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Self-circulating electrochemiluminescence chip for sensitive detection of circulating tumour nucleic acids in blood
Sensors & Actuators: B. Chemical 301 (2019) 127088
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Self-circulating electrochemiluminescence chip for sensitive detection of circulating tumour nucleic acids in blood
T
Ying Liua,1, Zhijin Fanc,1, Yuan Zhoua,1, Jingyan Lind, Yang Yangd, Li Yana, Yulin Lia, Ling Jianga, ⁎⁎ ⁎ Fan Yanga, Qiuyu Hua, Jun Yua, , Liuyuan Chena, Yuhui Liaoa,b,c, a
Department of Science and Education, Guiyang Sixth Hospital, Guiyang, China Molecular Diagnosis and Treatment Center for Infectious Diseases, Dermatology Hospital, Southern Medical University, Guangzhou, China c Program of Infection and Immunity, the Fifth Affiliated Hospital of Sun Yat-sen University, Zhongshan School of Medicine, Sun Yat-sen University, Guangdong, China d Shenzhen Key Laboratory of Pathogen and Immunity, State Key Discipline of Infectious Disease, Second Hospital Affiliated to Southern University of Science and Technology, Shenzhen Third People’s Hospital, Shenzhen, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Circulating tumour nucleic acids Electrochemiluminescence Liquid biopsy Tumour diagnosis
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.
1. 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 [1–4]. A diagnosis strategy based on CTNAs could serve as a liquid biopsy approach to supplement or replace tumour tissue biopsies [5–8]. The existing studies indicated that tumour patients generally have higher levels of CTNAs than healthy controls [9–11]. 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
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Corresponding author at: Department of Science and Education, Guiyang Sixth Hospital, Guiyang, China. Corresponding author. E-mail addresses: [email protected] (J. Yu), [email protected] (Y. Liao). 1 These authors contributed equally to this work. ⁎⁎
https://doi.org/10.1016/j.snb.2019.127088 Received 5 March 2019; Received in revised form 2 September 2019; Accepted 3 September 2019 Available online 04 September 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Sensors & Actuators: B. Chemical 301 (2019) 127088
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As shown in Fig. 1A, the capture probes were labelled at the terminal with biotin, which can be connected with streptavidin-modified magnetic beads to form the enrichment complex. The complex could be immobilized by a strong electromagnet while the blood was introduced. The CTNAs were captured by the enrichment complex. After a certain number of cycles, the signal probe was introduced (Fig. 1B). In this assay, the specific ECL signal was generated by a Ru (bpy)32+-polymer-amplified ECL strategy from our previous work [28]. The linear polymer-amplified ECL probe could recognize the CTNAs by the base-stacking hybridization model, which is adopted in the capture and signal generation process. An 8 nt DNA recognition domain was connected to the Ru(bpy)32+-polymer. It could not be hybridized to the capture probe without the target CTNAs because 8 nt is too short for nucleic acid hybridization at room temperature. However, a stabilization effect emerged when CTNAs were present. This effect provided the ECL switch for target CTNAs. Finally, the dissociative probe was cleaned with phosphate buffer solution (PBS), and the ECL signal was determined by the ECL detector (Fig. 1C) with the ECL substrate of tripropylamine (TPA).
mutations of CTNAs in serum. In this strategy, a magnet-controlled selfcirculating 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.
2.2. Detection limit and specificity of self-circulating ECL chip This section describes the evaluation of the detection limit performance of the self-circulating ECL chip with different concentrations of standard CTNAs (circulating miRNA21). The concentrations of circulating miRNA21 were mixed in PBS buffer and varied from 10 amol to 10 pmol. The ECL signals were observed and recorded in Fig. 2A. The ECL signal of CTNAs increased with the concentration of CTNAs. When the concentration of CTNAs decreased to 10 amol, the ECL intensity tended to coincide with that of the control group. Data analysis showed that the signal-to-noise ratio at 100 amol was 6.179 ± 0.792 (> 3), which demonstrated that the self-circulating ECL chip achieved a low detection limit of 100 amol. Thus, this platform possesses high efficiencies that could meet real-life demands. The linear regression analysis of the ECL intensities to verify the reliability of the experimental data indicated that the detection limit results have a good linear relation from 100 amol to 1 pmol, and an R2 value of 0.9937 was obtained. Thus, the high efficiency demonstrated that the proposed strategy is effective. To further evaluate the performance of the self-circulating ECL chip, a specificity experiment was performed. Herein, CTNAs and random sequences with identical concentrations of 100 fmol were detected by this platform. The corresponding signals are shown in Fig. 2B. The experimental groups of random sequences produced a very weak ECL signal, which was at the same level as that of the control group. However, intense signals were observed in the CTNA experimental group. This result indicated that this platform has adequate specificity to extract a differentiable signal from complex exractions of tumour cells and blood samples. Furthermore, the CTNAs were spiked into blood samples to construct simulated samples, and the corresponding detection limit was evaluated. The results in Fig. 2C indicated that the self-circulating ECL chip achieved a low detection limit of 500 amol in spiked blood samples. An R2 value of 0.9904 was obtained. These results confirmed the feasibility of this platform for the detection of CTNA samples in blood. However, this detection limit is higher than that of the standard samples in Fig. 2A. Considering that the excised blood sample was more complex than the standard sample, the spiked CTNAs exhibited obvious degradation. The results in Fig. S1 indicated that the absorption (260 nm) of the spiked CTNAs decreased with the incubation time. This result was consistent with the degradation of spiked CTNAs. Meanwhile, the ECL intensity of spiked CTNAs was lower than that of standard samples with the same concentration (Fig. S2), possibly because the DNA hybridization situation in blood was greatly changed. Then, we investigated the performance for spiked body fluid samples and evaluated the feasibility for complex samples. The results in Fig. 2D
