Magnetic aptamer-functionalized electrochemical nano-biosensor for the detection of circulating tumor DNA

2.1. Materials

FeCl₃·6H₂O, NaH₂PO₄, glucose, HAuCl₄⋅4H₂O, C₆H₅Na₃O₇, NaBH₄, KCl, trometamol (Tris), ethylene diamine tetraacetic acid (EDTA), K₃Fe(CN)₆, K₄Fe(CN)₆·3H₂O, NaCl, KH₂PO₄, and Na₂HPO₄ were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); polyethyleneimine (PEI) was purchased from Macklin Biochemical (Shanghai, China); tris-(2-carboxyethyl)-phosphine (TCEP) was supplied from Aladdin Reagent (Shanghai, China); phosphate buffered saline (PBS) was ordered from Servicebio Technology Co. Ltd. (Wuhan, China); absolute ethanol was obtained from Chengdu Chron Chemicals Co., Ltd. (Sichuan, China); bovine serum albumin (BSA) was purchased from Saiguo Biotech Co., Ltd. (Guangzhou, China). Human serum was provided by the Danyang People’s Hospital (Zhenjiang, China). All oligonucleotide sequences (Table 1) were supplied by Sangon Biotech Co., Ltd. (Shanghai, China).

2.2. Preparation and characterization of sheet-like α-Fe₂O₃/Fe₃O₄@Au MNCs

Firstly, α-Fe₂O₃ magnetic nanosheets (MNSs) were fabricated by the hydrothermal method. Typically, 0.541 g FeCl₃·6H₂O and 0.054 g NaH₂PO₄ were dissolved in 80 mL of deionized water. The obtained solution was transferred into a hydrothermal reactor (volume- 100 mL) for 24 h at 220°C. Thereafter, the reactor was naturally cooled, the mixture was centrifuged for 15 min at 10,000 rpm, and the product was alternately washed for thrice using absolute alcohol and deionized water. The product was dried for 12 h and ground; the α-Fe₂O₃ MNSs were obtained.

The sheet-like α-Fe₂O₃/Fe₃O₄ MNCs were prepared via the calcination process with α-Fe₂O₃ MNSs as the precursors. For this, 0.1 g α-Fe₂O₃ MNSs and 1.2 g glucose were uniformly mixed and placed into a crucible and calcined at 600°C for 4 h. It was then naturally cooled, and the solid was ground; the sheet-like α-Fe₂O₃/Fe₃O₄ MNCs were obtained.

The PEI containing terminal-NH₂ groups forms a covalent bond with Au, and keeps the positive charge on the surfaces of the nanosheets, which allows the gold nanoparticles (AuNPs) to carry the negative charge to load onto the surfaces. For this, 1.5 g PEI was dissolved in 150 mL ultrapure water to form a homogeneous solution; 50 mg sheet-like α-Fe₂O₃/Fe₃O₄ MNCs were added to the PEI solution, ultrasonically dispersed for 30 min, and then the suspension was magnetically stirred for 2 h in 90^oC water bath. Thereafter, it was centrifuged, the solid was washed with ultrapure water thrice, dried in a vacuum oven at 60°C, and the α-Fe₂O₃/Fe₃O₄-PEI MNCs were obtained.

α-Fe₂O₃/Fe₃O₄-PEI MNCs of 10 mg were dispersed in ultrapure water of 150 mL and dispersed for 10 min by ultrasound. Afterwards, 1 mL chloroauric acid solution (20 mg·mL^-1) was put into the suspension and further sonicated for 30 min in an ice-bath, and then 9 mL NaBH₄ solution (0.75 mg·mL^-1) was added into it and stirred for another 15 min till the suspension turned to black [42], and after centrifugal separation and dry in vacuum oven at 60°C, the sheet-like α-Fe₂O₃/Fe₃O₄@Au MNCs were obtained.

The phase identifications of the α-Fe₂O₃ MNSs and sheet-like α-Fe₂O₃/Fe₃O₄ MNCs were characterized by X-ray diffraction (XRD), their morphologies and compositions were investigated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Their magnetic measurements were determined by the vibrating sample magnetometer (VSM).

