Introduction

Layer-by-layer techniques are extensively employed to modify surfaces because the material quantity, electronic properties, and thickness of the films generated can be easily controlled [1]. Moreover, these modified surfaces can be used as biosensors and bioelectronic devices, because synergetic effects between the components of the multilayer arrangements improve the biocompatibility of the electrodic interface [2]. Furthermore, the study of new systems that interact with DNA biomolecules is interesting from the perspective of designing new bioelectrochemical interfaces [3–6].

Tetra-(triphenyl)-phosphoniumtolylporphyrin has been demonstrated to have electrostatic interactions with genomic calf thymus double-stranded DNA (CT DNA) in solution, at difference of other cationic porphyrins that intercalate between aromatic domains and grooves of DNA [7–9]. Alternately, CdTe quantum dots are semiconductor nanomaterials based on the II/IV family [10] with emission properties tunable by their size and with biocompatibility easily modulated by employing amino acids or polypeptides, such as cysteine or glutathione [11, 12].

Hybrid cationic porphyrins/CdTe quantum dots electrostatically assembled have been studied in solution by photophysical techniques [13], and their analytical applications were recently demonstrated in the detection of CT DNA, generated by hybrids acting as on-off fluorescent sensors [14–16].

To our knowledge, there are not examples of the properties of such hybrids assembled onto electrodic surfaces in CT DNA detection.

There are two main types of research concerning DNA detection. One of them requires DNA immobilization onto electrodic surface for using it for DNA sequencing, detecting DNA damage or for hybridization biosensors [6, 17]. This methodology can be used in the diagnosis of infectious diseases, genetic mutations, clinical medicine, and forensic applications [18–20]. On the other hand, the direct detection of native or double-stranded DNA, where immobilization is incompatible, is of interest for industrial biopharmaceutical laboratories where sensitive, simple, and rapid methods for DNA determination in solution are required [21].

DNA immobilization based on electrochemical techniques has been proposed as an alternative to conventional DNA detection techniques, because electrochemical techniques are cheaper, transportable, and easily miniaturized [17, 22–25].

Some groups have focused its efforts on CT DNA determination based on the increasing of the oxidation current of guanine by voltammetric techniques employing carbon paste or glassy carbon electrodes modified with nanomaterials as transducers [6, 26, 27]. Another way, in this work we present evidence on the feasibility of a hybrid system of porphyrins/quantum dots that presents a different behavior measured by electrochemical impedance spectroscopy (EIS) when CT DNA immobilization takes place. For that purpose, we use a common redox couple such as [Fe(CN)6]3−/[Fe(CN)6]4− to detect and differentiate CT DNA immobilization. This approach is an alternative procedure to voltammetric analysis, when voltammetric peaks observed for CT DNA detection are poorly defined.

From the broad spectra of electrochemical techniques, EIS has gained attention as a label-free DNA hybridization transduction [4, 5, 28] because phosphate groups of single-stranded or double-stranded DNA species can be easily discriminated in the presence of anionic redox species, such as [Fe(CN)6]3−/[Fe(CN)6]4− [17].

This work describes the characterization of hybrids {[Ttolyl(P-(C6H5)3)4]4+/CdTe} n by UV-Vis spectroscopy, SEM microscopy, cyclic voltammetry, and EIS, and their analytical performance in CT DNA determination by EIS. We provide evidence supporting the use of the {[Ttolyl(P-(C6H5)3)4]4+/CdTe} n system for the detection and quantification of CT DNA.

Materials and methods

Apparatus

UV-Vis spectroscopy was performed with a Scinco S-3100 spectrometer. Emission spectra were recorded with a Luminescence spectrometer Perkin Elmer LS 50 B irradiating at 350 nm in a quartz cuvette with an optical pass of 1.0 cm.