2. Results and discussion 2.1. Construction of self-circulating ECL chip The low abundance of CTNAs in blood presents a great challenge to clinical applications of CTNA-based liquid tumour biopsy. Thus, the enrichment assay could provide an efficient way to detect CTNAs. However, a large volume of blood was commonly acquired in the detection process to obtain sufficient CTNA samples (typically larger than 10 mL) [24,25]. However, the levels of CTNAs in plasma or serum samples vary drastically, and it is very difficult to differentiate tumour patients from healthy individuals [26,27]. The individual enrichment process of CTNAs in blood is usually ineffectual. Therefore, we constructed a self-circulating ECL chip (Fig. 1A) that could enrich the CTNAs with two operating modes: 1) Autologous blood transfusion and 2) a multiplex enrichment process. The autologous blood transfusion could provide feedback to specifically ischaemic patients, greatly reducing the sampling injury in critically ill patients. Furthermore, the efficiency of the enrichment process could be improved by extending the cycle time. The multiplex enrichment process could decrease the detection limit by increasing the contact time between the capture probe and target CTNAs. 2
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Fig. 1. Construction of self-circulating electrochemiluminescence (ECL) chip for circulating tumour nucleic acids (CTNAs). A. Self-circulating electrochemiluminescence (ECL) chip for enrichment of CTNAs. The chip could enrich the CTNAs with two operation modes: 1) Autologous blood transfusion. 2) Multiplex enrichment process. B. Process of capturing and stacking hybridization model. The specific ECL signal was generated by a linear Ru(bpy)32+-polymer amplified ECL strategy. An 8 nt DNA recognition domain was connected to the Ru(bpy)32+polymer. Base-stacking hybridization model as the ECL switch for target CTNAs. C. ECL signal generation procedure.
Fig. 2. A. Detection of standard CTNAs with different concentration through self-circulating ECL chip. The circulating miRNA21 was employed as the target CTNAs. B. Detection of different standard CTNAs with self-circulating ECL chip. RS1 and RS2 are random sequence. The CTNAs is circulating miRNA21. C. Detection of spiked blood samples with different concentration. The standard circulating miRNA21 was spiked in blood. D. Self-circulating ECL chip for spiked body fluid samples. ‘U’ is urine sample, ‘S’ is saliva sample, and ‘B’ is blood sample. All ‘C’ are control groups.
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Fig. 3. A. Detection of CTNAs with different signal probes. The signal probe of the nucleotide C joint site (‘G’ group) can respond to the wild-type of nucleotide G, and the signal probes at the nucleotide T (‘A’ group), G (‘C’ group) and A (‘T’ group) joint site can respond to point mutations to A, C and T. B. Point mutation ‘A’ detection of CTNAs with self-circulating ECL chip. C. Point mutation ‘C’ detection of CTNAs with self-circulating ECL chip. D. Point mutation ‘T’ Detection of CTNAs with self-circulating ECL chip. The ‘G’ group was the positive control, which was the wild-type nucleotide G.
tumour samples should have the capacity to cope with the complex physiological environment. Herein, we collected 40 blood samples from breast cancer patients. The medical record information is listed in Table S1. The results in Fig. 5A–D indicate that the experimental groups showed differentiable signals compared with the control group. Thus, these results proved that this platform possesses potential as an innovation in tumour molecular diagnosis technologies based on CTNAs. Furthermore, the response time and reproducibility results are shown in Figs. S3 and S4. These results confirmed the performance of this strategy.
revealed that this platform could obtain a stable ECL signal from complex spiked samples (blood, urine, saliva). Therefore, the feasibility of this platform for complex samples was confirmed. 2.3. Point mutations detection of CTNAs with self-circulating ECL chip As the preliminary work proved, the detection of mutated sequences linked to cancer in CTNAs (for example, KRAS and BRAF) could allow the specific diagnosis of related tumours. Benefiting from the recognition of the performance of the base-stacking hybridization model, a point mutation detection strategy with a self-circulating ECL chip was constructed for CTNAs. The point mutations of CTNAs were designed at the joint site between the signal probe and the capture probe. Four signal probes were also designed to recognize the variations A, T, C and G. The target CTNAs were the KRAS gene, which has three mutations at codons 12 exons, namely, 135A, 135C, 135 T [29–32]. Mutated KRAS (Kirsten rat sarcoma-2 virus) genes were relevant to lung cancer, colorectal cancer and ovarian cancer. The efficacies of the therapies were affected by mutations in this gene [33–37]. As shown in Fig. 3A, the ‘G’ group with the signal probe of a nucleotide C joint site can respond to the wild-type nucleotide G, and the ‘A’, ‘C’ and‘T’ groups with the signal probe for the nucleotide T (‘A’ group), G (‘C’ group) and A (‘T’ group) joint site can respond to point mutations of A, C and T. The results in Fig. 3B–D indicated that the self-circulating ECL chip could recognize different mutations. The wild-type nucleotide G was employed as the positive control.
