. 3, no any color change was observed when was observed. N ing et al. Journal of Analytical Science and Technology (2023) 14:5 Page 5 of 7 Optimization of experimental conditions and low limit of detection (LOD) of 267 particle/μL Several experimental conditions can affect the detection (LOD was calculated based on the 3σ/slope method). performance of the established colorimetric approach, Compared with the former sEVs detection methods, including the concentration of Exo-III enzyme, the con- the established approach showed a comparable detec- centration of MB. As a decisive parameter in constructing tion sensitivity and a wider detection range (Additional the signal recycles, the amount of Exo-III in the sensing file 1: Table S2). To test the specificity of the established system determines the efficiency of digestion. Therefore, approach, the method was applied for the detection of we firstly investigated the absorbance of the approach interferents, including ATP (adenosine triphosphate), when different concentrations of Exo-III were exploited CEA (carcino-embryonic antigen), CRP (C-reactionpro- in the system. From the result in Fig. 4A, the absorbance tein). From the result in Fig. 5C, the absorbance of the of the system enhanced with the concentration of Exo-III approach when detecting interferents showed no sig- ranged from 0 to 1 U/L, and no more increasements were nificant differences with the control group. An obvious observed when more than 1U/L Exo-III was added in the color change was observed when detecting sEVs. These system. Therefore, 1U/L Exo-III was selected for the sub - above experimental results proved the high selectivity of sequent experiments. Meanwhile, the optimized concen- the proposed approach in discrimination of sEVs from tration of MB was 200 nM (Fig. 4B). the Interference analytes. We then performed a recov- ery assay to study whether the proposed approach could Analytical performance of the established colorimetric be utilized in detecting sEVs in complicated biological approach samples. In the recovery assay, different concentrations To evaluate the detection performance of the established of sEVs were added in the 5% and 10% artificial serum approach, we have recorded the color of each group and samples, and the recovery rate was calculated as shown monitored the absorbance of the method when detect- in Table 1. Consequently, a quantitative recovery rate ing different concentrations of sEVs. From the result ranged from 99.1 to 106.6% were obtained, which is com- in Fig. 5A, a gradually darkening color was observed parable or superior to most of the existing Exo-III based when the concentration of sEVs increased. The obtained approaches. absorbance of the approach increased with the concen- tration of DNA sequences ranged from 10 particles/μL Conclusion to 10 particles/μL. The correlation equation between the In summary, we depict here a novel colorimetric method absorbance and the concentration of DNA sequences was for sensitive and accurate sEVs detection by integrat- determined Y = 0.1250*lgC − 0.003, with the correlation ing Exo-III assisted signal recycles and SDA medi- coefficient of 0.9786 (Fig. 5B). Meanwhile, the approach ated color generation. Based on the elegant design, the possesses a wide detection rage of 5 orders of magnitude method exhibited a wide detection range of 5 orders of Fig. 4 Optimization of experimental parameters. A Absorbance of the approach when the sensing system is incubated with different concentrations of Exo-III. B Absorbance of the approach with different concentrations of MB Ning et al. Journal of Analytical Science and Technology (2023) 14:5 Page 6 of 7 Fig. 5 Detection performance of the established approach. A Absorbance of the established approach for different concentrations of sEVs detection. B Correlation between the absorbance and the concentration of sEVs. C Absorbance of the approach when detecting interferents Table 1 Recovery assay to test the performance in artificial shows a robust capability in discriminating sEVs from serum interferents in complicated biological samples. All the results indicate the great potential of the approach in Serum Samples Calculations Recovery (%) clinical application and disease diagnosis. 5% 500 514 102.8 2500 2598 103.9 Supplementary Information 15,000 14,897 99.3 The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s40543- 023- 00370-9. 10% 500 533 106.6 2500 2587 103.4 Additional file 1. Table S1. Details of the sequences used in this research, 15,000 14,865 99.1 Supporting information. Table S2. Comparison of the established approach with former ones. Acknowledgements magnitudes, which is superior or comparable to most of The authors thank financial and technical support from the Natural Science the former reported colorimetric sEVs detection assays. Foundation of Chongqing Science and Technology Commission (cstc2021jcyj- msxmX0916) and the Natural Science Foundation of Chongqing Medical and In addition, a minimized background signal was obtained Pharmaceutical College (ygz2019302 and ygz2019305). by exploiting the SSB to reduce the ono-specific diges - tion of ssDNA by Exo-III enzyme. Moreover, this method N ing et al. Journal of Analytical Science and Technology (2023) 14:5 Page 7 of 7 Author contributions Waldenmaier M, Seibold T, Seufferlein T, Eiseler T. Pancreatic cancer small FY and HC is the supervisor of the team in all research steps including design- extracellular vesicles (exosomes): a tale of short- and long-distance com- ing, data analysis, and manuscript writing. LN, as the first author, has the main munication. Cancers (basel). 