Fluorescent aptasensor based on D-AMA/F-CSC for the sensitive and specific recognition of myoglobin

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Highlights

  • A novel fluorescent aptasensor for the specific detection of myoglobin (Mb) was developed.

  • The aptasensor was mainly composed of dabcyl-modified anti-Mb aptamer and 6- FAM-modified complementary short chain.

  • The developed aptasensor is inexpensive, easy to prepare and has high specificity and sensitivity for Mb.

  • The aptasensor was further applied to detect Mb levels in spiked human blood sera samples.

Abstract

A novel fluorescent biosensor based on dabcyl [(E)-4-((4-(dimethylamino) phenyl) diazenyl)benzoic acid] -modified anti-Mb aptamer (D-AMA) and 6-FAM(6-carboxyfluorescein) -modified complementary short chain (F-CSC)for the specific and sensitive detection of Mb levels is presented in this study. In PBS buffer solution, D-AMA bound to F-CSC, and then dabcyl quenched the fluorescence of 6-FAM. After adding Mb into the system, D-AMA bound to Mb and separated from F-CSC. The fluorescence of 6-FAM was restored after it separated from dabcyl. The assay exhibited high specificity and sensitivity toward Mb, with a low limit of detection of 0.07 ng/mL (S/N = 3) and linear relationships of 0.1–5 ng/mL. It was further applied to detect Mb levels in spiked human blood sera samples.

Introduction

Acute myocardial infarction (AMI) is one of the cardiovascular diseases that cause human morbidity and mortality worldwide [1]. Therefore, the accurate and rapid diagnosis of AMI is crucial and has an important clinical value in patient survival and successful prognosis. Myoglobin (Mb) is one of the important markers of early AMI, and it is the earliest biomarker released by cells after myocardial injury [2]. The normal concentration range of Mb in human serum is 6–100 ng/mL. When

AMI occurs, the Mb level increases to 600 ng/mL in a short time [3]. Hence, the rapid and accurate detection of Mb is of great importance for the early diagnosis of AMI.

At present, the main methods for detecting Mb include mass spectrometry [4], liquid chromatography [5], electrochemical method [6,7], fluorescence method [8], surface plasmon resonance method [9], colorimetric method [10], molecular imprinting [11,12], immunoassay [13,14], and chemiluminescence method [15]. Mass spectrometry has high sensitivity and specificity, however, it requires expensive instrument, and its database is not perfect. Thus, it has not been widely used. High-performance liquid chromatography has the advantages of good separation effect and high sensitivity. However, due to the low concentration of Mb in serum, samples need to be enriched and concentrated before detection. The operation is tedious and time-consuming and cannot meet the requirements of rapid determination of Mb. In addition, most of the above methods for detecting Mb are based on the mutual recognition of antibody and antigen. Though immunoassay has high selectivity and sensitivity toward Mb, the preparation of antibodies requires immune animal or cell experiments, which is cumbersome, time-consuming, and costly. Moreover, antibodies are susceptible to the influence of external conditions, especially temperature, and require strict storage conditions, greatly limiting the flexible application of these methods.

Aptamers are small single-stranded oligonucleotide sequences screened by SELEX (index enrichment ligand phylogenetic technology) [16]. They are a kind of small RNA or single-stranded DNA with specific three-dimensional conformation. Their length is generally 20–80 bases, and their relative molecular weight is 6–30kD. Given the structural diversity and spatial conformation of aptamers, some stable three-dimensional structures (e.g., hairpins, knots, convex rings, G-tetrads) can be formed by pairing with some complementary bases and electrostatic and hydrogen bonding interactions with target molecules. Moreover, aptamers can specifically recognize target molecules by binding them with high affinity. Therefore, small molecules, proteins, bacteria, viruses, and cells can be specifically recognized by aptamers [17]. Compared with antibodies, aptamers have high specificity, high affinity, no (or low) immunogenicity, low cost, easy synthesis and modification, easy fixation, wide target range, reusability, and long-term preservation.

The fluorescence method has low detection limit and high sensitivity. It is rapid, and requires simple instruments. Thus, a reliable, fast, highly selective, and sensitive analytical method for detecting Mb may be developed by combining fluorescence method with aptamer technique. Here, we developed a low-cost, simple, and accurate fluorescent biosensor based on dabcyl[(E)-4-((4-(dimethylamino)phenyl)diazenyl) benzoic acid]-modified anti-Mb aptamer (D-AMA) and 6-FAM (6-carboxyfluorescein)-modified complementary short chain (F-CSC) for the specific detection of Mb.

Section snippets

Materials and apparatus

Bovine sera albumin (BSA), human sera albumin (HSA), immunoglobulin A (IgA), and immunoglobulin G (IgG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cardiac troponin I (cTnI) was provided by Shanghai Linc-Bio Science Co., Ltd. (Shanghai, China). Myoglobin (Mb) was obtained from Shanghai Anyan Trade Co., Ltd. (Shanghai, China). Alpha-fetoprotein was purchased from Fitzgerald Company (USA). Human sera samples were provided by Xin’an International Hospital (Jiaxing, China). All other

Detection principle of the fluorescent aptasensor

In the PBS buffer solution, D-AMA and F-CSC were hybridized. The fluorescent group 6-FAM and the quenching group dabcyl contacted each other. The fluorescence of 6-FAM was quenched by dabcyl. When a certain concentration of Mb was added to the system, D-AMA was separated from F-CSC and bound to Mb because of its high affinity to Mb. The fluorescence of 6-FAM of F-CSC was restored after it separated from dabcyl of D-AMA (Scheme 1). The intensity of fluorescence recovery is proportional to the Mb

Conclusions

A simple fluorescent aptasensor based on the interaction between D-AMA and F-CSC was developed in this study. The developed aptasensor is inexpensive and easy to prepare and has high specificity and sensitivity for Mb. Given that the aptasensor displays good test performance, it has potential application value in the immediate detection of AMI.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This work was supported by the National Natural Science Foundation of China (No. 21677060 and 51503079), the Public Welfare Technology Research Project of Zhejiang Province (No. LGF18B050004 and LGC19B050007), the Natural Science Foundation of Zhejiang Province (No. LY16B050007 and LQ19B050002), the Program for Science and Technology of Zhejiang Province (No. 2018C37076) and the Science and Technology Plan Project of Jiaxing City, China (No. 2017AY33034 and 2018AY11002).

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