1H-NMR characterization was performed in a Bruker 400. Cyclic voltammetry and EIS were registered in a CH-Instrument 604C. Data from the impedance measurements were obtained by fitting with Z view® software. A one-compartment cell was employed with three electrodes, where indium tin oxide (ITO), Delta Technologies, with a resistance of 20–30 Ω and a geometric area of 1.5 cm2 was employed as a working electrode, Ag/AgCl (3 M KCl) was used as a reference electrode, and a platinum coil was used as an auxiliary electrode. Electrochemical impedance spectroscopy was measured between 100 kHz and 10 MHz with the application of E DC = +0.2 V. The sinusoidal potential pulse was 5 mV.

Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX) analyses were obtained from a high-resolution imaging via a Vegan 3 Tescan microscope coupled to a Bruker Quantax 400 energy dispersive X-ray detector.

Reagents

Diisobutylaluminium hydride (DIBAL-H) was acquired from Alfa Aesar Company. Bromotolylbenzonitrile, triphenylphosphine, cloroanile, boron trifluorideetherate, triethylamine, single-stranded deoxyribonucleic acid genomic from calf thymus (CT ssDNA), deoxyribonucleic acid genomic, type XV from calf thymus (CT DNA), deoxyribonucleic acid genomic from salmon testes (ST DNA), and Tween 20® were purchased from Sigma-Aldrich. The concentration of the starting standard was determined by its absorbance at 260 nm, assuming ε = 10,560 M−1 cm−1 for CT ssDNA, ε = 13,100 M−1 cm−1 [8] for CT DNA, and ε = 12,824 M−1 cm−1 for ST DNA [29].

Tris-(hydroxymethyl)-aminomethane and N-(2-hydroxyethyl)-ethylenediamine-N,N′,N′-triacetic acid (EDTA; molecular biology grade, Calbiochem), methanol, toluene, and n-hexane were obtained from Merck. Pyrrole was acquired from Sigma-Aldrich, and it was distilled prior to use. DIBAL-H was manipulated by avoiding contact with air and water.

Synthesis

5,10,15,20-tetra(triphenylphosphonium)tolylporphyrin was synthesized via a procedure described elsewhere [7]. Yield (32 %), 1H-NMR (CD3Cl + CD3OD): 8.76 (s, 8H, β-pyrrole), 8.05 (d, 8H, phenyl-CH2), 7.83–7.89 (m, 60H, P-phenyl), 7.46 (m, 8H, phenyl-CH2). FT-IR/cm−1 (in KBr): 3394, 3034 (m, =NH), 2859 (m, C–H on phenyl and pyrroles), 1618 (m, C = N imines), 1438 (C = C), 1111 (s, CH2-P). UV-Vis (CH3OH) 416 nm, log ε/M−1 cm−1 = 5.7 (π − π* transition Soret band), 514, 548, 591, 644 (π − π* transition, Q bands). CdTe (GSH) was synthesized according to a procedure described elsewhere [11, 30]. λ em = 582 nm (orange emission). The diameter size was estimated as ~3.28 nm by UV-Vis spectroscopy.

ITO pretreatment

ITO electrodes were activated by refluxing them at 105 °C in a solution consisting of NH4OH, H2O2, and H2O in a 1:1:5 proportion for 1.0 h, and the solution was agitated at 250 rpm. They were washed gently with bi-distilled water and then dried under a nitrogen stream.

Layer-by-layer electrostatic self-assemblies

ITO electrodes were immersed inside a solution of 0.1 mM tetra tolyl(triphenyl)phosphoniumporphyrin in a 10 mM Tris buffer, at a pH of 7.0 and with 8 % methanol, for 5 min. After the porphyrin layer deposition, the electrodes were washed with abundant water and dried with an N2 stream. After this, the electrodes were immersed inside a solution of CdTe (GSH) quantum dot in a 10 mM Tris buffer at a pH of 10 for 5 min. Then the electrode was cleaned with abundant deionized water and dried with an N2 stream. The process was repeated to obtain a higher number of multilayers. Every step was monitored by UV-Vis spectroscopy, cyclic voltammetry, and electrochemical impedance spectra in the presence of 2.0 mM [Fe(CN)6]3−/[Fe(CN)6]4− in 10 mM Tris at a pH of 7.0 and 0.1 M KCl.