2.5. Post-cure monitoring of CTNAs in tumour patients Finally, 10 patients with confirmed breast cancer (5 patients) and liver cancer (5 patients) were traced before and after exairesis, and blood samples were collected at two-week intervals. The results in Fig. 6A show that the patients with breast cancer presented higher levels of CTNAs before surgery. After the tumour tissue was removed, the ECL signal decreased over time. The patients with liver cancer showed a similar trend to that observed in patients with breast cancer. The results in Fig. 6B revealed that the patients with liver cancer also exhibited higher concentration levels before surgery. After the tumour tissue was removed, the ECL signal also decreased with time. Thus, the self-circulating ECL chip could be used for the post-cure monitoring of tumour patients. 3. Conclusions
2.4. Detection of CTNAs from cultured tumour cell lines and blood of tumour patients
A self-circulating ECL chip was constructed to detect the content and point mutations of CTNAs in serum. In this strategy, a magnetcontrolled self-circulating chip was constructed for enrichment of CTNAs in the blood, and autologous blood transfusion was performed for feedback. Meanwhile, the principle of base stacking was used for point mutation detection, and a satisfactory recognition performance for point mutations was achieved. Furthermore, an improved amplified ECL assay was employed for highly efficient signal generation, and a low detection limit of 100 amol and desirable specificity was achieved. The performance index for the analysis of clinical CTNA samples was investigated, and the results indicated that the self-circulating ECL chip reliably responded to CTNAs from the blood. Hence, the self-circulating ECL chip adequately met the strict clinical requirements for CTNA
Three tumour cell lines (A549, MCF-7, and HepG2) with high expression levels of miRNA21 were selected to investigate the capacity for the detection of CTNAs from tumour cells. The supernatants were collected after the tumour cell lines were cultured for three days. Then, the self-circulating ECL chip was operated. The results in Fig. 4A–C (30 tumour cell samples including 10 samples of A549, 10 samples of MCF7, and 10 samples of HepG2) indicated that recognizable ECL signals were obtained from the supernatants of tumour cells. The complexity and diversity of clinical blood samples from tumour patients present great challenges to the clinical diagnosis of tumours based on CTNAs [38,39]. Thus, an excellent diagnostic platform for 4
Sensors & Actuators: B. Chemical 301 (2019) 127088
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Fig. 4. A. Detection of CTNAs from cultured A549 cell lines. B. Detection of CTNAs from cultured MCF7 cell lines. C. Detection of CTNAs from cultured HepG2 cell lines. A549, MCF-7, and HepG2 cells have high miRNA21 expression levels. The supernatants were collected after the tumor cell lines were cultured for three days.
Fig. 5. Detection of CTNAs in blood of tumour patients. A. Sample ID from 1 to 10. B. Sample ID from 11 to 20. C. Sample ID from 21 to 30. D. Sample ID from 31 to 40. Forty blood samples were collected from breast cancer patients. The pathological information is listed in Table S1.
4. Experimental Section
detection and thus has potential as a new paradigm for liquid biopsy and diagnosis of tumours.
4.1. Reagents All chemical reagents, such as cis-Bis(2,2′-bipyridine)dichlororuthenium(II), 2,2′-bipyridine-4,4′-dicarboxylic acid, N,N’5
Sensors & Actuators: B. Chemical 301 (2019) 127088
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Fig. 6. A. Post-cure monitoring of CTNAs in breast cancer patients. B. Post-cure monitoring of CTNAs in liver cancer patients. The blood samples of breast cancer (5 patients) and liver cancer (5 patients) were traced before and after exairesis.
4.6. Experimental process for blood samples
dicyclohexylcarbodiimide (DCC), sodium hexafluorophosphate, N-(3(dimethylamino)propyl)-N’-ethylcarbodiimide hydrochloride (EDC), Nhydroxysuccinimide (NHS), and sodium borate, were obtained from Alfa Aesar Co., Ltd. Streptavidin magnetic beads were synthesized by New England BioLabs. Reagent-grade PLL was purchased from SigmaAldrich and used without further purification except where noted. Invitrogen synthesized all oligonucleotides.
The blood samples were obtained from tumour patients by collecting blood from an upper extremity vein. Optimal blood samples (2–3 mL) were collected from each tumour patient, and blood anticoagulants were necessary. Afterwards, the 2–3 mL blood samples were diluted with PBS to 10 mL and circulated by the ECL chip for 10 min. Finally, the ECL chip was washed with PBS three times, and TPA was added for ECL generation.
4.2. Ethics statement
Acknowledgements
All participants were given written or orally informed consent for this study. The experimental procedures were approved by the Ethics Committee of Guiyang Sixth Hospital.
This work was supported by the Science and Technology Planning Project of Guiyang [Funding Number: (2018)1-13], the National Natural Science Foundation of China (81972019,21904145).
4.3. Synthetic routes of Ru(bpy)32+-polymer
Appendix A. Supplementary data
Ru(bpy)32+ was activated by NHS and DCC in DMF. The Ru (bpy)32+-polymer was synthesized by employing PLL as the binding skeleton. The repetitive amino group of PLL was used as the binding site for Ru(bpy)32+. Twenty-five milligrams of PLL was added to excess Ru (bpy)32+ with a molar ratio of 1:10 between the amino group and Ru (bpy)32+. The mixture was incubated in DMF (20%) for 12 h at 37 °C. Finally, the products were purified using a Nanosep OMEGA tubular ultrafiltration membrane (50 K) from Pall Corp. (Port Washington, NY, USA). The purified Ru(bpy)32+-polymer was stored in a freezer (−20 °C).
