Amplified fluorescence detection of serum prostate specific antigen based on metal-dependent DNAzyme assistant nanomachine
Graphical abstract
An amplified fluorescence biosensing strategy for serum prostate specific antigen (PSA) was developed on the basis of Zn2+-dependent DNAzyme as the catalytic unit for the cleavage of hairpin substrate probe.
Introduction
DNAzymes, which can be obtained random from oligonucleotide libraries by in vitro screening, are nucleic acids holding a special structure, specific recognition function and high catalytic activity [[1], [2], [3], [4], [5]]. It can catalyze a series of reactions, such as the cleavage of DNA or RNA, DNA hydrolysis and DNA connection [[6], [7], [8], [9], [10]]. Compared with the traditional protease, DNAzymes have advantages of high stability, high catalytic efficiency and uneasy deactivation and thus have attracted increasing attention from researchers to be widely used in many fields [[11], [12], [13], [14], [15], [16], [17], [18]]. However, how to transform the recognition information between DNAzyme and the target into a detectable physical signal or even amplified signal has been a hotspot.
Magnetic beads (MB), which surface can be modified with a variety of functional groups (-OH, -SH, COOH, -NH2), are particularly suitable for modification of many biomolecules, such as DNA, enzymes or antibody. For example, the surface of MB is usually modified with streptavidin, which has good affinity to biotin-labeled molecules. Also it can accommodate a wide of pH and temperature range, and maintain the stability of avidin-biotin complex in the presence of a wide range of organic or denaturing reagents. Because of these characteristics, MB have been widely used in the field of biochemical analysis, such as the construction of biosensor [[19], [20], [21], [22], [23], [24], [25], [26], [27]], cell sorting [28], microbial rapid detection, and the separation and purification of nucleic acid [[29], [30], [31], [32], [33]].
Prostate Cancer (PCa), as one of serious urinary system tumors, greatly threats to men's healthy, which mortality rate ranked second in male cancer and second only to lung cancer. In 1980, Papsidero et al. [34], for the first time demonstrated the relevance of prostate specific antigen (PSA) and PCa. As a marker for the detection of PCa, serum prostate specific antigen (PSA) has been widely used in the early diagnosis of PCa. There are a number of popular routine methods for the detection of cancer biomarkers in serum, such as chemiluminescence, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and various spectral immunoassays [[35], [36], [37], [38], [39], [40], [41], [42]]. However, the traditional immunoassay methods have many disadvantages, including the operation process complex, expensive, time-consuming and susceptible to external factors, all of which limited its scope of application. Fluorescence analysis system with simple, convenient operational approach and satisfactory sensitivity has been widely applied for biosensing of various biomolecules. Recently, integrated with various nomaterials, fluorescence detection methods have been used to construct a series of novel biosensors, some of which have achieved remarkably sensitive and selective detection of tumor markers [[43], [44], [45], [46]]. Up to date, fluorescence biosensors based on nanomaterials and DNAzyme have been developed rapidly and penetrated into multiple analytical fields [[47], [48], [49], [50]], showing unique advantages such as be intuitive and easy to design. However, to further use it for clinical and practical testing, there are many aspects needed to be optimized. Based on the features of metal-dependent DNAzymes and magnetic nanoparticles, we proposed a novel approach for sensitive detection of PSA. By the surface modification technology, the biotin-antibody (biotin-Ab2) and DNAzyme were modified on the surface of the streptavidin-coated magnetic bead, according to a certain proportion, then specifically recognized different concentration of PSA in serum with high sensitivity benefited from the cyclic amplified strategy.