Native DNA adsorption onto {[Ttolyl(P-(C6H5)3)4]4+/CdTe} n

ITO electrodes modified with {[Ttolyl(P-(C6H5)3)4]4+/CdTe} n with n = 1, 3, and 5 were incubated for 30 min in solutions of CT DNA (calf thymus), ST dsDNA (salmon testes), or CT ssDNA (calf thymus) at 60 °C. After every incubation step, each electrode was gently washed with an aqueous solution of 0.05 % Tween® 20 and bi-distilled water and then dried with an N2 stream in order to eliminate any weakly adsorbed DNA molecules.

Results and discussion

Characterization of the {[Ttolyl(P-(C6H5)3)4]4+/CdTe} n assemblies

UV-Vis spectroscopy

Figure 1 shows the UV-Vis spectra of the {[Ttolyl(P-(C6H5)3)4]4+/CdTe} n hybrid. In all multilayer assemblies, the most intense peak appears at 426 nm and is associated with a π − π* electronic transition of cationic porphyrins, known as Soret band [30–32]. The absorbance of such band for these systems increased linearly with every new immersion step n, providing evidence that the porphyrins are being adsorbed in a layer-by-layer approach with the corresponding quantum dots [33–35]. The inset of Fig. 1 shows the Soret band for triplicate experiments, with a linear response related to the immersion steps, showing the accuracy of the methodology. It must be denoted that these spectra are slightly different to a previous work, where a smaller size of CdTe quantum dots was employed [30]. In the current work, only electronic transitions associated with porphyrins are present, and there is not exciton absorption of CdTe quantum dots (d = 3.28 nm) in the multilayer assembly.

Fig. 1
figure 1

UV-Vis spectra of the layer-by-layer assemblies of ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe} n , where n is the number of immersion steps, with a range from 1 to 5. Inset: absorbance at 426 nm vs n

Full size image

Cyclic voltammetry of {[Ttolyl(P-(C6H5)3)4]4+/CdTe} n assemblies

Figure 2 shows the behavior of the ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe} n in 2.0 mM Fe(CN)6 4−/Fe(CN)6 3−, where it can be observed clearly the presence of pinhole across the multilayer [30, 36]. The Fe(CN)6 4−/Fe(CN)6 3− couple presented a quasi-reversible behavior in the presence of the ITO electrode (n = 0), with a peak potential difference ΔE p of +0.27 V. However, in the presence of ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe}1–3 multilayer assemblies, Fe(CN)6 4−/Fe(CN)6 3− couple presented a higher ΔE p compared to that of the ITO electrode, corresponding to +0.36, +0.35, and +0.37 V, respectively. The current value diminished for every immersion step, demonstrating that the surface is changing when the multilayer assembly is formed. A more irreversible behavior of the probe Fe(CN)6 4−/Fe(CN)6 3− in the presence of ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe}4–5 was observed because its ΔE p was +0.60 V when n = 4, whereas the current associated with the Fe(CN)6 4−/Fe(CN)6 3− couple yielded a plateau shape at n = 5. These results can be associated with a sluggish electron transfer for every new immersion step performed for ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe} n systems in the presence of the [Fe(CN)6]3−/[Fe(CN)6]4− redox couple. The decrease in the current for the [Fe(CN)6]3−/[Fe(CN)6]4− probe and the more irreversible character of the peak potentials from n = 3 to n = 5 are due to the electrostatic repulsion between the anionic CdTe in the multilayer assembly and the [Fe(CN)6]3−/[Fe(CN)6]4−, which can be attributed to the increasing electrostatic repulsion and the increasing covering of the surface. A similar behavior was previously reported for other electrostatic multilayer assemblies [30, 36].

