An electrochemical CRISPR/Cas12a biosensing platform utilizing Fe₃O₄/α-Fe₂O₃ nanosheets for highly sensitive detection of EGFR-19Del

2.1. Preparation of Fe₃O₄/α-Fe₂O₃@Au NCs

Firstly, FeCl₃·6H₂O (0.541 g) and NH₄H₂PO₄ (16.6 mg) were dissolved in deionized water (100 mL). The solution of 80 mL was then put into a hydrothermal reactor and kept at 220°C for 24 hrs to obtain α-Fe₂O₃ nanosheets, and then, the precursor of α-Fe₂O₃ was partially reduced to Fe₃O₄ via high-temperature calcination (600°C, 4 hrs) with glucose as a reducing agent to prepare Fe₃O₄/α-Fe₂O₃ nanosheets. Subsequently, to improve the electrocatalytic properties of the Fe₃O₄/α-Fe₂O₃ nanosheets, sodium borohydride was used to reduce chloroauric acid to prepare Fe₃O₄/α-Fe₂O₃@Au NCs [37].

2.2. Construction of the biosensing platform

Construction of Fe₃O₄/α-Fe₂O₃@Au-hpS2 modified electrode: The hpS2 was prepared in TE buffer to a final concentration of 100 μM. Subsequently, hpS2 and TCEP (10 mM) were added in a molar ratio of 1:100 and allowed to react at room temperature for 1 hr to facilitate the reduction of disulfide bonds. The reaction mixture was then diluted with Phosphate-Buffered Saline (PBS) buffer (10 mM). The hpS2 (3 μM) solution (40 μL) was incubated with Fe₃O₄/α-Fe₂O₃@Au nanosheets (12 mg/mL) at 37°C for 3 hrs. After thorough cleaning with ultra-pure water, the excess water was removed to fabricate the Fe₃O₄/α-Fe₂O₃@Au-hpS2 modified electrode. The electrode modified with Fe₂O₃/α-Fe₃O₄@Au-hpS2/MCH was constructed by effectively blocking non-specific adsorption sites using MCH.

CRISPR/Cas12a cleavage activity: The Cas12a/crRNA double-stranded complex was formed by assembling 10 μL of a 200 nM Cas12a solution and 5 μL of a 400 nM crRNA in 5 μL of 10×NEBuffer 2.1 buffer, followed by incubation at 37°C for 30 mins. Subsequently, the cleavage activity was initiated by adding 10 μL of target double-stranded DNA (100 nM) and incubating the mixture with an additional 10 μL of Tris-HCl buffer at 37°C for another 30 mins. At this point, the Cas12a/crRNA complex reached a concentration of 50 nM. The Fe₃O₄/α-Fe₂O₃@Au-hpS2/MCH was then supplemented with a 40 μL solution of Cas12a/crRNA complex and tDNA, whereafter the execution of CRISPR/Cas12A-mediated cleavage reaction at 37°C for 1 hr. Subsequently, the obtained mixture was subjected to thermal inactivation of Cas12a enzyme at 65°C for 10 mins. After purification with ultra-pure water, dispersion was achieved by adding 40 μL ultra-pure water, and a volume of 9 μL solution was extracted for electrochemical detection. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were utilized to investigate the construction process of the biosensing platform.

2.3. Polyacrylamide gel electrophoresis

Preparation of 12% polyacrylamide gels: Gels for electrophoresis experiments were made by mixing 2.4 mL of 30% (29:1) acrylamide/bis-acrylamide solution, 1.6 mL of 5×TBE buffer, 4 mL of deionized water, 56 μL of ammonium persulfate (APS) and 8 μL of TEMED solution. Subsequently, 10 μL of each sample and 2 μL of bromophenol blue up-sampling buffer were loaded into a 12% polyacrylamide gel. The polyacrylamide gel electrophoresis (PAGE) was placed in 1×TBE run at a constant voltage of 120 V for 45 mins, and then the lane results were observed by UV analyzer.

