Electrochemical DNA Biosensor Based on Mercaptopropionic Acid-Capped ZnS Quantum Dots for Determination of the Gender of Arowana Fish

Abstract

A new electrochemical DNA biosensor based on mercaptopropionic acid (MPA)-capped ZnS quantum dots (MPA-ZnS QDs) immobilization matrix for covalent binding with 20-base aminated oligonucleotide has been successfully developed. Prior to the modification, screen-printed carbon paste electrode (SPE) was self-assembled with multilayer gold nanoparticles (AuNPs) and cysteamine (Cys). The inclusion of MPA-ZnS QDs semiconducting material in modified electrodes has enhanced the electron transfer between the SPE transducer and DNA leading to improved bioanalytical assay of target biomolecules. Electrochemical studies performed by cyclic voltammetry (CV) and differential pulsed voltammetry (DPV) demonstrated that the MPA-ZnS QDs modified AuNPs electrode was able to produce a lower charge transfer resistance response and hence higher electrical current response. Under optimal conditions, the immobilized synthetic DNA probe exhibited high selectivity towards synthetic target DNA. Based on the DPV response of the reduction of anthraquinone monosulphonic acid (AQMS) redox probe, the MPA-ZnS QDs-based electrochemical DNA biosensor responded to target DNA concentration from 1 × 10^-9 μM to 1 × 10^-3 μM with a sensitivity 1.2884 ± 0.12 µA, linear correlation coefficient (R²) of 0.9848 and limit of detection (LOD) of 1 × 10^-11 μM target DNA. The DNA biosensor exhibited satisfactory reproducibility with an average relative standard deviation (RSD) of 7.4%. The proposed electrochemical transducer substrate has been employed to immobilize the aminated Arowana fish (Scleropages formosus) DNA probe. The DNA biosensor showed linearity to target DNA from 1 × 10^-11 to 1 × 10^-6 µM (R² = 0.9785) with sensitivity 1.1251 ± 0.243 µA and LOD of 1 × 10^-11 µM. The biosensor has been successfully used to determine the gender of Arowana fish without incorporating toxic raw materials previously employed in the hazardous processing conditions of polypyrrole chemical conducting polymer, whereby the cleaning step becomes difficult with thicker films due to high levels of toxic residues from the decrease in polymerization efficacy as films grew.

Keywords: DNA biosensor; ZnS QDs; electrochemistry; gold nanoparticles; screen printed electrode.

Figure 1

The stepwise construction of the electrochemical DNA biosensor based on multilayer AuNPs, cysteamine linkers and MPA-capped ZnS QDs semiconducting nanoparticles.

Figure 2

(a) The electron transfer rates estimated from the Randles–Sevcik equation for bare SPE and SPEs modified with AuNPs, cysteamine linkers, ZnS QDs semiconducting nanoparticles and DNA molecules by using 1 mM AQMS redox indicator. (b) The sensitivity dependent AQMS i_pc response at each extended layer on the SPE at scan rate range between 30 mV s⁻¹ and 300 mV s⁻¹ in 0.01 M PBS at pH 7 containing 1 mM AQMS and 0.1 M KCl. (c) The DPV response of SPE after every modification with AuNPs, cysteamine, MPA-ZnS QDs, DNA probe, target DNA and non-complementary DNA and 1 h AQMS intercalation duration. The DPV measurement was conducted in 0.01 M PBS solution containing 0.1 M KCl at a scan rate of 100 mV s⁻¹.

Figure 3

(a) The cyclic voltammograms of AuNPs-SPEs with different amounts of immobilized AuNPs. The CV measurement was done in 0.1 M H₂SO₄ at a scan rate of 100 mV s⁻¹. (b) The differential pulse voltammograms of AuNPs-SPEs with various AuNPs loadings after exposure to 1 mM AQMS for 1 h. The DPV measurement was conducted in 0.01 M PBS (pH 7) containing 0.1 M KCl at a scan rate of 100 mV s⁻¹. (c) The DPV responses of MPA-ZnS QDs/AuNPs-SPE and DNA biosensor at different immobilized MPA-ZnS QDs amounts. The DPV measurements were carried out in 0.01 M PBS (pH 7) containing 0.1 M KCl and 1 mM AQMS for MPA-ZnS QDs/AuNPs-SPE and 0.01 M PBS (pH 7) containing 0.1 M KCl for DNA biosensor at the scan rate of 100 mV s⁻¹.

Figure 4

(a) The DNA biosensor response trend for a DNA hybridization period of 30–150 min by using 5 µM DNA probe and 5 µM target DNA. (b) The DNA biosensor response after exposure to 1 mM AQMS label from 15–75 min in 0.01 M PBS (pH 7). The DPV measurement was conducted in 0.01 M PBS (pH 7) containing 0.1 M KCl at the scan rate of 100 mV s⁻¹. The rehybridization profiles of the electrochemical DNA biosensor by using (c) temperature effect at 63.3, 68.3 and 78.3 °C and (d) NaOH regeneration solution at 0.01 M, 0.001 M and 0.0001 M.

Figure 5

(a) The DPV response trend of electrochemical DNA biosensor incorporated with various concentrations of cyteamine from 0.00-0.05 M. The DPV experiment was performed in 0.01 M PBS (pH 7) containing 0.1 M KCl at the scan rate of 100 mV s⁻¹. (b) Effect of different DNA probe concentrations from 0.5 µM to 6.0 µM on the detection of 5 µM target DNA. The DPV scanning was conducted in 0.01 M PBS containing 0.1 M KCl at pH 7 and scan rate of 100 mV s⁻¹ after the DNA biosensor was immersed in 1 mM AQMS for 1 h.

Figure 6

(a) Effects of pH from pH 5 to pH 10 and (b) PBS concentration from 0.5 mM to 50 mM containing 0.1 M KCl on the DNA biosensor response. The DPV response was recorded using a scan rate of 100 mV s⁻¹.

Figure 7

(a) The DPV current response of the DNA electrode towards the detection of 1 × 10⁻⁶ µM target DNA for seven weeks under the optimal conditions. (b) The DNA biosensor response towards 5 µM target DNA, mismatch DNA and non-complementary DNA with 3 h DNA hybridization time and 1 h AQMS intercalation duration. The DPV measurement was conducted in 0.01 M PBS (pH 7) containing 0.1 M KCl at the scan rate of 100 mV s⁻¹.

Figure 8

(a) DPV response of the electrochemical DNA biosensor at different target DNA concentrations. (b) The response curve of the electrochemical DNA biosensor established with different concentrations of target DNA from 1 × 10⁻¹⁰ µM to 1 × 10⁻¹ µM. The inset shows the linear calibration curve of the DNA biosensor from 1 × 10⁻⁹ µM to 1 × 10⁻³ µM target DNA.

Figure 9

DPV of electrochemical DNA biosensor toward Arowana DNA target concentrations of 1 × 10⁻¹³ to 1 × 10⁻⁶ µM. The biosensors were scanned in 10 mM PBS at pH 7 and 0.1 M KCl using a scan rate of 0.1 V s⁻¹.

Figure 10

Calibration curve of electrochemical DNA biosensor using 5 µM targets hybridized with different concentrations of target from 1 × 10⁻¹³ µM to 1 × 10⁻² µM. The biosensors were run in 10 mM PBS at pH 7 and 0.1 M KCl using a scan rate of 0.1 V s⁻¹.