Fig. 2
figure 2

Cyclic voltammetry of layer-by-layer assemblies of ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe} n , where n is the immersion step, with n ranging from 1 to 5, in the presence of 2.0 mM Fe(CN)6 4−/Fe(CN)6 3−, 10 mM Tris, and 0.1 M KCl

Full size image

This electrostatic effect can be confirmed when similar characterization is performed in the presence of a cationic redox probe, such as [Ru(NH)5Cl]2+ (see Figure S1), where it was observed that ΔE p slightly changes for every new immersion step from 0.16 V (n = 1) to 0.19 V (n = 5). The current value slightly decreases for every new immersion step. A similar behavior has been published previously for other electrostatic multilayer assemblies. The electroactive area for each ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe} n system showed slight variations compared to the electroactive area of the ITO electrode, providing evidence that electrostatic repulsions are playing an important role, more so than the disposable nanometer area with CdTe capped with glutathione.

Electrochemical impedance spectroscopy

Figure 3 shows the characteristic Nyquist plot obtained when the ITO electrode was immersed in an alternating manner in porphyrin and CdTe quantum dot solutions. An incomplete semicircle region (100 kHz to ~5000 Hz) associated with the inner capacitance of semiconductors was observed at higher frequencies [37, 38]. At frequencies lower than 5000 Hz, the electrochemical interfacial process can be observed, where the semicircle region can be associated with the resistance to the electron transfer between the [Fe(CN)6]3−/[Fe(CN)6]4− redox couple and a linear region associated with the mass transfer processes, the Warburg impedance.

Fig. 3
figure 3

Nyquist plots from electrochemical impedance spectra of different bi-layers onto ITO surfaces. Two millimolar Fe(CN)6 4−/Fe(CN)6 3−, 10 mM Tris, and 0.1 M KCl. Potential of +0.2 V applied vs Ag/AgCl. Amplitude pulse potential = 5 mV. Frequency range = 0.01 Hz–100 kHz

Full size image

Figure 4 shows Bode plots for every immersion step. It was observed that at high frequencies, the phase angle starts at ~ −45°, in the semiconductor zone, but all spectra show a peak process associated with the time constant of the [Fe(CN)6]3−/[Fe(CN)6]4− redox couple in the electrochemical interface. The module of impedance clearly shows the predominance of two resistances, a |Z| constant one associated with the solution resistance R s at frequencies lower than 5000 Hz, and the resistance to electron transfer at frequencies lower than 1 Hz, where the |Z| module reaches a plateau, associated with the resistance to the electron transfer of [Fe(CN)6]3−/[Fe(CN)6]4−. The presence of a double-layer capacitor was evident because the |Z| changes with frequency. Taking all of this information into consideration, the proposed equivalent circuit is displayed in Scheme 1. This circuit generates two time constants, as it can be observed in angle phase vs frequency plot, and demonstrates their combination zone of semiconductor and the electrochemical interface where the electrochemical reaction is occurring [38]. The χ-square of all the fits is lower than 0.002, confirming the good accuracy of the equivalent circuitry proposed. A constant phase element (CPE), instead of an ideal capacitor for double-layer capacitance, was employed because this type of impedance element is useful when the interface is not flat, such as the multilayer assemblies studied in this work. Z CPE = 1 / (T (iω))P, where the value of the P exponent changes from 0 to 1. Depending on this value, T can take resistance (~0) or capacitance (~1) units, showing the more resistive or capacitive behavior of the system [39]. Table 1 provides the values for the circuitry elements proposed. The predominant parameter that changes for every new immersion step is R ct. When n = 0 and n = 1, R ct exhibited a similar value; however, from n = 2 to n = 5, R ct increases for every new immersion step, demonstrating the electrostatic repulsion between [Fe(CN)6]3−/[Fe(CN)6]4− and the anionic outer layer CdTe in these assemblies. In a similar pattern, the relaxation time (τ = C dl R ct) for every multilayer increased in the range of 3.79 to 174.6 ms, showing a more sluggish process for [Fe(CN)6]3−/[Fe(CN)6]4− by increasing n. Inversely, the characteristic frequency for ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe} n in the presence of [Fe(CN)6]3−/[Fe(CN)6]4− diminishes with n in the range 42.0 to 0.82 s−1.