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4.4. Base-stacking hybridization model The key part of the base-stacking hybridization model is the 8 nt DNA recognition domain of the signal probe. A 50 nM signal probe solution was employed as the signal generation complex, which was mixed with 10 μL of magnetic beads (1 mg/mL) for 30 min. Then, the target was captured by a capture probe, and the signal probe was added to construct a base-stacking hybridization model at 37 °C. 4.5. ECL process As we previously reported [40], the magnetic beads used in this assay were labelled with streptavidin, which could recognize and bind the biotin on the capture probe. The signal probe, composed of the Ru (bpy)32+-polymer and recognition domain, was employed as the ECLgenerating group. In the presence of the target, the capture probe and signal probe constituted the base-stacking hybridization model, which can be adapted for nucleic acid detection. After the cleaning steps, the ECL signal was detected in the presence of the co-reactant tripropylamine. 6
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Martino, Dual-targeted therapy with trastuzumab and lapatinib in treatment-refractory, KRAS codon 12/13 wild-type, HER2positive metastatic colorectal cancer (HERACLES): a proof-of-concept, multicentre, open-label, phase 2 trial, Lancet Oncol. 17 (2016) 738–746. [32] Y. Zhang, L.X. Wang, F. Luo, B. Qiu, L.H. Guo, Z.Q. Weng, et al., An electrochemiluminescence biosensor for Kras mutations based on locked nucleic acid functionalized DNA walkers and hyperbranched rolling circle amplification, Chem. Commun. (Camb.) 53 (2017) 2910–2913. [33] M. Serresi, G. Gargiulo, N. Proost, B. Siteur, M. Cesaroni, M. Koppens, et al., Polycomb repressive complex 2 is a barrier to KRAS-driven inflammation and epithelial-mesenchymal transition in non-small-cell lung cancer, Cancer Cell 29 (2016) 17–31. [34] E. Manchado, S. Weissmueller, J.P. Morris, C.C. Chen, R. Wullenkord, A. Lujambio, et al., A combinatorial strategy for treating KRAS-mutant lung cancer, Nature 534 (2016) 647–651. [35] L.S. 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Ying Liu obtained her M.D. degree from Chiba University in 2013. She is now working as a director of science and education department at Guiyang Sixth Hospital, Guizhou, China. Her research interests aim at molecular diagnosis. Zhijin Fan graduated from South China Normal University in 2016 with a master's degree in biophysics. He is currently working in the Molecular Imaging Center of the Fifth Affiliated Hospital of Sun Yat-sen University. His research interests aim at the construction of cancer-related nanodiagnostic probes. Yuan Zhou obtained his bachelor degree of traditional Chinese medicine from Guiyang college of traditional Chinese medicine in 1998. He is now working as an associate chief physician at Guiyang Sixth Hospital, Guizhou, China. His research interests aim at orthopaedics. Jingyan Lin obtained her M.M. degree from Guanagzhou Medical University in 2013. She is now working as a staff at The Third People's Hospital of Shenzhen, Guangdong, China. Her research interests aim at immunity in infectious diseases. Yang Yang received his Ph.D degree from Chinese Center for Disease Control and Prevention in 2015 and now is a staff of Shenzhen Third People's Hospital, Guangdong,China. He mainly focused on the etiology and immunology of emerging infectious diseases. Li Yan obtained her bachelor degree of Nursing Science from Guizhou medical university in 2009. She is now working as a chief superintendent nurse at Guiyang Sixth Hospital, Guizhou, China. Her research interests aim at nursing management. Yulin Li obtained her bachelor degree of Nursing Science from Guizhou medical university in 2010. She is now working as a chief nurse at Guiyang Sixth Hospital, Guizhou, China. Her research interests aim at nursing research. Ling Jiang obtained her bachelor degree of Pharmacy from Guizhou medical university in 1996. She is now working as a chief pharmacist at Guiyang Sixth Hospital, Guizhou, China. Her research interests aim at pharmaceutical research. Fan Yang obtained his bachelor degree of narcology from Zunyi medical college in 2005. He is now working as a chief of anesthesia at Guiyang Sixth Hospital, Guizhou, China. His research interests aim at clinical anesthesia research. Qiuyu Hu obtained her bachelor degree of medicine from Guizhou medical university in 2013. She is now working as a staff of science and education department at Guiyang Sixth Hospital, Guizhou, China. Her research interests aim at statistical management. Jun Yu obtained her bachelor degree of medicine from Zunyi medical college in 1994. She is now working as a chief physician at Guiyang Sixth Hospital, Guizhou, China. Her research interests aim at cardiovascular medicine. Liuyuan Chen obtained his bachelor degree of management from Guizhou University in 2003. He is now working as a chief accountant at Guiyang Sixth Hospital, Guizhou, China. His research interests aim at hospital financial management. Yuhui Liao obtained his Ph.D degree from South China Normal University in 2016. He is now working as an associate research fellow of Southern Medical University and Sun Yatsen University. His research interests aim at molecular diagnosis and treatment for infectious diseases.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Self-circulating electrochemiluminescence chip for sensitive detection of circulating tumour nucleic acids in blood
T
Ying Liua,1, Zhijin Fanc,1, Yuan Zhoua,1, Jingyan Lind, Yang Yangd, Li Yana, Yulin Lia, Ling Jianga, ⁎⁎ ⁎ Fan Yanga, Qiuyu Hua, Jun Yua, , Liuyuan Chena, Yuhui Liaoa,b,c, a
Department of Science and Education, Guiyang Sixth Hospital, Guiyang, China Molecular Diagnosis and Treatment Center for Infectious Diseases, Dermatology Hospital, Southern Medical University, Guangzhou, China c Program of Infection and Immunity, the Fifth Affiliated Hospital of Sun Yat-sen University, Zhongshan School of Medicine, Sun Yat-sen University, Guangdong, China d Shenzhen Key Laboratory of Pathogen and Immunity, State Key Discipline of Infectious Disease, Second Hospital Affiliated to Southern University of Science and Technology, Shenzhen Third People’s Hospital, Shenzhen, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Circulating tumour nucleic acids Electrochemiluminescence Liquid biopsy Tumour diagnosis
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.