2.3. Construction and detection evaluation of the biosensor

The ssDNA-adapted ctDNA was dissolved in TE buffer to form a homogeneous solution of 100 μM. A certain amount of the solution was diluted to 1 μM, and the 1 μM ssDNA solution was added to 1 μL of TCEP (10 mM) according to the molar ratio of 1:100. It was then reacted for 1 h to prompt the binding of ssDNA with AuNPs through the 5’-end sulfhydryl group. Then, 15 μL of the ultrapure water suspension containing α-Fe₂O₃/Fe₃O₄@Au MNCs of 20 mg∙mL^-1 and 15 μL of TCEP-treated ssDNA were mixed and incubated for 13 h at 4°C, magnetically separated, and the supernatant was removed. The solid substances were washed with PBS to remove the uncombined ssDNA, the α-Fe₂O₃/Fe₃O₄@Au-ssDNA was obtained and dispersed in ultrapure water of 30 μL, then the suspension of 9 μL was dropped onto the glazed magnetic glass carbon electrode (MGCE) and dried, CV, EIS, and DPV techniques were employed to measure the current signal through an electrochemical workstation with MGCE, Pt electrode, and Ag/AgCl electrode as the working electrode, the counter, and the reference electrode, of which the scan voltage of CV was kept at -0.1 V-0.7 V, and the corresponding scan rate was controlled at 100 mV·s^-1; while, the frequency range of EIS was 0.1 Hz–10 kHz, and the signal amplitude was 5 mV.

Subsequently, the as-prepared α-Fe₂O₃/Fe₃O₄@Au-ssDNA was added into 30 μL of BSA solution (2.5 mg∙mL^-1), and incubated for 30 min at 4°C to shield the nonspecific sites to avoid any interference on the current signal, and the solid was magnetically separated and washed with PBS to remove the uncombined BSA. As expected, the resulting α-Fe₂O₃/Fe₃O₄@Au-ssDNA/BSA biosensors were constructed. For comprehending the amount of MGCE-α-Fe₂O₃/Fe₃O₄@Au-ssDNA probes on MGCE, their chrono coulometries were examined with or without hexaammineruthenium chloride (RuHex) at a scan rate of 0.01 V∙s^-1 to 0.1 V∙s^-1 for the peak anode currents (Ip) to the square root of the scanning rate ($\surd v$).

The detection process of ctDNA was as follows: the α-Fe₂O₃/Fe₃O₄@Au-ssDNA/BSA biosensors obtained in the previous step were added to a 30 μL ctDNA solution (1 μM), and incubated for 15 min at 65 °C to accomplish the capture of ctDNA. Finally, the solids were washed with PBS to remove free ctDNA, and then the α-Fe₂O₃/Fe₃O₄@Au-ssDNA/BSA/ctDNA nanocomposites were obtained by magnetic separation. The nanocomposites were dispersed into 30 μL of ultrapure water, and according to usual practice, the suspension of the same volume was dropped onto the glazed MGCE, dried, and the electrochemical signal was examined.

In the construction and detection processes, for enhancing the current signals, the concentration of α-Fe₂O₃/Fe₃O₄@Au MNCs was optimized; for greatest degree immobilization of ssDNA and economy, the concentration of ssDNA was investigated; for suitable capture of ctDNA, the incubation temperature and the incubation time were examined; for the line range of the detection, and the relationship of the signal or change signal with concentration of ctDNA was measured; at the same time, the reproducibility, stability, and selectivity were also investigated. All experiments were executed thrice, and the average values, error bars, recoveries, and their relative standard deviations were calculated.

3.1. Characteristics of α-Fe₂O₃ MNSs, α-Fe₂O₃/Fe₃O₄ MNCs and α-Fe₂O₃/Fe₃O₄@Au MNCs

The characteristics of α-Fe₂O₃ MNSs, α-Fe₂O₃/Fe₃O₄ MNCs, and α-Fe₂O₃/Fe₃O₄@Au MNCs have been exhibited in Figure 1. Figure 1(a) displays the SEM morphology of α-Fe₂O₃ nanomaterials fabricated via the hydrothermal process. The α-Fe₂O₃ nanomaterials were round-structured nanosheets, and their diameter and thickness were approximately 230 nm and 130 nm. The size distribution of α-Fe₂O₃ MNSs was uniform. Figure 1(b) reveals the TEM image of sheet-like α-Fe₂O₃/Fe₃O₄ MNCs via the calcination process; their average diameter and thickness were around 220 nm and 130 nm, respectively. As expected, the average diameter and thickness had remained almost unchanged compared with those of α-Fe₂O₃ nanosheets.

