A novel biosensor for the ultrasensitive detection of the lncRNA biomarker MALAT1 in non-small cell lung cancer

Long non-coding RNAs (lncRNAs) have been proposed as diagnostic biomarkers for the screening of non-small cell lung cancer and monitoring disease progression. Accordingly, new, rapid, and cost-effective lncRNA biosensors that can be used clinically are urgently needed. Herein, a novel effective and ultrasensitive electrochemical biosensor was developed based on a gold nanocage coupled with an amidated multi-walled carbon nanotube (Au NCs/MWCNT-NH2)-decorated screen-printed carbon electrode (SPCE). Because of its large surface area, superior conductivity, and excellent biocompatibility, this SPCE Au NCs/MWCNT-NH2 lncRNA biosensor showed a wide linear range (10–7–10–14 M) and low limit of detection limit (42.8 fM) coupled with satisfactory selectivity and stability. Compared to traditional RT-PCR, the proposed method exhibits acceptable stability, good selectivity, is simpler to operate, has faster detection, and uses less costly raw materials. In summary, this biosensor may be a powerful tool for detecting lncRNAs for efficient clinical prognosis and cancer diagnosis.

www.nature.com/scientificreports/ electrode (SPCE), which is small and inexpensive, as the working electrode offers the advantages of simple operation, portability, and miniaturization. However, few studies have examined the potential of SPCE-based biosensors for detecting lncRNAs. To improve the sensitivity of electrochemical RNA biosensors, signal amplification strategies based on functional nanocomposites have been proposed 17 , wherein large amounts of electrochemical mediators and natural micromolecules are loaded onto the nanomaterials 18,19 . Gold nanoparticles (Au NPs) are highly efficient nanomaterial-based catalysts that facilitate a large number of reactions, including the reduction of oxygen and alkene hydrogenation, and have been utilized in numerous electrochemical bioanalyses 20,21 . Given the success of NPs, considerable effort has recently been devoted to fabricating hollow metallic spheres with a larger specific surface area than their solid counterparts to achieve better catalytic effects 22 . Au nanocages (Au NCs) are innovative materials in the field of biosensing because of their hollow interior and porous walls 23,24 .
Recently, well-aligned, multi-walled carbon nanotubes (MWCNTs) were developed to improve the sensitivity of electrochemical detection 25,26 . MWCNTs are ideal materials for electrochemical biosensing because of their high electrical conductivity, large length-diameter ratio, large surface area, and excellent mechanical strength 27 . MWCNTs can be modified by incorporating hydrophilic primary amines to produce MWCNTs-NH 2 which show good dispersion in water and low cytotoxicity, and can easily be loaded with other nanomaterials or biomolecules 28,29 . Although Au NCs and MWCNTs have been used in electronic devices and supercapacitors, the combination of Au NCs with MWCNTs-NH 2 has not been explored. Furthermore, their use in the electrochemical detection of lncRNAs has not been investigated.
In this study, an effective, ultrasensitive SPCE-based electrochemical biosensor using an Au NCs/MWCNT-NH 2 nanostructure was used to produce a sensitive, inexpensive assay for detecting MALAT1 (Scheme 1). We found that Au NCs/MWCNTs-NH 2 can be loaded with a probe capable of specifically detecting MALAT1 and that this binding event alters the electrochemical properties of the biosensor. Furthermore, using SPCE as the working electrode in this biosensor enabled miniaturization, portability, and low cost. Compared to traditional RT-PCR, the proposed biosensor exhibited high sensitivity, acceptable stability, and good selectivity. This SPCEbased electrochemical strategy is also economical and rapid, and represents a quantitative alternative for detecting lncRNA levels in the clinic.

Materials and methods
Apparatus and materials. Electrochemical measurements were performed using a CHI 600E electrochemical workstation with anSPCE system composed of a carbon working electrode, carbon counter electrode, and Ag/AgCl reference electrode. Morphological characterization was performed using an FEI Nova 400 fieldemission scanning electron microscope (Hillsboro, OR, USA) and ZEISS LIBRA 200 transmission electron microscope (Oberkochen Germany). X-ray photoelectron spectroscopy was performed using an ESCALAB 250 photoelectron spectrometer (Thermo-VG Scientific, Waltham, MA, USA). MWCNTs were purchased from XF Scheme 1. Schematic representation of the SPCE electrochemical DNA biosensor.  Preparation of Au NCs. Au NCs were synthesized as previously described 30,31 . Briefly, 1 mg mL −1 polyvinylpyrrolidone was added to 5 mL of deionized water to produce a homogeneous solution, to which 500 mL of Ag nanocubes (3 nM) was added before being boiled for 10 min. Gold chloride (0.5 mM) was added to the flask at a rate of 45 mL h −1 , and the solution was refluxed for a further 30 min until the color of the reaction mixture was stable. After cooling to 25 °C, the sample was centrifuged, washed with saturated NaCl solution to remove AgCl, and washed three times with water to remove polyvinylpyrrolidone and NaCl.

