Enhanced photoelectrochemical aptasensing platform for TXNDC5 gene based on exciton energy transfer between NCQDs and TiO2 nanorods

The over expression of thioredoxin domain-containing protein 5 (TXNDC5) can promote the growth of castration-resistant prostate cancer (CRPC). A novel highly sensitive photoelectrochemical (PEC) aptsensor was developed for the detection of TXNDC5 by using the nanohybrids (TiO2 NRs/NCQDs) of nitrogen-doped carbon quantum dots (NCQDs) and TiO2 nanorods as the photo-to-electron conversion medium. TiO2 NRs/NCQDs nanohybrids were prepared by controlling the experimental condition. TiO2 NRs were self-assembled to form the nanopores with good photocurrent conversion efficiency. NCQDs possessed carboxyl groups (−COOH) and amino groups (−NH2) in the preparation process. −COOH and −NH2 groups played important roles for anchoring the capture probes (5′ primer and 3′ primer) through covalent binding. The ultrasensitive and stable detection for TXNDC5 was achieved by the specific recognition between the capture probes and the targets. The fabricated aptsensor showed excellent performance with a wide linear range (0.5 fmol/L ∼ 10 nmol/L) and a low detection limit of 0.1 fmol/L. This kind of aptsensor would provide a potential application for TXNDC5.

Scientific RepoRts | 6:19202 | DOI: 10.1038/srep19202 TiO 2 is one of the most studied semiconductor nanostructures [26][27][28] . The PEC performance of TiO 2 strongly depends on the dimensionality. Highly oriented TiO 2 single-crystalline NRs grown on the transparent conductive substrates have been considered as the optimum choice of the solar cell materials 29 . However, a major drawback of TiO 2 NRs is its ineffective utilization of the visible light as the irradiation source. Bandgap engineering by possible modification of TiO 2 -based materials is one of the plausible approaches to enhance the performance of TiO 2 NRs, such as QD-sensitized.
Herein, a PEC aptsensorbased on TiO 2 NRs/NCQDsnanohybrids as the PEC signal medium was constructed (Fig. 1). And the PEC property of TiO 2 NRs/NCQDs was studied under the visible light. This effort offered a promising method for the detections of TXNDC5 or other analytes.

Results and Discussion
Characterizations of TiO 2 NRs, NCQDs, TiO 2 NRs/NCQDs nanohybirds. Scanning electron microscope (SEM)images in Fig. 2A,B showed the morphology of as-prepared TiO 2 NRs. TiO 2 NRs products were assembled as hierarchical microspheres as shown. Figure 2C showed a single magnified broken flower-like microsphere. It can be seen that the microsphere was composited by many uniform TiO 2 NRs with an almost average diameter about 3-5 nm. Figure 2F was EDS result. It showed only Ti, O elements in the sample, which further confirmed the prepared TiO 2 was pure. High resolution transmission electron microscope (HRTEM) was used to reveal the small size of NCQDs (Fig. 2D). HRTEM image showed that the prepared NCQDs had the plentiful output with an average diameter of 3-5 nm. And they were monodisperse nanocrystals of near spherical morphology. Figure 2E showed SEM morphology for TiO 2 /NCQDs nanohybirds. TiO 2 NRs and as-assemble microsphere was not rigid and somewhat powder-like attachment can be distinguished on the surface. This suggested that NCQDs hybridized successfully with TiO 2 NRs. EDS result in Fig. 2G showed four elements Ti, O, C, N, which further confirmed the excellent hybridization of TiO 2 NRs and NCQDs. Figure 3A showed Fourier transform infrared (FT-IR) spectrum of NCQDs in the wavelength range of 500-4000 cm −1 . The peak at 1224 cm −1 indicated the characteristic stretch of C-N bond. C= O bending vibration was at 1390 cm −1 . The peak of 1625 cm −1 was associated with stretching vibrations of C-O band. The peak at 1730 cm −1 meant C= O stretching vibration. The weak peaks in the range of 2820-3050 cm −1 were related to C-H bond stretching vibrations. A broad peak centered at 3440 cm −1 suggested the presence of many hydroxyl groups on the NCQDs surfaces. All the above mentioned indicated that NCQDs had abundant carboxyl and amino groups on their surfaces. The doped amino and carboxyl groups of NCQDs can increase the hydrophilicity and the combination ability with the detection targets in aqueous systems 30 . Figure 3B presented X-ray diffraction (XRD) patterns of NCQDs. A wide peak (002) at about 23° corresponded to an interlayer spacing d of 0.385 nm, which