3.1. Characterization of modified materials on the electrode surface

Figure 2(a) showed the TEM image of the prepared Fe₃O₄/α-Fe₂O₃ nanosheets with an average diameter of 151 nm and thickness of 54 nm, respectively. Also, the size distribution has been shown in Figure S1A, B. The XRD pattern of Fe₃O₄/α-Fe₂O₃ nanosheets in Figure 2(b) was consistent with the Powder Diffraction File of Fe₃O₄ (JCPDS No. 19-0629) and α-Fe₂O₃ (JCPDS No. 33-0664), indicating the existent of α-Fe₂O₃ and Fe₃O₄. The characteristic peaks at 2θ of 24.1°, 33.2°, 35.6°, 40.8°, 49.4°, 54.0°, 62.4°, and 63.9° corresponded to the (012), (104), (110), (113), (024), (116), (214), and (300) crystal faces of α-Fe₂O₃. While the characteristic peaks at 2θ of 30.1°, 35.4°, 43.1°, 56.9°, and 73.9° corresponded to the (220), (311), (400), (511), and (533) crystal faces of Fe₃O₄. Additionally, the peak intensity ratio of Fe₃O₄/α-Fe₂O₃ nanosheets at 35.4° and 33.2° was higher than that of pure α-Fe₂O₃ because the presence of Fe₃O₄ was conducive to its characteristic peak at 35.4°. The average grain size of Fe₃O₄/α-Fe₂O₃ nanosheets was about 37 nm. The results confirmed the successful fabrication of Fe₃O₄/α-Fe₂O₃ nanosheets. Figure 2(c) showed the TEM image of Fe₃O₄/α-Fe₂O₃@Au NCs, AuNPs distributed on the surface of Fe₃O₄/α-Fe₂O₃ nanosheets, and the average diameter of AuNPs was about 11.3 nm. The size distributions of Fe₃O₄/α-Fe₂O₃@Au NCs and AuNPs have been plotted in Figure S1C, D, and E. The presence of Fe, O, and Au elements in the EDS spectra (Figure 2d) confirmed the successful preparation of the Fe₃O₄/α-Fe₂O₃@Au NCs, while the presence of other elements indicated that the DNA probes were successfully bound to the surface of the NCs. In addition, the elemental distribution mappings of the nanoprobes have been exhibited in Figure S2.

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Figure 2.

(a) The TEM image and (b) XRD pattern of Fe₃O₄/α-Fe₂O₃ nanosheets, (c) the TEM image of Fe₃O₄/α-Fe₂O₃@Au NCs, and (d) the EDS spectral analysis of Fe₃O₄/α-Fe₂O₃@Au-hpS2 nanoprobes. TEM: Transmission electron microscopy, XRD: X-ray diffraction, EDS: Energy dispersive spectroscopy.

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Furthermore, the chemical composition and electronic states of the elements in the NC were analyzed utilizing XPS, thus validating its successful synthesis (Figure 3). The full spectrum scanning of the NC has been displayed in Figure 3(a), with prominent peaks for Fe, C, Au, and O. The high-resolution spectrum of Fe 2p in Figure 3(b) exhibits two peaks at the binding energy (BE) of 710.70 and 724.40 eV, which correspond to Fe 2p_3/2 and Fe 2p_1/2, respectively. After peak differentiation imitating, the splitting peaks obtained at BE of 710.29 and 723.89 eV belonged to Fe(II), while the two at 712.14 and 725.64 eV were attributed to Fe(III), along with two satellite peaks at 718.01 and 732.54 eV. As depicted in Figure 3(c), after deconvolution, the C 1s spectrum had three peaks at the BE of 284.80, 285.54, and 288.46 eV, which could be ascribed to C–C/C–H, C–Oc, and O–C=O. Figure 3(d) illustrated two main peaks of the Au 4f region located at 83.84 and 87.52 eV, corresponding to Au(I) 4f_7/2 and Au(I) 4f_5/2 respectively. Additionally, it can be seen that the O 1s spectrum (Figure 3e) was composed of Fe–O, C–O/C–O=C, and H₂O observed at 529.89, 530.88, and 531.91 eV, respectively. In summary, Fe₃O₄/α-Fe₂O₃@Au NCs were successfully prepared.