1. 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 [1–4]. A diagnosis strategy based on CTNAs could serve as a liquid biopsy approach to supplement or replace tumour tissue biopsies [5–8]. The existing studies indicated that tumour patients generally have higher levels of CTNAs than healthy controls [9–11]. 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
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Corresponding author at: Department of Science and Education, Guiyang Sixth Hospital, Guiyang, China. Corresponding author. E-mail addresses: [email protected] (J. Yu), [email protected] (Y. Liao). 1 These authors contributed equally to this work. ⁎⁎
https://doi.org/10.1016/j.snb.2019.127088 Received 5 March 2019; Received in revised form 2 September 2019; Accepted 3 September 2019 Available online 04 September 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
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As shown in Fig. 1A, the capture probes were labelled at the terminal with biotin, which can be connected with streptavidin-modified magnetic beads to form the enrichment complex. The complex could be immobilized by a strong electromagnet while the blood was introduced. The CTNAs were captured by the enrichment complex. After a certain number of cycles, the signal probe was introduced (Fig. 1B). In this assay, the specific ECL signal was generated by a Ru (bpy)32+-polymer-amplified ECL strategy from our previous work [28]. The linear polymer-amplified ECL probe could recognize the CTNAs by the base-stacking hybridization model, which is adopted in the capture and signal generation process. An 8 nt DNA recognition domain was connected to the Ru(bpy)32+-polymer. It could not be hybridized to the capture probe without the target CTNAs because 8 nt is too short for nucleic acid hybridization at room temperature. However, a stabilization effect emerged when CTNAs were present. This effect provided the ECL switch for target CTNAs. Finally, the dissociative probe was cleaned with phosphate buffer solution (PBS), and the ECL signal was determined by the ECL detector (Fig. 1C) with the ECL substrate of tripropylamine (TPA).
mutations of CTNAs in serum. In this strategy, a magnet-controlled selfcirculating 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.
2.2. Detection limit and specificity of self-circulating ECL chip This section describes the evaluation of the detection limit performance of the self-circulating ECL chip with different concentrations of standard CTNAs (circulating miRNA21). The concentrations of circulating miRNA21 were mixed in PBS buffer and varied from 10 amol to 10 pmol. The ECL signals were observed and recorded in Fig. 2A. The ECL signal of CTNAs increased with the concentration of CTNAs. When the concentration of CTNAs decreased to 10 amol, the ECL intensity tended to coincide with that of the control group. Data analysis showed that the signal-to-noise ratio at 100 amol was 6.179 ± 0.792 (> 3), which demonstrated that the self-circulating ECL chip achieved a low detection limit of 100 amol. Thus, this platform possesses high efficiencies that could meet real-life demands. The linear regression analysis of the ECL intensities to verify the reliability of the experimental data indicated that the detection limit results have a good linear relation from 100 amol to 1 pmol, and an R2 value of 0.9937 was obtained. Thus, the high efficiency demonstrated that the proposed strategy is effective. To further evaluate the performance of the self-circulating ECL chip, a specificity experiment was performed. Herein, CTNAs and random sequences with identical concentrations of 100 fmol were detected by this platform. The corresponding signals are shown in Fig. 2B. The experimental groups of random sequences produced a very weak ECL signal, which was at the same level as that of the control group. However, intense signals were observed in the CTNA experimental group. This result indicated that this platform has adequate specificity to extract a differentiable signal from complex exractions of tumour cells and blood samples. Furthermore, the CTNAs were spiked into blood samples to construct simulated samples, and the corresponding detection limit was evaluated. The results in Fig. 2C indicated that the self-circulating ECL chip achieved a low detection limit of 500 amol in spiked blood samples. An R2 value of 0.9904 was obtained. These results confirmed the feasibility of this platform for the detection of CTNA samples in blood. However, this detection limit is higher than that of the standard samples in Fig. 2A. Considering that the excised blood sample was more complex than the standard sample, the spiked CTNAs exhibited obvious degradation. The results in Fig. S1 indicated that the absorption (260 nm) of the spiked CTNAs decreased with the incubation time. This result was consistent with the degradation of spiked CTNAs. Meanwhile, the ECL intensity of spiked CTNAs was lower than that of standard samples with the same concentration (Fig. S2), possibly because the DNA hybridization situation in blood was greatly changed. Then, we investigated the performance for spiked body fluid samples and evaluated the feasibility for complex samples. The results in Fig. 2D
2. Results and discussion 2.1. Construction of self-circulating ECL chip The low abundance of CTNAs in blood presents a great challenge to clinical applications of CTNA-based liquid tumour biopsy. Thus, the enrichment assay could provide an efficient way to detect CTNAs. However, a large volume of blood was commonly acquired in the detection process to obtain sufficient CTNA samples (typically larger than 10 mL) [24,25]. However, the levels of CTNAs in plasma or serum samples vary drastically, and it is very difficult to differentiate tumour patients from healthy individuals [26,27]. The individual enrichment process of CTNAs in blood is usually ineffectual. Therefore, we constructed a self-circulating ECL chip (Fig. 1A) that could enrich the CTNAs with two operating modes: 1) Autologous blood transfusion and 2) a multiplex enrichment process. The autologous blood transfusion could provide feedback to specifically ischaemic patients, greatly reducing the sampling injury in critically ill patients. Furthermore, the efficiency of the enrichment process could be improved by extending the cycle time. The multiplex enrichment process could decrease the detection limit by increasing the contact time between the capture probe and target CTNAs. 2
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Fig. 1. Construction of self-circulating electrochemiluminescence (ECL) chip for circulating tumour nucleic acids (CTNAs). A. Self-circulating electrochemiluminescence (ECL) chip for enrichment of CTNAs. The chip could enrich the CTNAs with two operation modes: 1) Autologous blood transfusion. 2) Multiplex enrichment process. B. Process of capturing and stacking hybridization model. The specific ECL signal was generated by a linear Ru(bpy)32+-polymer amplified ECL strategy. An 8 nt DNA recognition domain was connected to the Ru(bpy)32+polymer. Base-stacking hybridization model as the ECL switch for target CTNAs. C. ECL signal generation procedure.