agreed with the (002) lattice spacing of carbon-based materials with turbostratic disorder 31 . d value was larger than that of graphite (0.34 nm), which indicated that NCQDs possessed abundant oxygen-containing functional groups 32 . But this d value was same to the carbon nanoparticles from pyrolyzing ethanolamine 33 and hydrothermal carbonization of chitosan 34 . Figure 3C displayed XRD patterns of TiO 2 NRs, TiO 2 /NCQDs. Rutile phase TiO 2 was produced and it can be confirmed by XRD pattern. For TiO 2 NRs, seven diffraction peaks marked (1-7) can be respectively indexed to the (110), (101), (111), (210), (211), (220) and (002) plane. The addition of NCQDs had no obvious influence on the crystallinity and the phase purity of the resultant products. No diffraction peak resulted from NCQDs can be found, which may be attributed to the poor crystallinity and low content 35 . PEC performance mechanism proposition. Figure 4A     with short wavelength. The viewpoint was proved by the relatively weak photocurrent response even in the presence of AA in Fig. 4A (a). But the photocurrent of TiO 2 /NCQDs were apparently increased about 20 times (b), which proved the addition of NCQDs enhanced PEC behavior. This might be explained that h + /e − pair separation efficiency was distinctly enhanced through the electron injection between NCQDs and TiO 2 NRs. To further explore the PEC properties of TiO 2 NRs, NCQDs, TiO 2 /NCQDs, UV-vis absorption spectra were studied (Fig. 4B). Curve a showed a poor absorbancy of TiO 2 NRs, which was consistent with the fact of the wide band gap of 3.2 eV 36 . NCQDs showed a broad UV-vis absorption in the ultraviolet and visible region (curve b), which suggested that NCQDs may have high photocatalytic activity. A shoulder was shown, which was at about 350 nm and was similar to another report about NCQDs 37 . When TiO 2 and NCQDs combined with each other, the absorption intensity turned stronger and the absorption range became wide (curve c), which suggested that the composition can enhance the visible light absorbancy for both TiO 2 and NCQDs.
PL was a persuasive tool to illustrate the separation and recombination efficiency of photogenerated h + /e − pairs. It can provide the proof for the photogenerated h + /e − pair recombination process from another aspect 38 . Weaker PL intensity generally corresponded to stronger photocatalytic or PEC behavior. Curve a showed PL emission intensity of TiO 2 NRs, which was obvious stronger than those of NCQDs (curve b) and TiO 2 NRs/ NCQDs (curve c). It indicated a higher recombination efficiency of the photogenerated h + /e − pairs and a lower efficient electron injection from NCQDs to TiO 2 . PL intensity of NCQDs was lower than that of TiO 2 , which suggested that NCQDs owned better light absorbancy and photocurrent conversion efficiency. And also the addition of NCQDs into the nanohybrids decreased PL intensity which illustrated that NCQDs improved the photoelectrons transport and benefited the photoelectrons injection from occupied valence band (VB) to conduction band (CB). Therefore, NCQDs can enhance the PEC property of the wide band-gap TiO 2 although the emission peak showed a blue shift.
The PEC performance mechanism proposition was shown in Fig. 5. A VB and an empty CB were present in the semiconductors. The electron (e − ) was excited from VB to CB and the hole (h + ) was formed in the VB due to the presence of a band gap under the light irradiation. However, the recombination of the electron and the hole took place generally. For TiO 2 , the photogenerated electrons more efficiently transferred to CB of TiO 2 under the light irradiation. But subsequently the electrons were transferred directly into the VB of TiO 2 NRs without a transition through the excited state 39,40 or converted into other reactive oxidative species. And the wavelengths those were less than or equal to 380 nm can excite TiO 2 to produce the e − /h + pairs. However, when NCQDs were introduced into the composites system (TiO 2 /NCQDs), a mass of the visible light was converted to a shorter wavelength, which may increase the photogenerated e − /h + pairs to enhance the PEC activity.   44,45 . The shorter wavelengths that less than or equal to 380 nm can excite TiO 2 NRs to produce the e − /h + pairs. NCQDs can accept the photogenerated electrons from TiO 2 NRs and promote the separation of the photogenerated e − /h + pairs.