Fig. 4
figure 4

Bode plots of different layer-by-layer onto ITO surfaces. Two millimolar Fe(CN)6 4−/Fe(CN)6 3−, 10 mM Tris, and 0.1 M KCl. Potential of +0.2 V applied vs Ag/AgCl. Amplitude pulse potential = 5 mV. Frequency range = 0.01 Hz–100 kHz

Full size image
Scheme 1
scheme 1

Equivalent circuit employed. Csc ITO semiconductor capacitance, R s solution resistance, CPE dl constant phase element for electric double layer, R et resistance to electron transfer, W o1 Warburg diffusional impedance

Full size image
Table 1 Electrochemical impedance data obtained by fitting ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe (3.28 nm)} n , where n = 1–5, in the presence of 2 mM Fe(CN)6 4−/Fe(CN)6 3−, 10 mM Tris, and 0.1 M KCl. Potential of +0.2 V applied vs Ag/AgCl; amplitude pulse potential = 5 mV; frequency range = 0.01 Hz–100 kHz
Full size table

To corroborate the electrostatic effect of these assemblies, Ru(NH3)5 3+ was used as the redox couple for ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe} n . The ITO electrode shows a diffusional pattern only with a phase angle close to −45° against this redox couple. However, when ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe} n systems were immersed in the solution containing cationic ruthenium species, a semicircle was observed in the Nyquist plots, associated with the presence of the multilayer assemblies. Nonetheless, the diameter of the semicircle is almost constant between n = 1–4. Only when n = 5 there was an increase compared to the early step of modification (see Figure S2). The electron transfer resistance after fitting the spectra with a typical Randles circuitry shows a low dependence on the immersion step, suggesting that the predominantly anionic character of outer layer helps the external sphere electron to the transfer of Ru(NH3)5 3+ toward the ITO electrode. This pattern was observed in other electrostatic multilayer assemblies studied by EIS [21, 36].

Finally, the capacitance of the semiconductor zone changed significantly compared to the control with ITO, and it is three orders of magnitude order lower than the capacitance obtained from C dl.

Interactions of {[Ttolyl(P-(C6H5)3)4]4+/CdTe} n with genomic DNA

Assuming that the equivalent circuitry in Scheme 1 is valid, it was proposed to study the absorption of DNA species by {[Ttolyl(P-(C6H5)3)4]4+/CdTe} n considering R ct, taking into consideration its strong influence on DNA species as was explained above.

Optimization of the multilayer number

Figure 5 shows the R ct obtained from EIS of {[Ttolyl(P-(C6H5)3)4]4+/CdTe} n , where n = 1, 3, and 5, before and after incubation of these hybrids in 1.0 × 10−6 M of CT DNA and CT ssDNA, in separate experiments. No significant changes in R ct of the multilayer assembly were observed with n = 1 and n = 5. However, the R ct of the system {[Ttolyl(P-(C6H5)3)4]4+/CdTe}3 could differentiate CT ssDNA or CT DNA, demonstrating its ability to be immobilized even if the outer layer is negative CdTe quantum dots. The difference is probably associated with the repulsion of [Fe(CN)6]3−/[Fe(CN)6]4− redox couple with the different number phosphate groups in single- and double-stranded DNA. It can be seen from the results that the multilayer hybrid has a specific immersion step that is able to differentiate CT DNA, with n = 3 being the optimum multilayer number.

Fig. 5
figure 5

Resistance to charge transfer of ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe}1,3,5 after incubation in 1.0 × 10−6 M CT DNA obtained by EIS in the presence of 2 mM Fe(CN)6 4−/Fe(CN)6 3−, 10 mM Tris, and 0.1 M KCl. E dc = +0.2 V vs Ag/AgCl. Sinusoidal potential = 5 mV. Frequency range = 0.01 Hz–100 kHz