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Figure 1.

(a) SEM morphology of α-Fe₂O₃ MNSs, (b) TEM images and (c) XRD pattern of α-Fe₂O₃/Fe₃O₄ MNCs (d) TEM images of α-Fe2O3/Fe3O4@Au MNCs, and (e) hysteresis loops of α-Fe₂O₃/Fe₃O₄ and α-Fe₂O₃/Fe₃O₄@Au MNCs.

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The XRD pattern of α-Fe₂O₃/Fe₃O₄ MNCs has been exhibited in Figure 1(c), most diffraction peaks at 24.1°, 33.2°, 35.6°, 40.9°, 49.5°, 54.1°, 62.4°, and 64.0° correspond to those of the α-Fe₂O₃ standard PDF card (JCPDS No. 33-0664). However, the intensity ratio of diffraction peaks at 33.2° and 35.6° was 7:8, which was a larger difference compared with that of 10:7 in the α-Fe₂O₃ standard PDF card. The reason was that the diffraction peak at 35.5^o corresponding to Fe₃O₄ standard PDF card (JCPDS No. 03-0863), enhanced the diffraction at 35.5°, resulting in the change of intensity ratio for diffraction peaks at 33.2° and 35.6°, which confirmed the existence of Fe₃O₄ in the product, and that sheet-like α-Fe₂O₃/Fe₃O₄ MNCs were successfully prepared. Figure 1(d) displayed the TEM image of sheet-like α-Fe₂O₃/Fe₃O₄@Au MNCs. It could be seen that the particle size of AuNPs was around 8 nm, they attached to the surfaces of α-Fe₂O₃/Fe₃O₄@Au MNCs, and the distribution of AuNPs was uniform. AuNPs provided the linkage opportunity with ssDNA by the Au-S bond. The hysteresis loops of sheet-like α-Fe₂O₃/Fe₃O₄ and α-Fe₂O₃/Fe₃O₄@Au MNCs were measured and shown in Figure 1(e); their saturation magnetizations were 27.6 emu∙g^-1 and 9.9 emu∙g^-1. Obviously, the saturation magnetization of α-Fe₂O₃/Fe₃O₄@Au MNCs tremendously decreased compared with that of α-Fe₂O₃/Fe₃O₄ MNCs owing to the existence of Au, which suggested the successful loading of Au, which agreed with the conclusion from Figure 1(d).

