Signal-off photoelectrochemical biosensing platform based on hybridization chain-doped manganese porphyrin quenching on CdSe signal coupling with cyclic amplification for thrombin detection

Highlights

• A novel signal-off PEC biosensor based on DNA linear chain-doped MnPP quenching to QDs was reported.

• CdSe QDs and TiO₂ complex displayed strong PEC signal.

• A large amount of manganese porphyrin doped in linear DNA chain greatly amplified PEC signal change.

• DNA S1 and S2 specifically bind to thrombin to form the pliers structure with high selectivity.

Abstract

A novel signal-off photoelectrochemical (PEC) biosensing platform based on hybridization chain-doped manganese porphyrin quenching on PEC signal of CdSe quantum dots (QDs) coupling with cyclic amplification was developed for thrombin detection. Target thrombin firstly induced DNA enzyme-aided cyclic amplification reaction and produced abundant DNA I. Then DNA I hybridized with capture DNA on the TiO₂/CdSe QDs modified electrode to form DNA bridge, which triggered a linear chain hybridization reaction with S4 and S5. Numerous manganese porphyrin (MnPP) were doped in the long double-stranded DNA chain, which hindered electron transfer and resulted in obvious decrease of QDs PEC signal, achieving a sensitive detection of thrombin. This PEC biosensor exhibits excellent performance with a wide linear range from 1.0 f. to 100.0 nM and detection limit of 0.331 fM, and the recovery value ranged from 95.5% to 103.5%. The results suggest that this PEC biosensor has high sensitivity and precision, showing great application prospect in microbiological analysis of biomolecules and clinical medicine.

Introduction

As an emerging and continuously developing analysis technology, photoelectrochemical (PEC) analysis not only has the advantages of optical and electrochemical analysis methods [1], but is also preferable to traditional methods for higher sensitivity, lower background signal, simple operation and lower cost, so PEC analysis has been widely used in biomarker detection [[2], [3], [4], [5], [6]]. Quantum dots (QDs) with unique electrochemistry and photophysical properties have found more and more applications in many fields, especially in biological analysis [7]. Functional QDs can be labeled on various biological molecules, which can provide PEC signal to detect biological molecules [8]. PEC bioanalysis based on QDs has been widely applied in the field of biochemical analysis due to their unique sensing mode [9]. Moreover, increasing demand for clinical diagnosis and industrial analysis continues to drive the development of QDs in PEC sensor analysis [10].

Aptamer is a segment of oligonucleotide sequence that can bind to target with high affinity and specificity [11]. It has been widely used in molecular recognition, experimental diagnosis, disease treatment and drug research. Aptamer shows advantages in various analytical methods, such as colorimetry [12,13], fluorescence [14,15] and electrochemistry [16,17]. Among these sensing platforms, photoelectrochemical analysis has received more and more attention due to its unique advantages, such as sensitivity, simplicity, low cost, and miniaturization of devices.

Thrombin is a serine protease in blood, which can transform soluble fibrinogen into insoluble substance. Fibrin is associated with many diseases such as cardiovascular disease, inflammatory response, thromboembolic disease and anticoagulation therapy [18]. Therefore, quantitative detection of thrombin is very important in clinical research and diagnosis [19]. In order to further improve detection sensitivity, signal amplification methods include polymerase chain reaction [20], nanomaterials labeling [21], rolling ring amplification [22] and enzyme-assisted cyclic amplification [23], which have been widely included in aptamer-based thrombin detection. In fact, these newly developed methods can significantly amplify signal output and achieve highly sensitive thrombin detection. So far, most of the signal quenching in photoelectric detection is energy resonance transfer between quantum dots and gold nanoparticles. In this experiment we selected a new quenching agent‑manganese porphyrin. A new quenching agent is Manganese porphyrin (MnPP), that presents many advantages, such as low production cost, stable chemical properties, high catalytic performance and excellent biocompatibility. Porphyrin derivatives are a kind of planar compounds with a certain size and relatively few electrons. Due to its special structural features, it has been used as a double-stranded DNA binding agent [[24], [25], [26]]. MnPP, as an effective quenching agent in this work, can be doped in double-stranded DNA by tetragonal conjugation and electrostatic interaction, and hybridization chain reaction is used to increase the amount of MnPP.

In this experiment, PEC quenching effect of MnPP to CdSe QDs was coupled with DNA enzyme-assisted cyclic amplification and chain hybridization to design a new signal-off thrombin biosensor. Thrombin firstly specifically combined with its aptamer, and released S3 to establish relationship with target. Then S3 initiated DNA enzyme-assisted cyclic amplification to output a large amount of product chain I. DNA I act as a bridge to make S4/S5 DNA chain reaction be connected to the electrode, and the quenching agent MnPP was doped into the chain structure to reduce signal, realizing high sensitive detection of thrombin.