Full size image

SEM-EDX microscopy of ITO/{[Ttolyl(P-(C6H5)3)4]+/CdTe}3 after CT DNA incubation

The morphology of the ITO/{[Ttolyl(P-(C6H5)3)4]+/CdTe}3 system is very similar to that of our previous work [30]. However, here, we propose that CT DNA was effectively adsorbed onto the ITO/{[Ttolyl(P-(C6H5)3)4]+/CdTe}3, as can be observed in Fig. 6a. An organic layer is adsorbed onto the ITO electrode providing evidence for the presence of cationic porphyrin and CT DNA. However, as it is observed from backscattered electrons (see Fig. 6b), a homogeneous surface of spherical-like particles spreads over the ITO electrode. The quantum dots are agglomerated on the electrode surface, showing a mean size of 80 nm, which is larger than those obtained from UV-Vis measurements in solution [30]. The morphology pattern suggests that CT DNA was immobilized onto ITO/{[Ttolyl(P-(C6H5)3)4]+/CdTe}3.

Fig. 6
figure 6

SEM microscopy of ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe}3 after adsorption of CT DNA 1.0 × 10−6 M. a Mode. b Backscattering electrons

Full size image

Effect of CdTe (GSH) on CT DNA adsorption

To corroborate the effect of CdTe quantum dots on CT DNA immobilization, R ct was evaluated after different incubation times in 1.0 × 10−6 M CT DNA between a layer of porphyrin ascribed as ITO/{[Ttolyl(P-(C6H5)3)4]4+ and the hybrid ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe}3 (Fig. 7). It was observed that after incubation in the CT DNA solution, ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe}3 required 20 to 30 min to change the R ct, whereas ITO/{[Ttolyl(P-(C6H5)3)4]4+ required 90 min to change; hence, CdTe nanoparticles help in the rapid immobilization of 1.0 × 10−6 M CT DNA on ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe}3.

Fig. 7
figure 7

Time optimization with ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe}3 and ITO/[Ttolyl(P-(C6H5)3)4]4+ after incubation in 1.0 × 10−6 M CT DNA obtained by EIS in the presence of 2.0 mM Fe(CN)6 4−/Fe(CN)6 3−, 10 mM Tris, and 0.1 M KCl. Potential of +0.2 V applied vs Ag/AgCl. Sinusoidal pulse potential = 5 mV. Frequency range = 0.01 Hz–10 kHz

Full size image

As the outer layer of ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe}3 consists of negatively charged quantum dots, other non-covalent forces could be influencing the adsorption of CT DNA onto the multilayer assembly, as corroborated by the SEM micrograph. To this respect, it was recently reported that CdTe capped with glyphosate can immobilize CT DNA by non-covalent interactions, such as hydrogen bonding and DNA [40].

At this point, it is clear that a synergic effect between cationic porphyrin/CdTe immobilized onto ITO is helping in the adsorption of CT DNA. This effect on the multilayer assembly was corroborated by UV-Vis spectroscopy, because the Soret band of porphyrin diminishes its absorbance after immobilization of 1.0 × 10−6 M CT DNA (see Figure S3), as it could be expected from its behavior in solution with CT DNA [7–9].

Analytical performance of ITO/{[Ttolyl(P-(C6H5)3)4]+/CdTe}3

EIS is a valuable tool for studying the adsorption processes of DNA onto electrodic surfaces because negative charged redox couples can be repealed by the electrostatic action of phosphate groups present in the DNA backbone [17]. As it can be observed in Fig. 8, after CT DNA incubations, there was a significant increase in R ct at higher CT DNA concentrations. In contrast, after incubation in CT ssDNA and ST DNA, the R ct of ITO/{[Ttolyl(P-(C6H5)3)4]+/CdTe}3 varied slightly, from 1415 to1724 Ω and from 1415 to 1315 Ω, respectively. These results show that CT DNA is being expediently adsorbed onto ITO/{[Ttolyl(P-(C6H5)3)4]+/CdTe}3, whereas the CT ssDNA and ST DNA species are being poorly adsorbed.

