Self-circulating electrochemiluminescence chip for sensitive detection of circulating tumour nucleic acids in blood

Highlights

• This paper described a simple, accurate and reliable strategy for separation of and biomedical analysis of CTNAs.

• This platform achieved preferable specificity and a high sensitivity of 100 amol.

• The whole detection process realized a non-destructive detection mode of tumor fluid biopsy.

Abstract

Circulating tumour nucleic acids (CTNAs), cell-free nucleic acids released from tumour cells, have been employed as potential markers for the diagnosis and prognosis management of tumours. It is important to develop highly sensitive and reliable methods for the detection of CTNAs. Herein, a self-circulating electrochemiluminescence (ECL) chip was constructed for recognizing the point mutations of CTNAs in serum. This strategy relies on a magnet-controlled self-circulating chip for enrichment of CTNAs in blood samples, and autologous blood transfusion was performed for feedback. Meanwhile, the strategy of base stacking was employed as an effective indicator for the point mutation detection of CTNAs. Furthermore, an amplified ECL assay was employed as a highly efficient signal generation mode, and a low detection limit of 100 amol and desirable specificity were achieved. The performance evaluations for the analysis of clinical CTNA samples indicated that the self-circulating ECL chip reliably responded to CTNAs from the blood. Hence, this platform satisfied the strict clinical requirements for CTNA detection and thus has the potential to serve as a new paradigm for liquid tumour biopsy.

Introduction

Circulating tumour nucleic acids (CTNAs), cell-free nucleic acids released from tumour cells, have been employed as potential markers for tumour diagnosis and prognosis management [[1], [2], [3], [4]]. A diagnosis strategy based on CTNAs could serve as a liquid biopsy approach to supplement or replace tumour tissue biopsies [[5], [6], [7], [8]]. The existing studies indicated that tumour patients generally have higher levels of CTNAs than healthy controls [[9], [10], [11]]. However, the level of CTNAs in plasma or serum samples varies drastically, and it is very difficult to differentiate tumour patients from healthy individuals [8,12,13]. Furthermore, the low abundance of CTNAs is not within the performance range of existing detection methods, and a large volume of serum (typically greater than 10 mL) is required to obtain sufficient CTNA samples for the detection process [14]. Thus, it is very important to develop highly sensitive and reliable methods for the detection of CTNAs.

Detection strategies based on expression level and mutated sequence of CTNAs have been proven to serve as efficient assays of liquid tumour biopsies [11,15]. However, these strategies require a highly sensitive and specific approach to detect the low-abundance CTNAs and mutant genes among the high-abundance wild-type sequences in samples from tumour patients [11,16]. Polymerase chain reaction (PCR) and DNA sequencing have provided novel testing methodology for CTNAs [17,18]. DNA sequencing exhibits excellent performance for CTNA monitoring, but its implementation is too expensive for routine clinical analysis, and the time required (2–3 weeks) further limits its applications [19,20]. Meanwhile, PCR is not effective for the detection of point mutations in CTNAs [14,21,22]. Therefore, the development of a novel method that is more accurate and can detect CTNAs and mutations directly in serum, even in blood, is urgently needed.

To address the above issues, we constructed a self-circulating electrochemiluminescence (ECL) chip to detect the content and point mutations of CTNAs in serum. In this strategy, a magnet-controlled self-circulating chip was constructed for the enrichment of CTNAs in blood, and autologous blood transfusion was performed for feedback. Meanwhile, the principle of base stacking was employed to realize point mutation detection. In this work, an 8 nt DNA recognition domain was connected to the ECL complex, and it cannot be hybridized to the capture probe without target CTNAs, because 8 nt is too short for nucleic acid hybridization at room temperature [23]. However, a stabilization effect emerged when the target CTNAs were present. Benefiting from the recognition capacity of the base-stacking hybridization model, point mutation detection of CTNAs with a self-circulating ECL chip was also achieved. The point mutation of CTNAs was designed at the joint between the signal probe and capture probe. Four signal probes were designed to recognize the variations of A, T, C and G, and satisfactory recognition of point mutations was achieved.

Furthermore, ECL was employed as the efficient and widely used signal production mode. Compared with other assays, the ECL assay provided promising performance with low detection limit, a controllable reaction system, and a wide detection range. In particular, ECL does not require an additional light source for excitation; therefore, a higher signal-to-noise ratio can be achieved in the dark field than with photoluminescence. However, there is still substantial potential for the development of highly efficient ECL assays to achieve trace analysis. Our previous work reported a dendritic Ru(bpy)₃²⁺-polymer-amplified ECL strategy for the trace analysis of Zika virus [40]. In this work, the dendritic polymer was employed as the frame for the connection of Ru(bpy)₃²⁺. It achieved low detection limit and satisfactory specificity. In this work, an improved linear polymer-amplified ECL assay was employed as the highly efficient signal production mode, and a low detection limit of 100 amol and desirable specificity were achieved. The performance index for the analysis of clinical CTNA samples was also investigated, and the results indicated that the self-circulating ECL chip reliably responded to CTNAs from the blood. Compared with that of the dendric Ru(bpy)₃²⁺-polymer probe, the cost of the linear Ru(bpy)₃²⁺-polymer probe was much lower because the framework was commercial poly-L-lysine. However, the detection limit was higher than that of the dendric Ru(bpy)₃²⁺-polymer probe because the distal Ru(bpy)₃²⁺ of the linear Ru(bpy)₃²⁺-polymer probe could not effectively respond to the electrode. The detection limit was satisfactory for the point mutation detection of CTNAs. Hence, the self-circulating ECL chip could adequately meet the strict clinical requirements for CTNA detection and thus has the potential to serve as a new paradigm for liquid tumour biopsy.