A Novel Molecularly Imprinting Biosensor Including Graphene Quantum Dots/Multi-Walled Carbon Nanotubes Composite for Interleukin-6 Detection and Electrochemical Biosensor Validation

Human IL-6 standard grade antigen (1.0 mg·ml⁻¹), tumor necrosis factor alpha (TNF-α), human immunoglobulin G (IgG), bovine serum albumin (BSA), Mucin 1 (MUC1), Mucin 4 (MUC4), Mucin 16 (MUC16) and carcino-embryonic antigen (CEA) were purchased from Sigma-Aldrich and used by dilution in pH 7.4 phosphate buffer solution (PBS, 0.1 mol·l⁻¹).

SEM and XRD images were obtained by using ZEISS EVO 50 SEM analytical microscopy and Rigaku X-ray diffractometer, respectively. Gamry Reference 600 work-station (Gamry, USA) was performed for electrochemical measurements such as Differential pulse voltammetry (DPV), CV and EIS. In addition, glassy carbon electrodes (GCE) as working electrode were utilized and cleaned according to our previous study. ³⁸

The synthesis of GQD and f-MWCNTs were explained detailed in our previous works. ^39,40 Firstly, the thermal deoxidization of graphene oxide at 300 °C for 1 h provided graphene sheets. Then, the prepared graphene sheets (0.20 g) was subjected to oxidation process in H₂SO₄-HNO₃ (1:1, v/v) for 10 h and GQDs were collected and dried at 25 °C. Finally, 5.0 ml dispersion of GQDs and the prepared f-MWCNTs (0.5 mg·ml⁻¹) were mixed under vigorous stirring followed by centrifuged at 10000 rpm. Subsequently, as-obtained GQDs/f-MWCNTs composite were dried at 100 °C overnight, then dispersed in ultra-pure water at a concentration of 0.5 mg·ml⁻¹. ⁴¹

0.5 mg·ml⁻¹ of GQDs, 0.5 mg·ml⁻¹ of f-MWCNTs and 0.5 mg·ml⁻¹ of GQDs/f-MWCNTs dispersions (10.0 μl) were dropped onto the clean GCE surfaces. After that, solvent evaporation was performed by infrared lamp.

After the preparations of GQDs/GCE, f-MWCNTs/GCE and GQDs/f-MWCNTs/GCE, 100.0 mM pyrrole containing 25.0 mM IL-6 in 0.1 mol·l⁻¹ PBS was prepared in voltammetric cell for preparations of IL-6 imprinted GQDs/GCE, IL-6 imprinted f-MWCNTs/GCE and IL-6 imprinted GQDs/f-MWCNTs/GCE during 15 cycles at 100 mV·s⁻¹ (MIP/GQDs/GCE, MIP/f-MWCNTs/GCE and MIP/GQDs/f-MWCNTs/GCE). Scheme 1 only shows schematic preparation of MIP/GQDs/f-MWCNTs/GCE. In addition, the imprinting selectivity was investigated by using IL-6 non-imprinted GQDs/f-MWCNTs/GCE (NIP/GQDs/f-MWCNTs/GCE/GCE). After all IL-6 imprinted electrodes were swinged in bath including 1.0 M NaCl at 25 °C for 15 min to remove IL-6 proteins from electrode surface, all imprinted electrodes were dried with nitrogen gas. ⁴² All IL-6 imprinted electrodes were preserved without pressure fluctuations at 25 °C and electrochemical voltammograms were also obtained in an enclosed cabinet to prevent from external environmental influences.