3.2. Feasibility of the proposed biosensor

CV and EIS were employed to investigate the interfacial properties of the modified electrodes for each process of the biosensor, and the results have been demonstrated in Figure 2. From the CV examinations in Figure 2(a), after the α-Fe₂O₃/Fe₃O₄ nanosheets were self-assembled onto MGCE, the peak current of I_p (curve b) noticeably decreased compared with the bare electrode surface (curve a), which was due to the noteworthy steric hindrance formed by the nanosheets, hindering the accessibility of [Fe(CN)₆]^3-/4- to MGCE. In contrast, the current signal (curve c) of MGCE self-assembled α-Fe₂O₃/Fe₃O₄@Au MNCs remarkably increased compared with that of the α-Fe₂O₃/Fe₃O₄-modified electrode, and even surpassed that of the bare electrode (curve a). The reason was that AuNPs greatly improved the electrical conductivity of α-Fe₂O₃/Fe₃O₄@Au MNCs and effectively enhanced current signals, which also improved the detection sensitivity of the biosensor. When α-Fe₂O₃/Fe₃O₄@Au-ssDNA MNCs were assembled onto the MGCE, the append of ssDNA decreased the electroconductivity, resulting in the current signal (curve d) being reduced. When α-Fe₂O₃/Fe₃O₄@Au-ssDNA/BSA MNCs were further assembled onto the MGCE, the I_p was further decreased due to the poor electroconductivity of BSA (curve e). When ctDNA was captured onto probes and assembled onto the MGCE, the current signals (curve f) were decreased again. The corresponding EIS detections were examined and shown in Figure 2(b); the same principle was revealed, each step in the construction process similarly changed the transfer resistance (R_ct). As was well-known, the stronger the electrical conductivity of the nanocomposites, the weaker the transfer resistance. Therefore, the transfer resistance of α-Fe₂O₃/Fe₃O₄@Au MNCs became weaker, and that of α-Fe₂O₃/Fe₃O₄@Au-ssDNA, α-Fe₂O₃/Fe₃O₄@Au-ssDNA/BSA, and α-Fe₂O₃/Fe₃O₄@Au-ssDNA/BSA-ctDNA became stronger step by step. In the EIS image, the semicircular diameter of EIS for α-Fe₂O₃/Fe₃O₄@Au MNCs became small, and those of α-Fe₂O₃/Fe₃O₄@Au-ssDNA, α-Fe₂O₃/Fe₃O₄@Au-ssDNA/BSA, and α-Fe₂O₃/Fe₃O₄@Au-ssDNA/BSA-ctDNA also became larger step by step. All the results agreed with the CV examination principle.

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Figure 2.

(a) Unmodified MGCE, (b) MGCE/Fe₃O₄/α-Fe₂O₃, (c) MGCE/Fe₃O₄/α-Fe₂O₃@Au, (d) MGCE/Fe₃O₄/α-Fe₂O₃@Au-ssDNA, (e) MGCE/Fe₃O₄/α-Fe₂O₃@Au-ssDNA/BSA, and f MGCE/Fe₃O₄/α-Fe₂O₃@Au-ssDNA/BSA/CYFRA 21-1 DNA.

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The equivalent circuit for fitting the EIS curves was shown in the inset of Figure 2(b), wherein R_s was the solution resistance, R_ct was the charge transfer resistance of the solution redox probe, W₁ was the Warburg element, and C_d was the constant phase element. The electrochemical parameters of the EIS curves at different modification steps have been listed in Table 2. The time constant τ was related to the rate constant of the electrode reaction and the diffusion coefficient, which is commonly used to describe the rate of the electrode reaction and the charge transfer process. The formula for τ has been displayed in Eq. (1), and the results showed that the larger the resistance was, the smaller the value of τ was, which proved once again the success of the proposed electrochemical biosensor construction [43].

3.3. Analysis of the probe amount loaded on the electrode surface

The ssDNA attached to the magnetic α-Fe₂O₃/Fe₃O₄@Au nanocomposites through Au-S bonds played an important role in the detection of ctDNA by the electrochemical biosensor. Firstly, the CV curves (Figures 3a and b) of bare electrode and MGCE-α-Fe₂O₃/Fe₃O₄@Au-ssDNA at different scan rates (V) were examined to obtain their peak currents (Ip, μA), and their linear relationships were analyzed as (Figures 3c and d). Subsequently, based on the Randles-Sevcik Eq. (2), the electroactive surface area (A) of the modified electrode was calculated to be 0.0513 cm². The surface excess Γ₀ was obtained based on the integral Cottrell expression eq. (3) through the examination of chrono coulometry for MGCE-α-Fe₂O₃/Fe₃O₄@Au-ssDNA with or without RuHex, see Figure 3(e), which represented the amount of redox marker confined near the electrode surface. Finally, the electrode surface excess was converted to a ctDNA probe surface density of 1.15×10¹⁴ molecules⋅cm⁻² based on Eq. (4) [44-46].

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Figure 3.

(a) CV curves and (b) the corresponding liner relationships of anodic peaks for bare MGCE, (c) CV curves and (d) the corresponding liner relationships of anodic peaks for MGCE-α-Fe₂O₃/Fe₃O₄@Au-ssDNA, and the resulting chrono coulometry (e) for MGCE-α-Fe₂O₃/Fe₃O₄@Au-ssDNA with or without RuHex (The different coloured bands express the different scan rates).