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Figure 3.

XPS spectra of Fe₃O₄/α-Fe₂O₃@Au NCs nanocomposites: (a) Overview spectrum, (b) Fe 2p, (c) C 1s, (d) Au 4f, and (e) O 1s.

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3.2. Electrochemical characterization of the biosensor

Interface properties of the biosensor construction process were investigated using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and polyacrylamide gel electrophoresis (PAGE) techniques (Figure 4). In Figure 4(a), the curve a represents an unmodified electrode, while curve b corresponded to the deposition of Fe₃O₄/α-Fe₂O₃@Au on the electrode surface. The incorporation of AuNPs significantly enhanced the electrical conductivity, leading to an increase in redox current and a corresponding decrease in impedance. These results demonstrated the successful construction of a Fe₃O₄/α-Fe₂O₃@Au modified electrode. Subsequently, sulfhydryl-modified hpS2 was connected to Fe₃O₄/α-Fe₂O₃@Au by Au-S bonds, and the non-specific site was blocked with MCH to construct the Fe₃O₄/α-Fe₂O₃@Au/DNA/MCH modified electrode (curve c). Since the DNA phosphate backbone was negatively charged, there was electrostatic repulsion between it and Fe(CN)₆]^3-/4- in the solution, which made it more difficult for Fe(CN)₆]^3-/4- to reach the electrode surface, significantly reducing the redox current and increasing the charge transfer resistance. Curve d showed that the activated Cas12a protein was added to cut the hpS2. Because the nucleotide chain was cut into short fragments, the steric hindrance on the electrode surface decreased, and Fe(CN)₆]^3-/4- in the solution could reach the electrode surface more easily. Hence, the redox current increased, and the charge transfer resistance decreased.

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Figure 4.

(a) CV, (b) EIS, and (c) electrophoretic analysis of construction for CRISPR/Cas12a-based electrochemical biosensors. + stands for the factor exists, - means that it does not exist, CV: Cyclic voltammetry, EIS: Electrochemical impedance spectroscopy.

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The Nyquist diagram in Figure 4(b) corresponded to the CV diagram, and the Randles equivalent circuit model in the inset of Figure 4(b) was employed for the fitting of the EIS. The examined ligand density of the hpS2 probes was 1.7 (± 0.3) × 10¹² molecules per cm² via the chronocoulometric method [38,39]. The cleavage activity has been confirmed in Figure 4(c). The hpS2 was designed as the substrate for Cas12a/crRNA trans-cleavage. In the absence of Cas12a (lane 2), crRNA (lane 3), or tDNA (lane 4), the silencing of Cas12a trans-cleavage activity was observed. However, specific tDNA binding facilitated the cleavage activity mediated by crRNA-directed Cas12a (lane 5). The results above verified the successful construction of an electrochemical biosensing platform based on CRISPR/Cas12a.

3.3. Optimization of the detection conditions

To achieve optimal sensing performance, the key influencing factors in the experimental process were optimized [40,41]. The concentration of target dsDNA was set at 100 nM, and each experiment group was repeated three times to observe signal changes of differential pulse voltammetry (DPV). Initially, the DPV method was employed to investigate the effect of Fe₃O₄/α-Fe₂O₃ @Au NCs’ concentration in the electrolyte solution containing 5 mM Fe(CN)₆]^3-/4-. As illustrated in Figure S3, as the concentration of NCs increased from 4 to 16 mg/mL, the DPV current exhibited an upward trend due to the good conductivity of AuNPs. However, excessive deposition of NCs on the electrode surface would create significant spatial resistance that hindered electron transfer. Therefore, it was determined that the optimal concentration for Fe₃O₄/α-Fe₂O₃@Au NCs was 16 mg/mL.