2021;13(19):4844. role for experimental data collection, data gathering, preparation of results, Wang N, Jiang YJ, Zhang X, Lin HR, Cheng F, Li Q, Li CM, Huang CZ. Nano- and data analysis. YZ, YX, and ZD assist the data analysis. All authors read and surface energy transfer indicating Exo III-propelled stochastic 3D DNA approved the final manuscript. walkers for HIV DNA detection. Analyst. 2021;146(5):1675–81. Xie X, Xu W, Liu X. Improving colorimetric assays through protein Funding enzyme-assisted gold nanoparticle amplification. Acc Chem Res. No fund available. 2012;45(9):1511–20. Yahata S, Hirose M, Ueno T, Nagumo H, Sakai-Kato K. Eec ff t of sample concen- Availability of data and materials tration on nanoparticle tracking analysis of small extracellular vesicles Almost all details of experimental data are presented in the article or addi- and liposomes mimicking the physicochemical properties of exosomes. tional file. Chem Pharm Bull (tokyo). 2021;69(11):1045–53. Yang H, Xiao M, Lai W, Wan Y, Li L, Pei H. Stochastic DNA dual-walkers for ultra- fast colorimetric bacteria detection. Anal Chem. 2020;92(7):4990–5. Declarations Yeasmin S, Ammanath G, Ali Y, Boehm BO, Yildiz UH, Palaniappan A, Liedberg B. Colorimetric urinalysis for on-site detection of metabolic biomarkers. ACS Ethics approval and consent to participate Appl Mater Interfaces. 2020;12(28):31270–81. The manuscript does not contain clinical or trial studies on patients, humans, Zhao X, Luo C, Mei Q, Zhang H, Zhang W, Su D, Fu W, Luo Y. Aptamer-cho- or animals. lesterol-mediated proximity ligation assay for accurate identification of exosomes. Anal Chem. 2020a;92(7):5411–8. Competing interests Zhao X, Zeng L, Mei Q, Luo Y. Allosteric probe-initiated wash-free method The authors declare that they have no competing interests. for sensitive extracellular vesicle detection through dual cycle-assisted CRISPR-Cas12a. ACS Sens. 2020b;5(7):2239–46. Zhao X, Zhang W, Qiu X, Mei Q, Luo Y, Fu W. Rapid and sensitive exosome Received: 19 December 2022 Accepted: 16 January 2023 detection with CRISPR/Cas12a. Anal Bioanal Chem. 2020c;412(3):601–9. Zhao H, Chen X, Hu G, Li C, Guo L, Zhang L, Sun F, Xia Y, Yan W, Cui Z, Guo Y, Guo X, Huang C, Fan M, Wang S, Zhang F, Tao L. Small extracellular vesi- cles from brown adipose tissue mediate exercise cardioprotection. Circ Res. 2022a;130(10):1490–506. References Zhao X, Yuan Y, Liu X, Mao F, Xu G, Liu Q. A versatile platform for sensitive and Armacki M, Polaschek S, Waldenmaier M, Morawe M, Ruhland C, Schmid R, label-free identification of biomarkers through an exo-III-assisted cascade Lechel A, Tharehalli U, Steup C, Bektas Y, Li H, Kraus JM, Kestler HA, Kruger signal amplification strategy. Anal Chem. 2022b;94(4):2298–304. S, Ormanns S, Walther P, Eiseler T, Seufferlein T. Protein kinase D1, reduced in human pancreatic tumors, increases secretion of small extracellular Publisher’s Note vesicles from cancer cells that promote metastasis to lung in mice. Gas- Springer Nature remains neutral with regard to jurisdictional claims in pub- troenterology. 2020;159(3):1019-1035.e22. lished maps and institutional affiliations. Garcia-Martin R, Wang G, Brandao BB, Zanotto TM, Shah S, Kumar Patel S, Schil- ling B, Kahn CR. MicroRNA sequence codes for small extracellular vesicle release and cellular retention. Nature. 2022;601(7893):446–51. Gong S, Zhang S, Wang X, Li J, Pan W, Li N, Tang B. Strand displacement ampli- fication assisted CRISPR-Cas12a strategy for colorimetric analysis of viral nucleic acid. Anal Chem. 2021;93(45):15216–23. Hamidi SV, Perreault J. Simple rolling circle amplification colorimetric assay based on pH for target DNA detection. Talanta. 2019;201:419–25. Li R, Liu Q, Jin Y, Li B. G-triplex/hemin DNAzyme: an ideal signal generator for isothermal exponential amplification reaction-based biosensing platform. Anal Chim Acta. 2019;1079:139–45. Liu J, Ren L, Li S, Li W, Zheng X, Yang Y, Fu W, Yi J, Wang J, Du G. The biology, function, and applications of exosomes in cancer. Acta Pharm Sin B. 2021;11(9):2783–97. Malys MSS, Aigner C, Schulz SMM, Schachner H, Rees AJJ, Kain R. Isola- tion of small extracellular vesicles from human sera. Int J Mol Sci. 2021;22(9):4653. Marin MJ, Schofield CL, Field RA, Russell DA. Glyconanoparticles for colorimet - ric bioassays. Analyst. 2015;140(1):59–70. Masumoto M, Ohta S, Nakagawa M, Hiruta Y, Citterio D. Colorimetric paper- based sarcosine assay with improved sensitivity. Anal Bioanal Chem. 2022;414(1):691–701. Moller A, Lobb RJ. The evolving translational potential of small extracellular vesicles in cancer. Nat Rev Cancer. 2020;20(12):697–709. Urabe F, Kosaka N, Ito K, Kimura T, Egawa S, Ochiya T. Extracellular vesicles as biomarkers and therapeutic targets for cancer. Am J Physiol Cell Physiol. 2020;318(1):C29–39. van Niel G, Carter DRF, Clayton A, Lambert DW, Raposo G, Vader P. Challenges and directions in studying cell-cell communication by extracellular vesicles. Nat Rev Mol Cell Biol. 2022;23(5):369–82. Vogel U, Beerens K, Desmet T. A colorimetric assay for the screening and kinetic analysis of nucleotide sugar 4,6-dehydratases. Anal Biochem. 2022;655:114870.
Journal of Analytical Science and Technology – Springer Journals
Published: Jan 23, 2023
Keywords: Exonuclease III (Exo III); Single-stranded DNA binding protein (SSB); Strand displacement amplification (SDA); DNAzyme; Pancreatitis
Access the full text.
Sign up today, get DeepDyve free for 14 days.