Fig. 2. A. Detection of standard CTNAs with different concentration through self-circulating ECL chip. The circulating miRNA21 was employed as the target CTNAs. B. Detection of different standard CTNAs with self-circulating ECL chip. RS1 and RS2 are random sequence. The CTNAs is circulating miRNA21. C. Detection of spiked blood samples with different concentration. The standard circulating miRNA21 was spiked in blood. D. Self-circulating ECL chip for spiked body fluid samples. ‘U’ is urine sample, ‘S’ is saliva sample, and ‘B’ is blood sample. All ‘C’ are control groups.
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Fig. 3. A. Detection of CTNAs with different signal probes. The signal probe of the nucleotide C joint site (‘G’ group) can respond to the wild-type of nucleotide G, and the signal probes at the nucleotide T (‘A’ group), G (‘C’ group) and A (‘T’ group) joint site can respond to point mutations to A, C and T. B. Point mutation ‘A’ detection of CTNAs with self-circulating ECL chip. C. Point mutation ‘C’ detection of CTNAs with self-circulating ECL chip. D. Point mutation ‘T’ Detection of CTNAs with self-circulating ECL chip. The ‘G’ group was the positive control, which was the wild-type nucleotide G.
tumour samples should have the capacity to cope with the complex physiological environment. Herein, we collected 40 blood samples from breast cancer patients. The medical record information is listed in Table S1. The results in Fig. 5A–D indicate that the experimental groups showed differentiable signals compared with the control group. Thus, these results proved that this platform possesses potential as an innovation in tumour molecular diagnosis technologies based on CTNAs. Furthermore, the response time and reproducibility results are shown in Figs. S3 and S4. These results confirmed the performance of this strategy.
revealed that this platform could obtain a stable ECL signal from complex spiked samples (blood, urine, saliva). Therefore, the feasibility of this platform for complex samples was confirmed. 2.3. Point mutations detection of CTNAs with self-circulating ECL chip As the preliminary work proved, the detection of mutated sequences linked to cancer in CTNAs (for example, KRAS and BRAF) could allow the specific diagnosis of related tumours. Benefiting from the recognition of the performance of the base-stacking hybridization model, a point mutation detection strategy with a self-circulating ECL chip was constructed for CTNAs. The point mutations of CTNAs were designed at the joint site between the signal probe and the capture probe. Four signal probes were also designed to recognize the variations A, T, C and G. The target CTNAs were the KRAS gene, which has three mutations at codons 12 exons, namely, 135A, 135C, 135 T [29–32]. Mutated KRAS (Kirsten rat sarcoma-2 virus) genes were relevant to lung cancer, colorectal cancer and ovarian cancer. The efficacies of the therapies were affected by mutations in this gene [33–37]. As shown in Fig. 3A, the ‘G’ group with the signal probe of a nucleotide C joint site can respond to the wild-type nucleotide G, and the ‘A’, ‘C’ and‘T’ groups with the signal probe for the nucleotide T (‘A’ group), G (‘C’ group) and A (‘T’ group) joint site can respond to point mutations of A, C and T. The results in Fig. 3B–D indicated that the self-circulating ECL chip could recognize different mutations. The wild-type nucleotide G was employed as the positive control.
