Elsevier

Talanta

Volume 223, Part 2, 1 February 2021, 121730
Talanta

Ultrasensitive label-free detection for lung cancer CYFRA 21-1 DNA based on ring-opening polymerization

https://doi.org/10.1016/j.talanta.2020.121730Get rights and content

Highlights

  • The combination of ROP and EIS biosensing has been firstly used in the detection of small biological molecules.

  • The EIS method possesses real time monitor, label-free analysis, simple operation, high sensitivity and selectivity.

  • The strategy provides a new approach for early clinical diagnosis of lung cancer.

Abstract

Cytokeratin fragment antigen 21-1 (CYFRA 21-1) DNA is perceived as sensitive tumor marker for the diagnosis of non-small cell lung cancer and other tumor. Herein, linear chain poly(ε-caprolactone) (PCL) synthesized by ring-opening polymerization is applied to ultrasensitive label-free electrochemical impedance detection system for CYFRA 21-1 DNA. First, thiolated peptide nucleic acid (PNA) is self-assembled into the Au electrode surface through the formation of Au–S bonds, allowing the PNA to act as biomolecular probe and form PNA/DNA heteroduplex with the target DNA via specific hybridization. Then, PCL is conjugated to the immobilized DNA on the electrode via “carboxylate–Zr4+–phosphate” bridges. Finally, the electrochemical response of modified PNA/DNA/Zr4+/PCL electrode is determined by electrochemical impedance method to quantify of CYFRA 21-1 DNA. Under optimal conditions, this method exhibits highly sensitivity with a broad linear range (0.1 fM – 1 nM) (R2 = 0.995) and the limit of detection (LOD) is as low as 10.73 aM, which is equivalent to just 64 molecules in a 10 μL sample. What's more, the high selectivity, good anti-interference, label-free operation, and real-time monitoring in complex samples of the proposed strategy demonstrate its broad application for the early diagnosis and clinical monitoring.

Graphical abstract

A novel, label-free, real-time, and highly sensitive EIS detection system for quantitatively detecting CYFRA 21-1 DNA based on PCL polymer was developed.

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Introduction

Lung cancer is a malignant epithelial tumor with fastest growing, morbidity and mortality rates [[1], [2], [3]]. According to pathological type, lung cancer can be classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC is the most common type, accounting for about 85% of lung cancer [4,5]. The common treatment method is surgical resection, due to the low sensitivity of NSCLC to chemotherapy. However, certain indications and contraindication in the treatment still prevail, such as elderly patients or with distant metastasis [6]. In view of this, an early diagnosis of NSCLC has a positive impact on treatment and survival rates, and its sensitive detection is essential. Cytokeratin fragment antigen 21-1 (CYFRA 21-1) is presently an important biomarker for NSCLC [[7], [8], [9]]. So far, numerous techniques have been developed for detecting CYFRA 21-1 DNA, including polymerase chain reaction (PCR), flow cytometry (FCM), immunocytochemistry, western blotting, and northern blotting [[10], [11], [12], [13], [14], [15]]. Whereas, these detection methods suffer from issues in terms of time-consuming operation, high cost, low sensitivity, poor portability, and low enzyme-thermostability [2]. Therefore, the development of a highly sensitive method for CYFRA 21-1 DNA detection will benefit the crucial diagnosis and treatment of NSCLC.

Electrochemical method has appealed extensive attention due to its rapid response, high sensitivity, small dimensions, and low cost, including voltammetry, amperometry, and impedance spectroscopy [[16], [17], [18]]. Electrochemical impedance spectroscopy (EIS) is used to determine biomolecules and genes by converting biological signals into measurable impedance in biological field [[19], [20], [21]]. In general, to amplify signal and thus enhance its sensitivity and linear range, it can be used in combination with different polymerization methods such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer polymerization (RAFT), ring opening polymerization (ROP), and so on. However, ATRP and RAFT have disadvantages of expensive reagents, pH sensitivity, and the necessity for high concentrations of transition metal complexes [22]. Conversely, ROP can be conducted under mild reaction conditions, and is a mature method for the synthesis of linear polymers due to less side reaction, high yields, and needless ratio of equivalent [[23], [24], [25]]. Herein, we employ poly(ε-caprolactone) (PCL) synthesized from ε-caprolactone (ε-CL) and 2, 2-bis(methylol)propionic acid (bis-MPA) by using ROP. Bis-MPA acts as an initiator, where two hydroxyl groups on each bis-MPA molecule can respectively initiate ROP to form PCL polymer chains, which dramatically enhance the detection signal.

In this paper, for the first time, we have applied PCL obtained by ROP to present a label-free EIS method for lung cancer CYFRA 21-1 DNA detection. In this approach, the thiolated peptide nucleic acid (PNA), a DNA analogue with a polypeptide-like skeleton, is used as a bioprobe due to some particular properties such as non-degradability of protease or nucleases [26,27]. First, PNA was self-assembled on the surface of gold electrode via spontaneous formation of Au–S bonds and specifically recognized CYFRA 21-1 DNA through Watson-Crick base pairing to form heteroduplex. Then PCL macromolecules were associated to the electrode surface through the connection of Zr4+ linker. Such polymer chains imparted dramatically EIS signals than that of DNA alone, so this process effectively amplified EIS signal responses to the target DNA (tDNA). The modified electrode was then measured electrochemically to determine the tDNA concentration of the analyte. Our results show that the impedance signal has a linear relationship with the logarithm of CYFRA 21-1 DNA in the concentration range of 0.1 fM – 1 nM. Furthermore, the label-free EIS method exhibits high selectivity, low susceptibility to interference, excellent stability, and the capacity of detecting the analyte in complex biological matrices. It is demonstrated that this strategy has tremendous potential for early diagnosis.

Section snippets

Reagents

Toluene was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Zirconium dichloride oxide octahydrate (ZrOCl2·8H2O) was obtained from J&K Scientific Ltd. (Beijing, China). 2-Ethylhexanoate (Sn(Oct)2), 2, 2-bis(methylol)propionic acid (bis-MPA), and ε-caprolactone (ε-CL) were provided by Aladdin Reagent Co., Ltd. (Shanghai, China). Methanol, ethanol and dichloromethane solutions were provided by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Mercapto-1-hexanol (MCH) was

Principles of the proposed strategy and ROP

Briefly, our ultrasensitive label-free EIS strategy for tDNA detection depends on specific recognition and complexing of PNA probe and ROP polymer. The establishing process is shown schematically in Scheme 1. A self-assembly monolayer (SAM) of PNA was deposited on the gold electrode surface via Au–S bonds in the first place. Then MCH, as masking agent, was used to seal off the Au surface that was not bonded to PNA, thereby preventing nonspecific adsorption of tDNA. The CYFRA 21-1 DNA was

Conclusion

To sum up, a label-free EIS method for ultrasensitive CYFRA 21-1 DNA detection has been developed successfully for the first time by exploiting the DNA-specificity of neutral PNA and the long-chain polymer PCL. The proposed strategy reveals satisfactory analytical capacity within shorter detection time, and the LOD can be down to 10.73 aM under optimal experimental conditions. Furthermore, it displays satisfactory selectivity and reliability in distinguishing DNA with mismatched bases and

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.

Acknowledgements

This work was supported by the project of tackling of key scientific and technical problems in Henan Province (192102310033) and National Natural Science Foundation of China (21974068).

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