Moreover, the concentration of hpS2 emerged as a crucial parameter that exerted an impact on both the sensitivity and stability of the biosensing platform (Figure 5a). The peak current of MB served as an indicator for quantifying its concentration. The high concentration of hpS2 could result in electrode surface congestion, thereby impeding CRISPR/Cas12a cleavage activity. Thus, this experiment quantified the variations in current exhibited by hpS2 across a concentration range spanning from 1 μM to 3 μM. The DPV signal of MB was observed to be enhanced as the concentration of hpS2 increased from 1 μM to 2 μM, as illustrated in Figure 5(a). However, the current signal exhibited a subsequent decrease with an increase in the concentration. Consequently, 2 μM was determined to be the optimal concentration. The concentrations of Cas12a and crRNA were crucial factors directly impacting trans-cleavage efficiency. The maximum ΔI% was observed at the concentration of 40 nM as the Cas12a concentration increased. Henceforth, it was selected as the optimal Cas12a concentration (Figure 5b). In Figure 5(c), trans-cleavage activity increased with higher crRNA concentrations. Nevertheless, when exceeding 40 nM, ΔI% slightly decreased. Thus, 40 nM was selected as the optimal crRNA concentration. Furthermore, the effect of cleavage time was also investigated. It achieved stability after 60 min. Hence, 60 min was determined as the optimum condition (Figure 5d). It’s worth noting that ΔI% = (I - I₀)/I × 100%, where I was the peak MB current obtained after optimization of hpS2 concentration, and I₀ was the current value expressed by the sensor when each condition was optimized.

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Figure 5.

Optimization: (a) Concentration of hpS2, (b) Cas12a, (c) crRNA, and (d) cleavage time.

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3.4. Analytical performance of constructed biosensor towards EGFR-19Del

Under optimized experimental conditions, the performance of the biosensing platform was evaluated utilizing the DPV method, as depicted in Figure 6. The alteration in tDNA concentration could influence the trans-cleavage activity of CRISPR/Cas12a and subsequently resulted in variations in electrical signals on the electrode surface. Within the range of 10 pM to 100 nM, ΔI% was linearly correlated with the logarithm of the tDNA concentration (described by the equation ΔI% = 19.35 lg C (nM) + 52.99 with an R² value of 0.997). The limit of detection (LOD) was determined as 0.8 pM (LOD=3σ/slope), while the limit of quantification (LOQ) was found to be 2.7 pM (LOQ=10σ/slope). In comparison with previously reported methods (Table 1 and Table S2), this CRISPR-based electrochemical biosensor platform exhibited superior sensitivity with a wider linear range as well as lower LOD, thereby serving as a valuable reference for detecting other nucleic acid sequences using CRISPR-based biosensors.

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Figure 6.

(a) DPV curves of the biosensing platform for the detection of different concentrations of tDNA and (b) the linear relationship between ΔI% and the logarithm of tDNA concentration. DPV: Differential pulse voltammetry. tDNA: target DNA.

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3.5. Selectivity, stability, and reproducibility of the biosensing platform

The selectivity of the biosensing platform was investigated by examining its ability to discriminate between tDNA and non-target sequence, with a concentration of 1 nM for tDNA and 100 nM for the non-target sequences. The current change (ΔI%) of the tDNA was significantly higher than that of the non-complementary sequences, indicating its good selectivity (Figure 7a). The detection of tDNA of 100 pM and 10 nM was performed using a set of five electrodes, yielding RSD values of 3.05% and 4.61%, respectively. These results demonstrated excellent reproducibility (Figure 7b). Moreover, the stability was investigated by recording the DPV current responses for consecutive 10 days. The current fluctuated slightly without significant difference, and the current response value remained still 97.3% of the initial value after 10 days, which indicated the biosensing platform had excellent stability (Figure 7c).

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Figure 7.

(a) The selectivity, (b) reproductivity, and (c) stability of the CRISPR/Cas12a-based biosensing platform.

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3.6. Analysis of EGFR-19Del in human serum samples

To assess the capability of the biosensing platform for biological sample analysis [42], we conducted tests on serum-labeled samples with various concentrations (Table 2). The serum was diluted 20-fold using a diluent of 20 mM PBS (pH 7.4). The experimental findings revealed that the RSD values ranged from 1.23% to 6.55%, while the recovery rate fell within the range of 94.73% to 114.50%. These results strongly suggested that the biosensor platform had great potential for clinical sample detection.