2.5. Post-cure monitoring of CTNAs in tumour patients Finally, 10 patients with confirmed breast cancer (5 patients) and liver cancer (5 patients) were traced before and after exairesis, and blood samples were collected at two-week intervals. The results in Fig. 6A show that the patients with breast cancer presented higher levels of CTNAs before surgery. After the tumour tissue was removed, the ECL signal decreased over time. The patients with liver cancer showed a similar trend to that observed in patients with breast cancer. The results in Fig. 6B revealed that the patients with liver cancer also exhibited higher concentration levels before surgery. After the tumour tissue was removed, the ECL signal also decreased with time. Thus, the self-circulating ECL chip could be used for the post-cure monitoring of tumour patients. 3. Conclusions
2.4. Detection of CTNAs from cultured tumour cell lines and blood of tumour patients
A self-circulating ECL chip was constructed to detect the content and point mutations of CTNAs in serum. In this strategy, a magnetcontrolled self-circulating chip was constructed for enrichment of CTNAs in the blood, and autologous blood transfusion was performed for feedback. Meanwhile, the principle of base stacking was used for point mutation detection, and a satisfactory recognition performance for point mutations was achieved. Furthermore, an improved amplified ECL assay was employed for highly efficient signal generation, and a low detection limit of 100 amol and desirable specificity was achieved. The performance index for the analysis of clinical CTNA samples was investigated, and the results indicated that the self-circulating ECL chip reliably responded to CTNAs from the blood. Hence, the self-circulating ECL chip adequately met the strict clinical requirements for CTNA
Three tumour cell lines (A549, MCF-7, and HepG2) with high expression levels of miRNA21 were selected to investigate the capacity for the detection of CTNAs from tumour cells. The supernatants were collected after the tumour cell lines were cultured for three days. Then, the self-circulating ECL chip was operated. The results in Fig. 4A–C (30 tumour cell samples including 10 samples of A549, 10 samples of MCF7, and 10 samples of HepG2) indicated that recognizable ECL signals were obtained from the supernatants of tumour cells. The complexity and diversity of clinical blood samples from tumour patients present great challenges to the clinical diagnosis of tumours based on CTNAs [38,39]. Thus, an excellent diagnostic platform for 4
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Fig. 4. A. Detection of CTNAs from cultured A549 cell lines. B. Detection of CTNAs from cultured MCF7 cell lines. C. Detection of CTNAs from cultured HepG2 cell lines. A549, MCF-7, and HepG2 cells have high miRNA21 expression levels. The supernatants were collected after the tumor cell lines were cultured for three days.
Fig. 5. Detection of CTNAs in blood of tumour patients. A. Sample ID from 1 to 10. B. Sample ID from 11 to 20. C. Sample ID from 21 to 30. D. Sample ID from 31 to 40. Forty blood samples were collected from breast cancer patients. The pathological information is listed in Table S1.
4. Experimental Section
detection and thus has potential as a new paradigm for liquid biopsy and diagnosis of tumours.
4.1. Reagents All chemical reagents, such as cis-Bis(2,2′-bipyridine)dichlororuthenium(II), 2,2′-bipyridine-4,4′-dicarboxylic acid, N,N’5
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Fig. 6. A. Post-cure monitoring of CTNAs in breast cancer patients. B. Post-cure monitoring of CTNAs in liver cancer patients. The blood samples of breast cancer (5 patients) and liver cancer (5 patients) were traced before and after exairesis.
4.6. Experimental process for blood samples
dicyclohexylcarbodiimide (DCC), sodium hexafluorophosphate, N-(3(dimethylamino)propyl)-N’-ethylcarbodiimide hydrochloride (EDC), Nhydroxysuccinimide (NHS), and sodium borate, were obtained from Alfa Aesar Co., Ltd. Streptavidin magnetic beads were synthesized by New England BioLabs. Reagent-grade PLL was purchased from SigmaAldrich and used without further purification except where noted. Invitrogen synthesized all oligonucleotides.
The blood samples were obtained from tumour patients by collecting blood from an upper extremity vein. Optimal blood samples (2–3 mL) were collected from each tumour patient, and blood anticoagulants were necessary. Afterwards, the 2–3 mL blood samples were diluted with PBS to 10 mL and circulated by the ECL chip for 10 min. Finally, the ECL chip was washed with PBS three times, and TPA was added for ECL generation.
4.2. Ethics statement
Acknowledgements
All participants were given written or orally informed consent for this study. The experimental procedures were approved by the Ethics Committee of Guiyang Sixth Hospital.
This work was supported by the Science and Technology Planning Project of Guiyang [Funding Number: (2018)1-13], the National Natural Science Foundation of China (81972019,21904145).
4.3. Synthetic routes of Ru(bpy)32+-polymer
Appendix A. Supplementary data
Ru(bpy)32+ was activated by NHS and DCC in DMF. The Ru (bpy)32+-polymer was synthesized by employing PLL as the binding skeleton. The repetitive amino group of PLL was used as the binding site for Ru(bpy)32+. Twenty-five milligrams of PLL was added to excess Ru (bpy)32+ with a molar ratio of 1:10 between the amino group and Ru (bpy)32+. The mixture was incubated in DMF (20%) for 12 h at 37 °C. Finally, the products were purified using a Nanosep OMEGA tubular ultrafiltration membrane (50 K) from Pall Corp. (Port Washington, NY, USA). The purified Ru(bpy)32+-polymer was stored in a freezer (−20 °C).