Zoom In Zoom Out Reset image size

IL-6-free plasma samples provided from a Blood Bank in Turkey were prepared in four different ways in order to determine the selectivity of the developed biosensor against IL-6 protein. 3.0 ml of methanol was poured onto a 0.5 ml aliquot of each plasma samples followed by centrifugation at 20000 rpm for 20 min. Subsequently, the supernatants were prepared by analysis by diluting with 0.1 M PBS, pH 7.4. ³¹

The inter-connected tubular formation of f-MWCNTs verified specific structure of carbon nanotubes (Fig. 1A). ⁴³ GQDs' diameters of 5–9 nm and the uniform dispersion of GQDs were showed on Fig. 1B. In addition, the wrap of f-MWCNTs with GQDs was demonstrated on SEM image of GQDs/f-MWCNTs composite (Fig. 1C). Hence, the aggregation of nanomaterials (GQDs and f-MWCNTs) was prevented by the conjugated GQDs/f-MWCNTs composite which was stable in aqueous solutions.

Zoom In Zoom Out Reset image size

The morphologies of GQDs, f-MWCNTs and GQDs/f-MWCNTs composite were investigated by TEM. Figure 2A demonstrates GQDs having uniform shape and their average diameter was found to be 5–8 nm. A typical TEM image of acid-purified MWCNTs is shown in Fig. 2B. After the synthesis of GQDs on f-MWCNTs under strong stirring, Fig. 2C confirmed the successful conjugation between GQDs and f-MWCNTs.

Zoom In Zoom Out Reset image size

Raman spectroscopy was used to show specific bands of nanomaterials. D band and G bands were shown at ∼1340 cm⁻¹ and ∼1600 cm⁻¹, respectively (Fig. 3A). The peak at ∼1340 cm⁻¹ indicated disordered structure and local defects. The peak at ∼1600 cm⁻¹ is corresponded sp² bonds of carbon atoms relating to graphitization. I_D/I_G ratio of about 0.85 (5000 a.u./5882 a.u.) is corresponded to characteristic Raman spectra of GQDs. ⁴⁴ I_D/I_G ratio of GQDs/f-MWCNTs composite is 0.81 (6000 a.u./7401 a.u.) and this value is higher than that of f-MWCNTs (6000 a.u./7895 a.u. = 0.76), indicating the formation of GQDs/f-MWCNTs composite via π–π interaction. The XRD peak at 24.4° (002) indicated GQDs formation (Fig. 3B). ⁴⁵ Interlayer space of 0.39 nm in comparison with graphite (0.33 nm) was attributed to the presence of oxygen in GQDs. The peak at 24.4° (002) and a minor peak at 42.6° (100) emerged on XRD pattern of f-MWCNTs. Finally, the successful synthesis of GQDs/f-MWCNTs was confirmed by presence of the peaks at 24.3° and 42.6°.

Zoom In Zoom Out Reset image size

UV–vis spectroscopy was utilized to investigate the optical properties of GQDs, f-MWCNTs and GQDs/f-MWCNTs composite (Fig. 4A). π–π* transition of aromatic C=C bonds corresponding to at about 355 nm was confirmed by blue spectrum of GQDs. ⁴⁶ Two bands at about 225 and 335 nm also indicated π–π* and n–π* transition, respectively (black spectrum of f-MWCNTs). Finally, similar bands were seen for GQDs/f-MWCNTs composite (red spectrum) in comparison with f-MWCNTs.

Zoom In Zoom Out Reset image size

FTIR (Fig. 4B) was used for demonstrating functional groups of nanomaterials. C=O stretching at 1550 cm⁻¹, C–O stretching at 1275 cm⁻¹, −CH₂ asymmetric stretching at 2980 cm⁻¹, symmetric stretching at 2985 cm⁻¹ and O–H stretching at 3325 cm⁻¹ showed characteristic stretching bands of GQDs (blue spectrum). ⁴⁷ Black spectrum of f-MWCNTs belonging to ύ_OH at 3325 cm⁻¹, ύ_C=O at 1585 cm⁻¹ and ύ_OH at 1031 cm⁻¹ verified −COOH and –OH functional groups. C=O symmetric stretching (1710 cm⁻¹) of −COOH groups, C=C stretching aromatic vibration (1530 cm⁻¹), −CH₂ bending vibration (1530 cm⁻¹ and 650 cm⁻¹) and C–O stretching (1185 cm⁻¹) indicated the presence of GQDs/f-MWCNTs composite. Furthermore, asymmetric and symmetric −CH₂ stretching bands at 2938 and 2881 cm⁻¹ shifted to red area in comparison with GQDs. Hence, this red shift confirmed the formation of GQDs/f-MWCNTs.