Section snippets
Reagents and materials
Albumin from Bovine Serum (BSA), Thioglycolic acid (TGA), imidazole, Nhydroxysuccinimide(NHS) and N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC) were acquired from Aladdin-Reagent Co., Ltd. (Shanghai, China). EDC and NHS were used immediately after dissolved in sterile water. Streptavidin magnetic beads (MB, ∼100 nm diameter) was purchased from Tiandz Co., Ltd. (Beijing, China). Human prostate-specific antigen (PSA), two mouse antihuman total PSA monoclonal antibodies
The design of amplified sensing system for PSA
The catalytic reaction of DNAzyme depends on the specific cofactor (such as metal ions), which provides a platform for the construction of specific sensing systems. As Zn2+-dependent DNAzyme possesses highly catalytic property, it was selected in our design as the catalytic unit for sensing system and adopted the cofactors Zn2+ to maintain its functionality. Moreover, magnetic beads were used as a carrier and combined with sandwich immune structure, thus the DNAzyme-based cyclic amplified
Conclusion
In summary, a novel signal amplified biosensing strategy for fluorescence detection of serum PSA was developed. In this biosensing approach, Zn2+-dependent DNAzyme was selected as the catalytic unit for the cleavage of hairpin substrate probe, magnetic beads modified by antibody and enzyme strand as a composite probe. Upon increasing the concentration of PSA, the fluorescence signal increases corresponding. It shows a high selectivity and a low detection limit of 0.05 ng mL−1. This biosensing
Acknowledgements
Financial support was provided by the National Natural Science Foundation of China (Grant Nos.: 21505065, 21675074), the “Innovation Team Development Plan” of the Ministry of Education Rolling Support (IRT_15R31).
References (60)
- et al.
Amplified detection of T4 polynucleotide kinase activity based on exonuclease cleavage-induced DNAzyme releasing strateg
Sens. Actuators, B: Chem.
(2014) - et al.
Mesoporous silica thin film mechanized with a DNAzyme-based molecular switch for electrochemical biosensing
Chem. Commun.
(2015) - et al.
A portable chemiluminescence imaging immunoassay for simultaneous detection of different isoforms of prostate specific antigen in serum
Biosens. Bioelectron.
(2016) - et al.
Ni(OH)2/NGQDs-based electrochemiluminescence immunosensor for prostate specific antigen detection by coupling resonance energy transfer with Fe3O4@MnO2 composites
Biosens. Bioelectron.
(2018) - et al.
A new label-free electrochemical immunosensor based on dendritic core-shell AuPd@Au nanocrystals for highly sensitive detection of prostate specific antigen
Biosens. Bioelectron.
(2018) - et al.
Versatile DNAzyme-based amplified biosensing platforms for nucleic acid, protein, and enzyme activity detection
Anal. Chem.
(2013) - et al.
Aptazyme-gold nanoparticle sensor for amplified molecular probing in living cells
Anal. Chem.
(2016) - et al.
Zn2+-Ligation DNAzyme-driven enzymatic and nonenzymatic cascades for the amplified detection of DNA
J. Am. Chem. Soc.
(2012) A novel biosensor comprises at least one fluorophore and at least two quenchers, and is capable of selectively and specifically detecting the presence of an ion in the presence of other ions
Nat. Biotechnol.
(1997)- et al.
Biostable L-DNAzyme for sensing of metal ions in biological systems
Anal. Chem.
(1997)
Biosens. Bioelectron.
Lead-dependent DNAzyme with a two-step mechanism
Biochem
Illumination dependent optical properties of plasmonic nanorods coupled to thin-film cavities
Microchim. Acta
Lower expression of the TWIK-related acid-sensitive K+ channel 2 (TASK-2) gene is a hallmark of aldosterone-producing adenoma causing human primary aldosteronism
Endocrin
Specific genotype detection of factor V Leiden mutation from polymerase chain reaction amplicons based on label-free electrochemical genosensor
Elec. Chem. Comm.
Smart mesoporous SiO2 nanoparticles for the DNAzyme-induced multiplexed release of substrates
J. Am. Chem. Soc.
Functional DNA-containing nanomaterials: cellular applications in biosensing, imaging, and targeted therapy
Acc. Chem. Res.
A smart DNAzyme-MnO2 nanosystem for efficient gene silencing
Angew. Chem. Int. Edit
A nanoscale DNA–Au dendrimer as a signal amplifier for the universal design of functional DNA-based SERS biosensors
J. Am. Chem. Soc.
Target responsive DNAzyme cross-linked hydrogel for visual quantitative detection of Lead
Anal. Chem.
Label-Free colorimetric aptasensor based on nicking enzyme assisted signal amplification and DNAzyme amplification for highly sensitive detection of protein
Anal. Chem.