Thus, from the schematic diagram of the process, it can be seen that the VB and CB energy levels of NCQDs lied above those of TiO 2 NRs. Under the visible light irradiation, the electrons in the VB of NCQDs obtained enough energy and injected to the CB. The photogenerated electrons can be easily injected from NCQDs into TiO 2 NRs via the interface injection. However, the holes on the VB of TiO 2 NR scan transfer to NCQDs. As a reducing agent, AA acted as an electron donor to trap the holes in the VB of TiO 2 , which inhibited the e − /h + recombination and improved the photocurrent response 46 . After that, the electrons were transported through ITO film and conducted through the external circuit to the counter electrode.
Therefore, NCQDs in the nanocomposites facilitated the transfer of the electrons from TiO 2 NRs and the electrons can be shuttled freely along the conducting paths in NCQDs. The combination of TiO 2 NRs/NCQDs benefited for the charge separation and for hindering e − /h + recombination 47,48 . And what's more, N-doping was another main reason for the enhancement of the photocatalytic activity of TiO 2 /NCQDs. N-doping can lower the work function of carbon nanomaterials and the lower work function of NCQDs produced much smaller barrier between NCQDs and TiO 2 . The inference was also proved by UV-vis and PL measurement as shown in Fig. 4B,C. The above results illuminated that NCQDs can help for the transfer of the photogenerated electrons and reduce the recombination rate of e − /h + pairs.
Characterizations of the fabricated PEC aptsensor. In order to realize better detecting effect, the optimization of the experimental conditions was carried out. The application amount of TiO 2 NRs, the composition methods (three methods) of TiO 2 NRs and NCQDs, the application amount of NCQDs composited with TiO 2 NRs, pH values and the application amount of 3′ and 5′ primers were investigated in this study (Fig. 6). The obtained optimum experimental conditions were the application amount of TiO 2 NRs = 15 μ L (Fig. 6A), the composition method was method 1 (Fig. 6B), the application amount of NCQDs = 22 mg/mL (Fig. 6C), pH = 7.4 (Fig. 6D) and the application amount of 3′ and 5′ primers = 15 μ L (Fig. 6E), respectively.
The fabrication procedure of the PEC aptsensor could also be monitored by the photocurrent responses under the optimal experimental condition (Fig. 7A). PB solution contained 0.1 mol/L of AA to improve photocurrent conversion efficiency. Curve a showed there was almost non photocurrent response for bare ITO electrode. The photocurrent remarkably enhanced when TiO 2 /NCQDs nanohybrids were modified on ITO electrode (curve b). This indicated that the composition of NCQDs with TiO 2 NRs conspicuously increased the photocurrent. Because of the composition of NCQDs and TiO 2 NRs, the electron transfer efficiency enhanced much and the recombination probability between the electrons and the holes was depressed apparently. And also the composition manner was also investigated (Fig. 6B). TiO 2 /NCQDs prepared by method 2 showed poor PEC property with a photocurrent about 1.5 μ A. TiO 2 /NCQDs prepared by method 3 displayed better PEC performance with a photocurrent about 4.6 μ A. Only method 1 can make the photocurrent of TiO 2 /NCQDs improve obviously up to about 11.9 μ A. The temperature and duration time using in method 1 might result in the effective combination between TiO 2 and NCQDs. This effective combination might make the electrons injection much easier between CB and VB. Later on, when the primers of TXNDC5 were modified on the electrode, the photocurrent reduced (curve c). The bases of the primers owned− NH 2 groups and TiO 2 /NCQDs have − COOH groups. When the primers were incubated on the modified ITO electrode surface, the interaction between the amino groups of primers and the carboxyl group of TiO 2 /NCQDs would occur under the activation of EDC/NHS amidization protocol. TiO 2 /NCQDs can immobilize the primers probes on ITO electrode through the interaction. But the bases can not transfer the electrons and absorb the visible light, which would result in the obstruction of the absorption of the light and the suppression of the electrons transfer. This would cause the decrease of the photocurrent and the experimental results proved this speculation. Therefore, the PEC aptsensor was fabricated as expected.