In addition, Figs. 5A and 5B show SEM images of MIP/GQDs/f-MWCNTs/GCE and NIP/GQDs/f-MWCNTs/GCE, respectively. Polymer layer with porous structure and polymer layer without porous structure were obtained for MIP/GQDs/f-MWCNTs/GCE (Fig. 5A) and NIP/GQDs/f-MWCNTs/GCE (Fig. 5B), respectively. Thus, we can say that molecularly imprinting technique provided the specific cavities to IL-6 protein.

Zoom In Zoom Out Reset image size

Thanks to the high specific surface area of GQDs the more certain peaks were perceived for GQDs/GCE (curve b of Fig. 6A) while for bare GCE the anodic and cathodic peaks potential difference (ΔEp) was obtained as 120 mV (curve a of Fig. 6A). ³⁹ Even, the higher electrochemical responses and reversible peaks were obtained by using GQDs/f-MWCNTs/GCE (ΔEp = 59.5 mV) (curve c of Fig. 6A). The increased electrode surface area and the synergistic effect between graphene quantum dots and multi-walled carbon nanotubes can cause the enhanced performances. In addition, effective surface areas were calculated in presence of 1.0 mM [Fe(CN)₆]³⁻ solution containing 0.1 M KCl by Randles–Sevcik equation of i_p = 2.69 × 10⁵ A n^3/2 D^1/2 C v^1/2. The effective surface areas of the electrodes were found by using i_p − v^1/2 graphics (n = 6) which were formed to use current signal values against different scan rates. Consequently, the electrochemical active areas of bare GCE, GQDs/GCE and GQDs/f-MWCNTs/GCE were calculated as 0.170 ± 0.002 cm², 0.515 ± 0.009 cm² and 0.981 ± 0.005 cm², respectively. ⁴⁸

Zoom In Zoom Out Reset image size

Charge transfer resistance (Rct) values are 175 ohm (curve c of bare GCE), 75 ohm (curve b of GQDs/GCE) and 25 ohm (curve a of GQDs/f-MWCNTs/GCE), respectively by EIS spectra (Fig. 6B). Hence, the highest conductivity was obtained by GQDs/f-MWCNTs/GCE. Two obvious reasons explain the optimal electrochemical responses which are caused by GQDs/f-MWCNTs/GCE:

• (1)  
  GQDs demonstrated a highly size-dependent electrocatalytic activity. ⁴⁹ Nonetheless, owing to oxidation processes, obvious degree of graphitization was destroyed. GQDs also tend to aggregation. ⁵⁰ Due to these reasons, GQDs/f-MWCNTs can not only prevent the stacking of GQDs but also has larger surface area. f-MWCNTs can also accelerate charge transfer in electrochemical reactions due to their conductivity. ⁵¹
• (2)  
  GQDs/f-MWCNTs composite has important functional groups and conjugation. Synergistic effect between GQDs and f-MWCNTs facilitates the electron transfer on GCE. ⁴¹ Hence, the highest sensitivity was achieved for IL-6 protein detection by GQDs/f-MWCNTs/GCE.