Simultaneous imaging of Zn2+ and Cu2+ in living cells based on DNAzyme modified gold nanoparticle
Anal. Chem.
Binding-induced autonomous disassembly of aptamer-DNAzyme supersandwich nanostructures for sensitive
Nanoscale
Raman spectroscopic detection of sub-picomolar DNA by coupling silver catalyzed silver deposition with circular strand-replacement polymerization on magnetic nanoparticles
Chem. Commun.
Highly sensitive colorimetric cancer cell detection based on dual signal amplification
ACS Appl. Mater. Interface.
An electrochemiluminescence biosensor for Kras mutations based on locked nucleic acid functionalized DNA walkers and hyperbranched rolling circle amplification
Chem. Commun.
Alkaline phosphatase responsive anodic electrochemiluminescence of CdSe nanoparticles
Anal. Chem.
Label-Free colorimetric aptasensor based on nicking enzyme assisted signal amplification and DNAzyme amplification for highly sensitive detection of protein
Anal. Chem.
Multiplexed assay for proteins based on sequestration electrochemistry using the protein binding electroactive magnetic microbeads
Anal. Sci.
Nanosphere based one-step strategy for efficient and nondestruc-tive detection of circulating tumor cells
Biosens. Bioelectron.
Cited by (19)
DNAzyme-based ultrasensitive immunoassay: Recent advances and emerging trends
2024, Biosensors and BioelectronicsSensitive determination of prostate-specific antigen with graphene quantum dot-based fluorescence aptasensor using few-layer V<inf>2</inf>CT<inf>x</inf> MXene as quencher
2023, Spectrochimica Acta - Part A: Molecular and Biomolecular SpectroscopyIntegrating DNA nanostructures with DNAzymes for biosensing, bioimaging and cancer therapy
2022, Coordination Chemistry ReviewsDNA nanomachine activation and Zn<sup>2+</sup> imaging in living cells with single NIR irradiation
2022, Analytica Chimica ActaCitation Excerpt :DNA nanomachine confines DNA reaction strands in nanostructures, and accelerates cascade hybridization reactions in response to tiny stimulus [1–4]. Conjugating DNAzyme catalytic reactions with DNA walkers that move along designed orbits, DNAzyme-based nanomachines could achieve substantial signal enhancement in short time and have become an useful technique for biosensing and bioimaging [5–9]. Most reported DNAzyme-based nanomachines have “always-on” design which automatically operates in response to detection targets as stimuli [10–13].
Enzyme-free dual-amplification assay for colorimetric detection of tetracycline based on Mg<sup>2+</sup>-dependent DNAzyme assisted catalytic hairpin assembly
2022, TalantaCitation Excerpt :In this study, we developed an enzyme-free dual-amplification assay as an accurate, sensitive, and selective sensing system for the colorimetric detection of TC in milk based on Mg2+-dependent DNAzyme (MNAzyme) assisted catalytic hairpin assembly (CHA). The MNAzyme, a nucleic acid-based enzymatic molecule, can cyclic catalytic cleavage of the specific substrates sequences to achieve signal amplification with the help of Mg2+ as boosting agent [25–27]. Recent extensive studies had been reported that the MNAzyme was combined with other amplification strategies to improve the detection sensitivity [28–32].
Proximity binding induced nucleic acid cascade amplification strategy for ultrasensitive homogeneous detection of PSA
2021, Analytica Chimica ActaCitation Excerpt :Meanwhile, the linear regression equation was expressed as Ic (a.u.) = 1979.27 + 583.24 lgC (PSA, ng mL−1) and the limit of detection (LOD) was 0.73 pg mL−1 on the basis of the standard 3σ rule. The results demonstrate that the proposed approach provides better performance for fluorescence detection of the PSA with a low enough LOD comparing with previous studies [3–5,7–10,41–45]. In addition, comparing to our previous enzyme-based amplification method for PSA testing [46], this method not only has a lower detection line, but also avoids some disadvantages of using enzymes, such as the specific buffer solution and reaction temperature, easily inactivated, expensive, harsh reaction conditions and complex reaction system.