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