Analytical performance characteristics. In order to investigate the possibility of the aptsensor applied for TXNDC5 analysis, quantitative detection of TXNDC5 was operated under the optimal conditions. The process began after incubating various concentrations of TXNDC5 on ITO electrode. The detection results were given in Fig. 7B,C. The concentration of TXNDC5 affected the strength of the photocurrent response and a stronger response was achieved at high TXNDC5 concentration. This indicated that the proposed PEC platform showed good detection performance so that it can be used for the TXNDC5 quantitative detection. A calibration graph was plotted under the optimal conditions (Fig. 7D). A positive relationship can be deduced between the photocurrent response signal change and the target concentration. PEC signal change increased linearly with the logarithm of the TXNDC5 concentration from 0.5 fmol/L to 10.0 nmol/L. The linear equation was obtained as ∆I (μ A) = 2.901 + 0.901 lgc TXNDC5 (pmol/L) and the correlation coefficient was 0.991. A detection limit of 0.1 fmol/L was obtained for the reported aptsensor. The proposed PEC aptsensor therefore showed a wide linear range and a low detection limit for the determination of TXNDC5. The low detection limit may be ascribed to good separated excitation energy of TiO 2 /NCQDs and the specific recognition between the capture probe and the target.
Stability and reproducibility were two important parameters affecting the practical application of an aptsensor. Figure 7E showed the photocurrent responses of the PEC aptsensor with the visible-light irradiation repeated every 20 s. The irradiation process was repeated 20 on/off cycles over 800 s. During every on/off cycle, the photocurrent did not show any obvious change. This indicated that the photocurrent response was very stable and this strategy was appropriate to construct the PEC sensors. A series of six electrodes were fabricated and used to determinate 500 pmol/L of TXNDC5. All of the tests were carried out under the same conditions. The relative standard deviation (RSD) for TXNDC5 was 1.8%, so the reproducibility of the proposed PEC aptsensor was good.

Conclusions
This work proposed a novel PEC aptsensor for the rapid and ultrasensitive detection of TXNDC5. The specific detection was realized by specific recognization between the capture probe and the target. When exposed to the visible light, the signal generator of TiO 2 NRs/NCQDs nanohybrids expressed significantly enhanced PEC property. The combination of NCQDs improved the charge separation efficiency and the charge transfer ability, and suppressed the h + /e − recombination effectively. The developed aptsensor displayed the ultra-sensitivity and good stability. Thus, it provided good detection effect for TXNDC5 and might provide a feasible platform to determinate of other analyses.

Regents.
Phosphate buffered solution (PB, 0.067 mol/L KH 2 PO 4 and 0.067 mol/L Na 2 HPO 4 ) were used as the electrolyte for all electrochemistry measurements. All other chemical reagents were analytical reagent grade and directly used without further purification. The ultrapure water (resistivity of 18.25 MΩ •cm) and pipette tips were put into a LDZX-30KBS pressure steam sterilizer (Shanghai Shenan Medical Instrument) and sterilized at 121 °C for 40 min. After cooling to room temperature, the tips were stored in a 4 °C refrigerator.