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3.4. Optimization of the key factors for the electrochemical biosensor

For obtaining the largest current signals, the concentration of α-Fe₂O₃/Fe₃O₄@Au MNCs for the immobilization of ssDNA was optimized, as shown in Figure 4(a). With the increase of α-Fe₂O₃/Fe₃O₄@Au concentration in ultrapure water, the electrochemical response increased, obviously, the increase of Au with the high electrical conductivity indeed promoted charge transfer rate, resulting in the increase of the current signal, and the concentration of α-Fe₂O₃/Fe₃O₄@Au MNCs increased to 10 mg∙mL^-1, the electrochemical response reached largest; with the sequential increase of α-Fe₂O₃/Fe₃O₄@Au concentration, the electrochemical response decreased, the reason for which was that with the increase of α-Fe₂O₃/Fe₃O₄@Au, their agglomeration increased, leading to the increase of the steric hindrance and the decrease of the electrochemical response. Therefore, 10 mg∙mL^-1 of α-Fe₂O₃/Fe₃O₄@Au was applied as the optimal condition.

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Figure 4.

(a) Optimization on the construction conditions of α-Fe₂O₃/Fe₃O₄@Au MNCs and (b) ssDNA concentrations, (c) hybridization temperature, and (d) hybridization time.

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To a great degree, immobilization of ssDNA onto the surfaces of α-Fe₂O₃/Fe₃O₄@Au and the effect of ssDNA concentration were investigated, see Figure 4(b). With the increase of ssDNA concentration, the electrochemical response gradually reduced and achieved the stable value at 3 μM, which suggested that the binding of ssDNA onto α-Fe₂O₃/Fe₃O₄@Au MNCs had achieved the saturation state at ssDNA of 3 μM, and the larger ssDNA could not be immobilized onto the surfaces of α-Fe₂O₃/Fe₃O₄@Au nanocomposites, in other words, many ssDNA stayed in the solution, more ssDNA was meaningless. So, the concentration of ssDNA was selected at 3 μM.

For better capture of ctDNA by the α-Fe₂O₃/Fe₃O₄@Au-ssDNA/BSA, the incubation temperature and the incubation time of ctDNA in the suspension containing α-Fe₂O₃/Fe₃O₄@Au-ssDNA/BSA probes were detected, see Figures 4(c, d). From Figure 4(c), it can be seen that with the incubation temperature of ctDNA increasing from 37°C to 65°C, the electrochemical response gradually reduced and achieved the minimum value at 65°C; this suggested that the capture dose of ctDNA increased with the increase of the incubation temperature, and the electrical conductivity of the electrochemical system decreased. With the further increase of incubation temperature, the electrochemical response became large. On the contrary, the reason for this was that a higher temperature made the ctDNA inactive; the inactivated ctDNA could not be captured. Therefore, the electrical conductivity of the electrochemical detection system might not be decreased, and the electrochemical response becomes large. Therefore, the incubation temperature of ctDNA was selected at 65°C. Similarly, when the incubation temperature of ctDNA was 65°C, with the incubation time of ctDNA increasing from 5 to 15 min, the electrochemical response gradually reduced and achieved the minimum value at 15 min; and with the further extension of incubation time, the electrochemical response also became large. The reason for the similar rule was that under the incubation temperature of 65°C, the extension of the incubation time could contribute to the capture of ctDNA. However, the overlong time similarly resulted in the inactivation of ctDNA, which could result in the decrease of the electrical conductivity, so the incubation time of 15 min was selected.

To summarize, the optimized parameters of the electrochemical system for the detection of ctDNA were 10 mg∙mL^-1 of α-Fe₂O₃/Fe₃O₄@Au concentration, 3 μM of ssDNA concentration, 65^oC of ctDNA incubation temperature, and 15 min of incubation time.

