A highly sensitive biosensor based on Au NPs/rGO-PAMAM-Fc nanomaterials for detection of cholesterol

Figures & data

Figure 1 Characterization of rGO-PAMAM-Fc nanomaterials.

Notes: (A) ¹H NMR spectrum of PAMAM. (B) FT-IR spectra of (a) rGO, (b) Fc, (c) PAMAM, (d) rGO-PAMAM-Fc nanomaterials. (C) UV–vis spectra of (a) rGO, (b) Fc, (c) PAMAM, (d) rGO-PAMAM-Fc nanomaterials. (D) Raman spectrum of (a) rGO, (b) rGO-PAMAM-Fc nanomaterials. (E) AFM of rGO-PAMAM-Fc nanomaterials. (F) Cyclic voltammogram response of various electrodes: (a) bare GE, (b) MEA/GE, (c) rGO-PAMAM-Fc/MEA/GE from −0.2 to 0.6 V with a scan rate of 100 mV/s in PBS containing 5 mM [Fe(CN)₆]^3−/4−.

Abbreviations: AFM, atomic force microscope; Fc, ferrocene; GE, gold electrode; NMR, nuclear magnetic resonance; NPs, nanoparticles; PAMAM, polyamidoamine; rGO, reduced graphene oxide; UV, ultraviolet.

Figure 2 Electrochemical behaviors of the biosensor.

Notes: (A) CV response of (a) bare GE, (b) MEA/GE, (c) rGO-PAMAM-Fc/MEA/GE, (d) (rGO-PAMAM-Fc)₂/MEA/GE, (e) (rGO-PAMAM-Fc)₃/MEA/GE, (f) (rGO-PAMAM-Fc)₄/MEA/GE, (g) (rGO-PAMAM-Fc)₅/MEA/GE, (h) Au NPs/(rGO-PAMAM-Fc)₅/MEA/GE, (i) ChOx&ChEt/Au NPs/(rGO-PAMAM-Fc)₅/MEA/GE in [Fe(CN)₆]^3−/4− solution from −0.4 to 0.8 V with a scanning rate of 100 mV/s. (B) EIS of (a) bare GE, (b) MEA/GE, (c) rGO-PAMAM-Fc/MEA/GE, (d) (rGO-PAMAM-Fc)₂/MEA/GE, (e) (rGO-PAMAM-Fc)₃/MEA/GE, (f) (rGO-PAMAM-Fc)₄/MEA/GE, (g) (rGO-PAMAM-Fc)₅/MEA/GE, (h) Au NPs/(rGO-PAMAM-Fc)₅/MEA/GE, (i) ChOx&ChEt/Au NPs/(rGO-PAMAM-Fc)₅/MEA/GE in PBS containing 5 mM Fe(CN)₆³⁻/⁴⁻ at 0.24 V (vs SCE) with a frequency range of 0.1–100 kHz. (C) Current response toward 5 µM cholesterol by LSV with the different modifications, including bare GE, ChOx&ChEt/rGO-PAMAM-Fc/GE, ChOx&ChEt/Au NPs/rGO-PAMAM/GE, ChOx&ChEt/Au NPs/PAMAM-Fc/GE, ChOx&ChEt/Au NPs/PAMAM/GE and ChOx&ChEt/Au NPs/(rGO-PAMAM-Fc)/GE. Error bars represent the standard error of the mean (n=3 electrodes).

Abbreviations: CV, cyclic voltammetry; EIS, electrochemical impedance spectroscopy; Fc, ferrocene; GE, gold electrode; LSV, linear sweep voltammetry; NPs, nanoparticles; PAMAM, polyamidoamine; rGO, reduced graphene oxide; SCE, saturated calomel electrode.

Figure 3 Characterization of the ChOx&ChEt/Au NPs/rGO-PAMAM-Fc biosensor.

Notes: SEM images of (A) bare electrode, (B) rGO-PAMAM-Fc/GE, (C) Au NPs/rGO-PAMAM-Fc/GE, (D) ChOx&ChEt/Au NPs/rGO-PAMAM-Fc/GE, and (E) Ag/ChOx&ChEt/ Au NPs/rGO-PAMAM-Fc/GE. (F) EDS image of Ag/ChOx&ChEt/Au NPs/rGO-PAMAM-Fc/GE.

Abbreviations: EDS, energy dispersive spectrometer; Fc, ferrocene; GE, gold electrode; NPs, nanoparticles; PAMAM, polyamidoamine; rGO, reduced graphene oxide; SEM, scanning electron microscope.

Figure 4 Characterization of the modified electrodes.

Notes: (A) Chronocoulometric curves of (a) GE, (b) rGO-PAMAM-Fc/GE and (c) Au NPs/rGO-PAMAM-Fc/GE for the reduction of 5 mM K₃[Fe(CN)₆] in 0.1 mol/LPBS. (B) Raman spectrum of (a) rGO/GE, (b) ChOx&ChEt/Au NPs/rGO-PAMAM-Fc/GE. (C) CV for ChOx&ChEt/Au NPs/rGO-PAMAM-Fc/GE in PBS containing 5 mM Fe(CN)₆³⁻/⁴⁻ (scan rate: 10–500 mV/s, scan range: −0.2–0.6 V). (D) The relationship between the square root of the scan rate and the peak current curves, I_pa and I_pc.

Abbreviations: CV, cyclic voltammetry; Fc, ferrocene; GE, gold electrode; NPs, nanoparticles; PAMAM, polyamidoamine; rGO, reduced graphene oxide.

Figure 5 Optimization of the determining conditions.

