Elsevier

Analytica Chimica Acta

Volume 1008, 30 May 2018, Pages 96-102
Analytica Chimica Acta

Amplified fluorescence detection of serum prostate specific antigen based on metal-dependent DNAzyme assistant nanomachine

https://doi.org/10.1016/j.aca.2017.12.041Get rights and content

Highlights

  • A novel signal amplified biosensing strategy for fluorescence detection of serum PSA was developed on the basis of DNAzyme.

  • Zn2+-dependent DNAzyme was selected as the catalytic unit for the cleavage of hairpin substrate probe.

  • This biosensing approach provided a new strategy for protein biomarkers immunoassay and clinical application.

Abstract

An amplified fluorescence biosensing strategy for serum prostate specific antigen (PSA) was developed on the basis of DNAzyme. In presence of cofactor Zn2+, Zn2+ -dependent DNAzyme could cleave the hairpin substrate probes which were dispersed in solution and generate remarkable fluorescent signal. Taking advantage of the magnetic beads as a carrier, one target protein could bring plentiful hairpin substrate probes on to the electrode through a sandwich structure (Ab1/PSA/biotin-Ab2). Moreover, during the cleavage process of as formed DNAzyme, DNAzyme did not be destroyed and could further react with other hairpin probes, then generated continuous fluorescent signal. Benefited by this amplified strategy, the limit of detection (LOD) was low to 0.05 ng mL−1, which was much lower than our previous reports. This method could be applied to detect different protein biomarkers in serum without corresponding aptamers by changing the corresponding antibodies and thus showed a remarkable prospect in clinical application.

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.

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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).

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