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.127088. References [1] A.M. Aravanis, M. Lee, R.D. Klausner, Next-generation sequencing of circulating tumor DNA for early cancer detection, Cell 168 (2017) 571–574. [2] T. Reinert, L.V. Scholer, R. Thomsen, H. Tobiasen, S.R. Vang, I. Nordentoft, et al., Analysis of circulating tumour DNA to monitor disease burden following colorectal cancer surgery, Gut 65 (2016) 625–634. [3] V. Swarup, M.R. Rajeswari, Circulating (cell-free) nucleic acids - A promising, noninvasive tool for early detection of several human diseases, FEBS Lett. 581 (2007) 795–799. [4] M. Tomasetti, M. Amati, J. Neuzil, L. Santarelli, Circulating epigenetic biomarkers in lung malignancies: from early diagnosis to therapy, Lung Cancer 107 (2017) 65–72. [5] J.C.M. Wan, C. Massie, J. Garcia-Corbacho, F. Mouliere, J.D. Brenton, C. Caldas, et al., Liquid biopsies come of age: towards implementation of circulating tumour DNA, Nat. Rev. Cancer 17 (2017) 223–238. [6] L. Sorber, K. Zwaenepoel, V. Deschoolmeester, P.E.Y. Van Schil, J. Van Meerbeeck, F. Lardon, et al., Circulating cell-free nucleic acids and platelets as a liquid biopsy in the provision of personalized therapy for lung cancer patients, Lung Cancer 107 (2017) 100–107. [7] G. Siravegna, S. Marsoni, S. Siena, A. Bardelli, Integrating liquid biopsies into the management of cancer, Nat. Rev. Clin. Oncol. 14 (2017) 531–548. [8] L. Gorgannezhad, M. Umer, M.N. Islam, N.T. Nguyen, M.J.A. Shiddiky, Circulating tumor DNA and liquid biopsy: opportunities, challenges, and recent advances in detection technologies, Lab Chip 18 (2018) 1174–1196. [9] H. Schwarzenbach, D.S.B. Hoon, K. Pantel, Cell-free nucleic acids as biomarkers in cancer patients, Nat. Rev. Cancer 11 (2011) 426–437. [10] C.H. Lawrie, S. Gal, H.M. Dunlop, B. Pushkaran, A.P. Liggins, K. Pulford, et al., Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma, Brit. J. Haematol. 141 (2008) 672–675. [11] J. Das, I. Ivanov, L. Montermini, J. Rak, E.H. Sargent, S.O. Kelley, An electrochemical clamp assay for direct, rapid analysis of circulating nucleic acids in serum, Nat. Chem. 7 (2015) 569–575. [12] N. Krishnamurthy, E. Spencer, A. Torkamani, L. Nicholson, Liquid biopsies for Cancer: coming to a patient near you, J. Clin. Med. 6 (3) (2017). [13] E. Heitzer, P. Ulz, J.B. Geigl, Circulating tumor DNA as a liquid biopsy for cancer,
4.4. Base-stacking hybridization model The key part of the base-stacking hybridization model is the 8 nt DNA recognition domain of the signal probe. A 50 nM signal probe solution was employed as the signal generation complex, which was mixed with 10 μL of magnetic beads (1 mg/mL) for 30 min. Then, the target was captured by a capture probe, and the signal probe was added to construct a base-stacking hybridization model at 37 °C. 4.5. ECL process As we previously reported [40], the magnetic beads used in this assay were labelled with streptavidin, which could recognize and bind the biotin on the capture probe. The signal probe, composed of the Ru (bpy)32+-polymer and recognition domain, was employed as the ECLgenerating group. In the presence of the target, the capture probe and signal probe constituted the base-stacking hybridization model, which can be adapted for nucleic acid detection. After the cleaning steps, the ECL signal was detected in the presence of the co-reactant tripropylamine. 6
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Ying Liu obtained her M.D. degree from Chiba University in 2013. She is now working as a director of science and education department at Guiyang Sixth Hospital, Guizhou, China. Her research interests aim at molecular diagnosis. Zhijin Fan graduated from South China Normal University in 2016 with a master's degree in biophysics. He is currently working in the Molecular Imaging Center of the Fifth Affiliated Hospital of Sun Yat-sen University. His research interests aim at the construction of cancer-related nanodiagnostic probes. Yuan Zhou obtained his bachelor degree of traditional Chinese medicine from Guiyang college of traditional Chinese medicine in 1998. He is now working as an associate chief physician at Guiyang Sixth Hospital, Guizhou, China. His research interests aim at orthopaedics. Jingyan Lin obtained her M.M. degree from Guanagzhou Medical University in 2013. She is now working as a staff at The Third People's Hospital of Shenzhen, Guangdong, China. Her research interests aim at immunity in infectious diseases. Yang Yang received his Ph.D degree from Chinese Center for Disease Control and Prevention in 2015 and now is a staff of Shenzhen Third People's Hospital, Guangdong,China. He mainly focused on the etiology and immunology of emerging infectious diseases. Li Yan obtained her bachelor degree of Nursing Science from Guizhou medical university in 2009. She is now working as a chief superintendent nurse at Guiyang Sixth Hospital, Guizhou, China. Her research interests aim at nursing management. Yulin Li obtained her bachelor degree of Nursing Science from Guizhou medical university in 2010. She is now working as a chief nurse at Guiyang Sixth Hospital, Guizhou, China. Her research interests aim at nursing research. Ling Jiang obtained her bachelor degree of Pharmacy from Guizhou medical university in 1996. She is now working as a chief pharmacist at Guiyang Sixth Hospital, Guizhou, China. Her research interests aim at pharmaceutical research. Fan Yang obtained his bachelor degree of narcology from Zunyi medical college in 2005. He is now working as a chief of anesthesia at Guiyang Sixth Hospital, Guizhou, China. His research interests aim at clinical anesthesia research. Qiuyu Hu obtained her bachelor degree of medicine from Guizhou medical university in 2013. She is now working as a staff of science and education department at Guiyang Sixth Hospital, Guizhou, China. Her research interests aim at statistical management. Jun Yu obtained her bachelor degree of medicine from Zunyi medical college in 1994. She is now working as a chief physician at Guiyang Sixth Hospital, Guizhou, China. Her research interests aim at cardiovascular medicine. Liuyuan Chen obtained his bachelor degree of management from Guizhou University in 2003. He is now working as a chief accountant at Guiyang Sixth Hospital, Guizhou, China. His research interests aim at hospital financial management. Yuhui Liao obtained his Ph.D degree from South China Normal University in 2016. He is now working as an associate research fellow of Southern Medical University and Sun Yatsen University. His research interests aim at molecular diagnosis and treatment for infectious diseases.
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