Irreversible peaks at about +0.70 V belonging mixture containing pyrrole and IL-6 protein diminished gradually (Fig. 7A). Especially, the irreversible peaks disappeared at 15th cycle on GQDs/f-MWCNTs/GCE. These disappearing peaks indicated the formation of IL-6 imprinted polymer layer on electrode surface. Differential pulse voltammograms (DPVs) were also recorded to show imprinting selectivity. As seen on curve a of Fig. 7B, any current signal was not observed in only 0.1 mol l⁻¹ PBS (pH 7.4). However, In presence of 0.2 pg ml⁻¹ IL-6 protein, MIP/GQDs/f-MWCNTs/GCE showed the clear current signal at +0.20 V in (curve c of Fig. 7B). Finally, NIP/GQDs/f-MWCNTs/GCE showed the smaller current signal (curve b of Fig. 7B). Hence, we can indicate that molecularly imprinting technique can provide obvious sensitivity and selectivity in sensor studies. Also, the effect of nanomaterials was investigated on signal of molecularly imprinting sensor. The highest catalytic effect was observed by using MIP/GQDs/f-MWCNTs/GCE (curve a of Fig. 7C) in comparison with MIP/GCE (curve c of Fig. 7C) and MIP/GQDs/GCE (curve b of Fig. 7C).

Zoom In Zoom Out Reset image size

Mole ratio IL-6 molecule to pyrrole effect

Firstly, mole ratio IL-6 protein to pyrrole effect was investigated on signal of MIP/GQDs/f-MWCNTs/GCE (Fig. 8A). When 100.0 mM pyrrole and 25.0 mM IL-6 protein were used for biosensor preparation, the optimal biosensor signals were observed. However, non-specific interactions between pyrrole and IL-6 protein were observed at higher concentrations of monomer ratios and the optimal biosensor signals deformed. Thus, 25.0 mM: 100.0 mM (1:4) was chosen as ideal ratio.

Zoom In Zoom Out Reset image size

Removal (regeneration) time effect

Scan cycle effect

pH effect

Linearity range

The linearity and correlation between MIP/GQDs/f-MWCNTs/GCE and IL-6 protein was shown on Fig. 9. Inset of Fig. 9 showed the calibration equation "y (μA) = 50.029× (pg ml⁻¹) −0.1701." 0.010 pg ml⁻¹ and 0.0030 pg ml⁻¹ were found as quantification limit (LOQ) and LOD, respectively. LOQ and LOD were calculated by Eqs. 1 and 2:

$$\begin{eqnarray}&&LOQ=10.0\,{\rm{S}}/{\rm{m}}\end{eqnarray} \tag{ 1 }$$

$$\begin{eqnarray}&&LOD=3.3\,{\rm{S}}/{\rm{m}}\end{eqnarray} \tag{ 2 }$$

Where S is standard deviation of intercept and m is slope of calibration equation. When we consider linearity range and LOD of the developed biosensor in this study, some comparisons are performed according to other techniques in literature (Table I). Firstly, the MIP/GQDs/f-MWCNTs/GCE is the more sensitive than other biosensors. In addition, MIP/GQDs/f-MWCNTs/GCE is simple, fast, selective and stable biosensor. Finally, when GQDs/f-MWCNTs composite was prepared, a small amount of chemicals was used. Thus, we can say that environmentally friendly biosensor was presented in the literature. The developed biosensor, which is alternative to the other biosensors, can be used for clinical applications.

Zoom In Zoom Out Reset image size

Table I. Comparison of the biosensor with other methods.

Biosensor/Method	Linear Range	LOD	References
Impedimetric	5.00–100.0 pg ml−1	1.60 pg ml−1	52
Impedimetric	1.0 pg ml−1–15.0 μg ml−1	0.33 pg ml−1	53
DPV	0.0−100.0 pg ml−1	1.0 pg ml−1	54
AgNP–TiP	0.0005−10.0 ng ml−1	0.1 pg ml−1	55
SERS	1.0 pg ml−1−1.0 μg ml−1	1.0 pg ml−1	56
LFIA	2.0−500.0 pg ml−1	0.37 pg ml−1	57
Aptamer-coated AuNPs	3.3−125.0 μg ml−1	1.95 μg ml−1	58
GO thin film	1.0−300.0 pg ml−1	1.0 pg ml−1	59
MIP/GQDs/f-MWCNTs/GCE	0.01−2.0 pg ml−1	0.0030 pg ml−1	This study

AgNP–TiP: Silver nanoparticle–hollow titanium phosphate sphere; SERS: Surface-enhanced Raman scattering; LFIA: lateral flow immunoassay; AuNPs: Gold nanoparticles; GO: Graphene oxide.