3.5. Analytical performance

The DPV response technique was employed to assess the analytical property of the constructed electrochemical system for ctDNA detection, as revealed in Figure 5(a). Under the optimized conditions, the DPV responses were examined in Figure 5(a). It was not difficult to find that the DPV response gradually decreased while the ctDNA concentration increased from 10 pM to 1 μM, which suggested that the biosensors belonged to the “turn off” type detection mechanism. While, the current signal exhibited the robust linear relation with the logarithm of ctDNA concentration, as depicted in Figure 5(b), and the linear equation was I = - 2.62 lgC_ctDNA+138.35 with the higher variance (R²) of 0.998, Sum of Squares for Error (SSE) of 0.2549, Standard Errors (SE) of 0.0488 and 0.19967 for slope and intercept, and SSE/SE of 1.277. The LOD of 229 fM for the biosensor was calculated using equation of LOD=3 σ/m (σ referred the standard deviation (SD=±0.20) of the blank group response values (n=10) detected on different electrodes in five days; m denoted the slope (2.62) of the calibration curve; the confidence level was 95%); while, the LOQ of 763 fM for the biosensor was calculated using equation of LOQ=10 σ/m. Compared with other detection approaches (Table 3), the constructed nano-biosensor revealed a lower LOD and a wider linear range, especially, the operational process was simplified, and the operational period was also shortened.

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Figure 5.

DPV curves (a) of the biosensors for the detection of various standard solutions with various ctDNA concentrations, the corresponding linear relationship (b) and the corresponding inset for the non-linear relationship

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3.6. Analyses of reproducibility, stability, specificity, and spiked sample in clinical serum

The reproducibility was important for the biosensor; it reflected the reliability of measurement data, and its investigation was indispensable. Five electrodes dropped 9 μL of α-Fe₂O₃/Fe₃O₄@Au-ssDNA/BSA-ctDNA suspension incubated with a ctDNA concentration of 1 μM were recorded with DPV under the same optimal conditions, and the detections were repeated for thrice by various persons at different times. The peak currents were similar with RSD values of 1.13% for Person No. 1, 2.25% for Person No. 2, 1.61% for Person No. 3, and 1.68% for the total samples (Figure 6a), illustrating superior reproducibility of the biosensor. As everyone knows, the stability of the biosensor could reflect the resting period, which would offer a guarantee for the detection. The α-Fe₂O₃/Fe₃O₄@Au-ssDNA/BSA probes were dispersed in ultrapure water to form a suspension of 100 pg∙mL^-1 and stored in 4^oC refrigerator. Subsequently, the ctDNA was examined every 2 days. The detection errors of the response peak currents remained at -0.97%–1.47% in 14 days, as depicted in Figure 6(b), indicating excellent stability of the biosensors. The specificity was a key to evaluating whether the biosensors could or not be applied in the detection of ctDNA. The α-Fe₂O₃/Fe₃O₄@Au-ssDNA/BSA suspensions were incubated with 1 μM of ctDNA, SBM-ctDNA, DBM-ctDNA, TBM-ctDNA, and NC-ctDNA, respectively. And the peak currents were examined and displayed in Figure 6(c). As predicted, the peak currents for the detections of SBM-ctDNA, DBM-ctDNA, TBM-ctDNA, and NC-ctDNA were enhanced; only the peak current for the detection of ctDNA was decreased, revealing that only ctDNA could be captured onto the probes, suggesting the promising high selectivity.

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Figure 6.

(a) Reproducibility and (b) stability of the biosensors for ctDNA detection, and the (c) selectivity with 1 μM of ctDNA, SBM-ctDNA, DBM-ctDNA, TBM-ctDNA, and NC-ctDNA (The different colors indicate different storage times).

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The various concentrations of ctDNA in human serums (10 pM– 10⁶ pM) were prepared, and they were incubated with α-Fe₂O₃/Fe₃O₄@Au-ssDNA/BSA probes, as the experimental section described their peak currents were detected and listed in Table 4. The RSD values for them were 0.19%–1.15%, and their recovery rates for the detection of ctDNA were 96.26%–102.67%. All the data revealed that the detection of ctDNA using the biosensor was accurate and reliable, and suggested the significant clinical application value in the detection of ctDNA for the biosensors. At present, we are collecting the clinical samples; in the future, we will detect the concentrations of ctDNA for healthy persons and patients, and compare the results detected by the gold method. According to our estimation, all the work will be finished in about a year, and the related results will be reported.