Notes: (A) Optimization of the number of layers of rGO-PAMAM-Fc nanomaterial film on GE. (B) Effect of different molecular generations of PAMAM in rGO-PAMAM-Fc nanoparticles toward cholesterol. (C) Effect of temperature of deposition reaction on the current response to cholesterol. (D) Effect of electrodeposition time of Au NPs on the modified electrode. (E) Effect of glycine buffer pH on the current response to cholesterol. (F) Effect of concentration of Ag⁺ in the glycine buffer. (G) Effect of time of deposition reaction on the current response to cholesterol. The current response was recorded using LSV measurements in 0.1 mol/L HNO₃ solution containing 0.6 mol/L KNO₃ from −0.2 to 0.6 V at 0.1 V/s scanning rate. Error bars are the standard error of the mean (n=3 electrodes).

Abbreviations: Fc, ferrocene; GE, gold electrode; LSV, linear sweep voltammetry; NPs, nanoparticles; PAMAM, polyamidoamine; rGO, reduced graphene oxide.

Table 1 Comparison of some characteristics of different electrodes for the determination of cholesterol

Nanomaterial	Sensing element	Linear range (mM)	LOD (μM)	Reference
nNi0-MWCNT/ITO	ChOx	0.25–12.93	30	Ali et al (2013)^Citation40
PANI-MWCNT	ChOx	1.29–12.93		Dhand et al (2008)^Citation41
Nano-Pt/MWCNT-PANI	ChOx	0.002–0.5	0.8	Xu et al (2016)^Citation36
Au NPs/PEDOT-PSS/SPCE	ChOx	0.1–6	90	Phongphut et al (2013)^Citation52
MWNT(SH)-Au/Chi-IL	ChOx	0.5–5.0		Gopalan et al (2009)^Citation42
Au nanowire	ChOx, ChEt	0.01–0.06		Aravamudhan et al (2007)^Citation5
SnO2 NPs	ChOx	0.26–10.36	130	Ansari et al (2009)^Citation43
Au NPs-MWCNTs	ChOx	0.01–5.0	4.3	Zhu et al (2013)^Citation44
G/Ti(G)-3DNS/CS	ChOx	0.05–8.0	6	Komathi et al (2016)^Citation1
Pt/TMOS sol–gel/p (DB)	ChOx	0.06–3.0		Ding et al (2014)^Citation45
PSBTz/β-CD	ChOx	150–22,500		Soylemez et al (2014)^Citation46
Graphene oxide	ChOx	0.0005–0.0465		Dey and Raj (2013)^Citation47
Cu2O NPs	ChOx, ChEt	0.259–11.64		Singh et al (2013)^Citation48
ZnO-CuO/ITO	ChOx, ChEt	0.5–12	500	Batra et al (2015)^Citation49
CHIT/CeO2-NG	ChOx	0.004–5	133	Du et al (2017)^Citation50
NiONPs-CHIT/ZnO-ZnHCF/Au	ChOx	0.56–7.9	110	Narang et al (2013)^Citation51
Au NPs/rGO-PAMAM-Fc	ChOx, ChEt	0.0004–15.36	0.01	This work

Abbreviations: Fc, ferrocene; GE, gold electrode; LOD, limit of detection; NPs, nanoparticles; PAMAM, polyamidoamine; rGO, reduced graphene oxide; PEDOT, poly(3,4-ethylenedioxythiophene); ITO, indium tin oxide; PANI, Polyaniline; PEDOT-PSS, poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid; SPCE, screen-printed carbon electrode; 3DNS, 3Dnanostacks; CS, chitosan; TMOS, tetramethoxysilane; DB, diaminobenzene; PSBTz, poly(2-(2-octyldodecyl)-4,7-di(selenoph-2-yl)-2H-benzo triazole); β-CD, β-cyclodextrin; CHIT, chitosan; NG, grapheme.

Figure 6 Performance of the biosensor toward cholesterol.

Notes: (A) Current response of the ChOx&ChEt/Au NPs/rGO-PAMAM-Fc biosensor toward cholesterol concentration ranging from 1.0×10⁻⁷ to 15.36 mM. (B) Linear relation between current response and logarithm of cholesterol concentration. The current response was recorded using LSV measurements in 0.1 mol/L HNO₃ solution containing 0.6 mol/L KNO₃ from −0.2 to 0.6 V at a scanning rate of 0.1 V/s. Error bars are the standard error of the mean (n=3 electrodes). (C) The LSV measurement curve toward different concentration of cholesterol.

Abbreviations: Fc, ferrocene; GE, gold electrode; LSV, linear sweep voltammetry; NPs, nanoparticles; PAMAM, polyamidoamine; rGO, reduced graphene oxide.

Figure 7 Current of the biosensor in the presence of cholesterol and some interfering substances. Error bars are the standard error of the mean (n=3 electrodes). P>0.05.

Scheme 1 Principle of ChOx&ChEt/Au NPs/rGO-PAMAM-Fc biosensor detecting cholesterol.

Abbreviations: Fc, ferrocene; LSV, linear sweep voltammetry; NPs, nanoparticles; PAMAM, polyamidoamine; rGO, reduced graphene oxide.

Table 2 Determination and recovery of cholesterol in human serum samples

Sample	Added (μM)	Found (μM)	Recovery (%)	RSD (%)
	720.04	706.09	98.06
Human serum	408.28	415.57	101.79
	266.87	282.41	105.82	4.35
	1,235.70	1,173.67	94.98
	622.73	645.34	103.63

Note: Error bars are the standard error of the mean (n=3 electrodes).

Abbreviation: RSD, relative SD.

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