Precision and accuracy

Recovery

Selectivity

IL-6 is a member of pluripotent cytokines including in interferons, interleukins, lymphokines and tumor necrosis factors. Proinflammatory cytokines greatly stimulate MUC1, MUC4 and MUC16 expressions in various cellular contexts. Common protein interferences (IgG, BSA and CEA) were also selected. Some of these proteins are present in the blood (even in nonpathological conditions). In this work, prepared eight different solutions (0.20 pg ml⁻¹ IL-6, TNF-α, IgG, BSA, MUC1, MUC4, MUC16 and CEA in presence of 0.1 mol l⁻¹ PBS, pH 7.4) were prepared to evaluate the selectivity of the MIP/GQDs/f-MWCNTs/GCE. According to Fig. 10A, the highest sensor signals (ΔI = 10.00 μA) were observed towards 0.20 pg ml⁻¹ IL-6. On the other hand, MIP/GQDs/f-MWCNTs/GCE demonstrated low non-specific sensor signals towards TNF-α, IgG, BSA, MUC1, MUC4, MUC16 and CEA. The results depicted that the MIP/GQDs/f-MWCNTs/GCE was 3.33, 4.00, 5.00, 6.67, 10.00, 20.00 and 40.00 times selective towards IL-6 in comparison with TNF-α, IgG, BSA, MUC1, MUC4, MUC16 and CEA (Table V). In addition, the specificity of imprinting was investigated by using NIP/GQDs/f-MWCNTs/GCE (Fig. 10B). The selectivity coefficients for NIP/GQDs/f-MWCNTs/GCE in respect to TNF-α, IgG, BSA, MUC1, MUC4, MUC16 and CEA were obtained to be 2.67, 3.33, 4.00, 5.00, 6.67, 10.00 and 20.00, respectively. According to imprinting factors (k'), IL-6 imprinted biosensor can recognize IL-6 protein 1.25 times more selective than TNF-α, 1.20 times more selective than IgG, 1.25 times more selective than BSA, 1.33 times more selective than MUC1, 1.50 times more selective than MUC4, 2.00 times more selective than MUC16 and 2.00 times more selective than CEA (Table V). As seen from the results, molecular imprinting process brought high selectivity to IL-6 imprinted biosensor.

Zoom In Zoom Out Reset image size

Table V. The selectivity coefficients (k) and relative selectivity coefficients (k') values of MIP/GQDs/f-MWCNTs/GCE and NIP/GQDs/f-MWCNTs/GCE.

	MIP		NIP
	ΔI	k	ΔI	k	k'
IL-6	10.00	—	2.00	—	—
TNF-α	3.00	3.33	0.75	2.67	1.25
IgG	2.50	4.00	0.60	3.33	1.20
BSA	2.00	5.00	0.50	4.00	1.25
MUC1	1.50	6.67	0.40	5.00	1.33
MUC4	1.00	10.00	0.30	6.67	1.50
MUC16	0.50	20.00	0.20	10.00	2.00
CEA	0.25	40.00	0.10	20.00	2.00

k = ΔI_analyte/ΔI_interference and k' = k_MIP/k_NIP (k' was calculated to obtain the specificity of imprinting for developed biosensor). ⁶²

Stability

When one MIP/GQDs/f-MWCNTs/GCE was used for 60 d, DPVs and observed current signals were recorded (Fig. 11). According to DPVs, the current signals having about 10.0 μA (RSD: 0.16%) were obtained. Thus, the prepared MIP/GQDs/f-MWCNTs/GCE has good stability for clinical applications.

Zoom In Zoom Out Reset image size

Repeatability

Reproducibility

Reusability