Synthesis of Au NCs/MWCNTs-NH 2 .
First, an oxidizing acid was used to introduce active carboxyl groups at the ports or defects in MWCNTs to synthesize carboxyl-modified MWCNTs (MWCNTs-COOH) 28 .
To achieve this, MWCNTs (500 mg) and concentrated nitric acid (68%, 150 mL) were mixed and stirred at 70 °C for 4 h. After cooling to 25 °C, the mixture was filtered through cellulose filter membranes (450-nm) and then cleaned with ionized water. The products were dried in a vacuum freeze dryer at 40 °C for 12 h. Next, MWC-NTs-NH 2 were prepared via a condensation reaction between the -COOH and -NH 2 groups by adding 0.

Results and discussion
Characterization of Au NCs/MWCNT-NH 2 composite. Scanning electron microscopy (SEM) images showed that Au NCs were well-dispersed and highly uniform (Fig. 1A). The Au-NPs were orange-purple, whereas Au NCs were blue. The ultraviolet absorption spectrogram is shown in Fig. 1B. SEM observations revealed typical morphological features for the MWCNT-NH 2 , with an average length and diameter of approximately 15 and 20 μm, respectively (Fig. 1C,D). These results support that the Au NCs and MWCNT-NH 2 were successfully synthesized. As determined by transmission electron microscopy (TEM), the Au NCs were around 40-50 nm in size and had hollow structures ( Fig. 2A). After the MWCNTs-NH 2 and Au NCs were mixed, large numbers of Au NCs were successfully linked to the surface of MWCNT-NH 2 (Fig. 2B,C) www.nature.com/scientificreports/ MWCNT-NH 2 . Moreover, the corresponding lattice fringes were visible in the high-resolution TEM images. These images showed that the MWCNT-NH 2 wall was 4.545 nm thick and the fringe lattice was 0.522 nm thick, corresponding to the (111) crystal plane (Fig. 2D). The interplanar spacing was approximately 0.236 nm (Fig. 2E), supporting the d-spacing of the (111) lattice plane for Au. Furthermore, the electron diffraction pattern of small individual NPs had a ring-like area (Fig. 2F), with interplanar distances corresponding to the face-centered cubic phase of Au. X-ray photoelectron spectroscopy was performed to analyze the surface chemical composition of the Au NCs/MWCNT-NH 2 . Peaks at 285.4, 398.6, 532.0, and 711 eV were associated with C1s, O1s, N1s, and Au 4f., respectively (Fig. 3A). In the C1s spectrum of Au NCs/MWCNT-NH 2 (Fig. 3B), the absorption peaks at 284.6 and 285.0 eV corresponded to the sp2 and sp3 hybrid graphite-like structural carbons on MWNTs, respectively 33 . The peak at 286.6 eV corresponded to the C-NH binding energy, indicating the presence of -NH 2 functional groups on the MWCNT-NH 2 surface. Moreover, Au 4f. peaks were observed at 84.0 and 87.6 eV, which corresponded to Au 4f 7/2 and 4f 5/2 , respectively (Fig. 3C), confirming the presence of metallic Au on the Au NCs/ MWCNT-NH 2 composites.

Electrochemical behavior of modified electrodes. CV is the most effective and convenient method
for investigating electrochemical processes occurring at electrode interfaces. CV was performed on different modified electrodes in 1.0 mM [Fe (CN) 6 ] 3− / 4− solution containing 0.1 M KCl at a scan rate of 100 mV s −1 (Fig. 4A). Compared to bare SPCEs (curve a), Au-NPs (curve b), Au NCs (curve c), and Au-NP/MWCNTs-NH 2 (curve d), the biosensor using Au NCs/MWCNTs-NH 2 as a label exhibited a much greater electrochemical response (curve e). This phenomenon may be attributed to two factors: (1) novel hollow Au NCs provide a higher specific surface area, more exposed active sites, and therefore exhibit excellent electrocatalytic activity  www.nature.com/scientificreports/ and (2) MWCNT-NH 2 is an attractive support for Au NCs, resulting in a synergistic combination with strong electrocatalytic performance. Next, the capture probes were assembled on the surface of Au NCs/MWCNTs-NH 2 (curve f) and hybridized with the target lncRNA (curve g). The peak current continuously decreased as the negatively charged RNA biomolecules blocked [Fe(CN) 6 ] 3−/4− electron transfer. This noticeable decrease in the peak current indicated that the RNA biosensor had been constructed successfully. EIS analysis is an effective method for investigating the electrochemical properties of electrodes after each stage of modification. When Au-NPs attached to the bare SPCE (Fig. 4B, curve a), the electron transfer resistance (Ret) decreased (curve b). After electrode modification with Au NCs, a lower Ret was observed (curve c), which we ascribed to the ability of the hollow structure to more effectively promote electron transfer. Au NCs/ MWCNTs-NH 2 showed the lowest Ret (curve d). When nonconducting RNA molecules were introduced and loaded onto the nanomaterial surface, the Ret increased significantly (curves e, f). Based on the consistency of the EIS data with the CV data, we had successfully fabricated the SPCE-based biosensor.