Fig. 8
figure 8

Nyquist plot of ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe}3 after incubation at different concentrations of genomic DNA: curve 1 (0 DNA); 2 (CT ssDNA 0.6 × 10−6 M); 3 (1.0 × 10−6 M ST DNA); 4–8 CT DNA at concentrations: 4 (1.0 × 10−10 M); 5 (1.0 × 10−9 M); 6 (1.0 × 10−8 M); 7 (1.0 × 10−7 M); and 8 (1.0 × 10−6 M)

Full size image

Figure 9 shows a representative R ct signal response as a function of the concentration of CT DNA. R ct increases linearly with the logarithm of CT DNA, as can be expected for an affinity experiment when a broad range of concentrations are studied, such as in this work [41]. No correlations with the concentrations of the corresponding CT ssDNA and ST DNA were observed in the range of concentrations studied. These results suggest that ITO/{[Ttolyl(P-(C6H5)3)4]+/CdTe}3 can specifically bind to CT DNA instead of other typical genomic DNA species. The reproducibility of the present method was excellent: the standard deviation for four replications at a concentration of 1.0 × 10−8 M was 7.68 %.

Fig. 9
figure 9

R ct of ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe}3 by triplicate experiments in the presence of 2.0 mM Fe(CN)6 4−/Fe(CN)6 3− obtained by EIS after genomic DNA incubations. Ten millimolar Tris, pH = 7.0, and 0.1 M KCl. Potential of +0.2 V applied vs Ag/AgCl. Amplitude sinusoidal pulse = 5 mV. Frequency range = 0.01 Hz–100 kHz

Full size image

The impedimetric sensor for CT DNA was compared with other sensors for this biomolecule. As observed in Table 2, the proposed sensor has a broader concentration range, and a lower LOD compared to other reports [6, 14, 21, 41], making the proposed ITO/{[Ttolyl(P-(C6H5)3)4]+/CdTe}3 hybrid system a good candidate for the determination of genomic CT DNA.

Table 2 Comparison of the analytical parameters for CT DNA determination
Full size table

Finally, cyclic voltammetry after DNA incubations showed an irreversible oxidation associated with the typical guanine oxidation at +1.2 V (see Figure S4). The signal associated with this process was broad and poor defined at low CT DNA concentration, and it is not present after CT ssDNA incubation, validating the idea of the utilization of EIS to study the CT DNA adsorption onto the ITO/{[Ttolyl(P-(C6H5)3)4]+/CdTe}3 system.

Conclusion

Hybrid {[Ttolyl(P-(C6H5)3)4]4+/CdTe} n specimens, where n = 1–5, were prepared by immersion steps, and they were characterized by UV-Vis spectroscopy, cyclic voltammetry, and EIS.

The immersion steps of the hybrid system and the time to immobilize DNA species were optimized by EIS. It was found that ITO/{[Ttolyl(P-(C6H5)3)4]+/CdTe}3 has synergic effects on the immobilization of CT DNA by electrochemical impedance spectroscopy, cyclic voltammetry, and UV-Vis spectroscopy. ITO/{[Ttolyl(P-(C6H5)3)4]+/CdTe}3 was demonstrated as selectively adsorbing CT DNA instead of other genomic species with a linear range of 1.0 × 10−10 to 1.0 × 10−6 M and a LOD of 6.0 × 10−11 M using EIS. SEM-EDX microscopy of ITO/{[Ttolyl(P-(C6H5)3)4]+/CdTe}3 after incubation in CT DNA showed a film on the CdTe capped with glutathione, and backscattering microscopy confirmed that CdTe was spread over all of the surface. These results suggest that CT DNA could be specifically immobilized by both electrostatic attraction (cationic porphyrin) and hydrogen bonds CdTe (GSH).

This work is a first approach for the use of hybrid cationic porphyrin/quantum dot assemblies to adsorb biomolecules onto electrodic surfaces; this interface brings about multiple binding sites to CT DNA via non-covalent interactions. Overall, the results indicate that the newly described {[Ttolyl(P-(C6H5)3)4]4+/CdTe} n system is an excellent method for the determination of CT DNA.