Preparation of TXNDC5. TXNDC5 was prepared according to our previous work 3 . TXNDC5 mRNA was reverse-transcribed by ReverTra Ace qPCR RT kit and SYBR Green PCR kit (Toyobo, Osaka, Japan). The primers for the amplification of TXNDC5 were as follows: forward primer 5′ -CTC TGG GCC TTG AAC ATT-3′ and reverse primer 5′ -CCC TCA GTG ACT CCA AA-3′ . The sequence of TXNDC5 was as follows: 5′ -CTC TGG GCC TTG AAC ATT CCG AAA CTG TCA AGA TTG GCA AGG TTG ATT GTA CAC AGC ACT ATG AAC TCT GCT CCG GAA ACC AGG TTC GTG GCT ATC CCA CTC TTC TCT GGT TCC GAG ATG GGA AAA AGG TGG ATC AGT ACA AGG GAA AGC GGG ATT TGG AGT CAC TGA GGG-3′ . The specificity of the qRT-PCR assay was evaluated by melting curve analysis. It showed that the TXNDC5 amplification product generated a melting peak at 81.20 ± 0.34 °C without primer-dimers or nonspecific products. 15 μ L of 5′ primer (10 μ mol/L) and 15 μ L of 3′ primer (10 μ mol/L) were diluted respectively with 323 μ L ultrapure water (0 °C). Then the mixtures were oscillated for 40 min under 0 °C. The concentration of the diluted primers was much greater than that of TXNDC5.
Preparation of TiO 2 NRs. TiO 2 NRs were prepared according to the method in the literature with slight modifications 35 . 5 g of TiCl 3 aqueous solution and 4 g of NaCl were added into 10 mL of distilled water under stirring. The solution was put into a Teflon-lined stainless steel autoclave with a capacity of 50 mL. The autoclave was sealed and heated at 100 °C for 12 h. After cooling to room temperature, the products were washed with distilled water and absolute ethanol for several times. Then the products dried under vacuum for use.

Preparation of NCQDs.
NCQDs were prepared as the method described in the literature with slight modifications 30 . 2 g of DTPA was placed in 20 mL ultrapure water and stirred vigorously to form a turbid liquid. Then, the formed liquid turned into the colorless solution by heating. The heating process (100 °C) was continued until the colorless solution became a yellow clustered solid, indicating the formation of NCQDs. The yellow solid was dissolved in 20 mL ultrapure water. After that, the yellow crude NCQDs solution was centrifuged at 8000 r/min for 15 min to remove the unreacted DTPA. Finally, the obtained supernatant was freeze-dried to get the pure solid of NCQDs.
Preparation of TiO 2 NRs/NCQDs. Method 1:TiO 2 NRs/NCQDs nanohybrids were prepared as described in the literature with slight modifications 35 . 5 g of TiCl 3 aqueous solution, 4 g of NaCl and 10 ~ 28 mg of NCQDs powder were added into 10 mL of distilled water under stirring. The following procedure was same to the preparation of TiO 2 NRs. TiO 2 NRs/NCQDs nanohybrids were prepared as method 2, 3 (ESI †).
Fabrication of the PEC aptsensor. The fabrication procedure of the PEC aptsensor was shown in Fig. 1.
Firstly, ITO conductive glass was cut (1.0 cm × 2.5 cm, rectangle) as the working electrode, washed orderly by ultrasonication for 30 min with acetone, ethanol and ultrapure water respectively, and dried by pure nitrogen.
Secondly, 10 μ L of TiO 2 /NCQDs nanohybrids were dropped on ITO electrode and dried in a 4 °C refrigerator. Thirdly, 15 μ L of diluted 5′ primer was dropped on TiO 2 /NCQDs/ITO electrode surface using an EDC/NHS amidization protocol and incubated in a 4 °C refrigerator for 4 h. After that, 15 μ L of 3′ primer was dropped on 5′ primer/TiO 2 /NCQDs/ITO electrode using an EDC/NHS amidization protocol and incubated in a 4 °C refrigerator for 4 h. At last, TXNDC5 ssDNA with different concentrations were dropped on 3′ primer/5′ primer/TiO 2 / NCQDs/ITO electrode surface respectively and incubated at 4 °C for 4 h. Measurement procedure. The PEC measurements were performed immediately after the incubation of TXNDC5 ssDNA. A conventional three-electrode system was used in all the PEC experiments. A platinum wire was the auxiliary electrode. A KCl-saturated calomel electrode (SCE) was the reference electrode. The modified ITO electrode was used as the working electrode. A commercial LED light (430 nm) was used as the irradiation energy in all the PEC tests. PB containing 0.1 mol/L of ascorbic acid (AA) was used as an electrolyte solution for all the PEC measurements. The bias voltage was 0.1 V. Both light duration and no light duration were 20 s. All the PEC experiments were operated at room temperature.