Optimization of experimental conditions.
A key issue is how the reaction solution pH affects the sensor response; therefore, we evaluated how the reaction solution pH affected the biosensor performance. Figure S1a shows a CV plot of the fully assembled biosensor exposed to 10 fM mL −1 MALAT1 in 2 mL of 0.1 M PBS at varying pH (6.0-8.5). The peaked reached a maximum at pH 7.0; therefore, PBS at a pH of 7.0 was used as the optimum working buffer in subsequent experiments. The effect of the time used for nucleic acid hybridization to the biosensor was also examined. The probe RNA/Au NCs/MWCNT-NH 2 /SPCE was incubated with lncRNA solution (100 pM) for 20, 40, 60, 80, and 100 min, and the CV was recorded (Fig. S1b). The reduction in peak current reached a maximum at an extended hybridization time of 80 min. The peak current then increased because of the strict effect of hybridization events. These data demonstrate that the hybridization reaction was mostly complete after 80 min; accordingly, we used this hybridization time in subsequent experiments.
Analytical performance of lncRNA biosensor. To investigate the sensitivity of the proposed method for lncRNA detection, DPV was used to measure the electrochemical response after hybridizing an ss-DNA capture probe with the MALAT1 lncRNA target under optimized experimental conditions. The reduction in the peak current in response to the MALAT1 target increased linearly as the target concentration increased (10 -8 to 10 -14 M; Fig. 5). The regression equation was ΔI (μA) = − 0.076logC-5.23 (R 2 = 0.902). The detection limit was calculated as 42.8 fM with an S/N ratio of 3 (where S is the relative standard deviation of a blank solution). We also analyzed the performance of several types of biosensors that have been used to detect non-coding RNAs www.nature.com/scientificreports/ (Table 1). This analysis showed that the proposed biosensor had a satisfactory detection limit and linear range. The biosensor also had high sensitivity and a low limit-of-detection, which we attributed to the superior conductivity and large specific surface area of Au NCs/MWCNT-NH 2 s. Therefore, this biosensor is a promising platform for capturing large numbers of target lncRNAs and facilitating the electron transfer process. Moreover, this new, easy to use SPCE is a meaningful step in the field of biosensing.
Biosensor specificity, reproducibility, and stability. To investigate the specificity of the proposed method for assaying target lncRNAs, its selectivity was assessed using samples containing various potential interfering substances. These substances included HOTAIR, H19, miRNA126, and target lncRNA MALAT1 with a single-base mismatch target (1MT) or a three-base mismatch target (3MT), as well as a MALAT1 mixture. A 50-fold increase in selected RNAs (50 pM; HOTAIR, H19, and miRNA126) caused minimal current responses, similar to that in the blank test; however, the presence of a much lower concentration (1 pM) of the perfectly matched target MALAT1 lncRNA and its mixture resulted in a significantly higher current response (Fig. 6). Additionally, the peak current was higher with 1MT compared to 3MT. These results demonstrate that the proposed biosensor has ideal specificity for detecting lncRNAs and, therefore, great potential for clinical use.
To assess the reproducibility of the biosensor, five electrodes were prepared to detect lncRNA. The relative standard deviation of measurements was less than 4.6% (n = 5). In addition, the stability of the RNA biosensor was evaluated after storage for 14 days at 4 °C, with measurements taken every 2 days. The CV peak current of the RNA biosensor decreased gradually, with the final peak current retaining 88.54% of the initial current after 21 days. Therefore, the reproducibility and stability of the proposed biosensor were acceptable for detecting lncRNA. www.nature.com/scientificreports/ Analytical and clinical applicability of the biosensor. The practical feasibility of the proposed RNA SPCE biosensor for clinical application was investigated using the standard method of adding lncRNA analytes to human serum samples (n = 6). Using the optimal experimental conditions, the electrochemical signals from different serum samples were determined using DPV, and the recovery rate was calculated (see Table 2). Satisfac-    www.nature.com/scientificreports/