Meng Du

Immobilization-free direct electrochemical detection for DNA specific sequences based on electrochemically converted gold nanoparticles/graphene composite film

A direct electrochemical DNA sensor was constructed based on gold nanoparticles/graphene film. A precursor graphene film was fabricated on glassy carbon electrode (GCE) using both electrochemically reduced graphene oxide (ERGNO) and chemically reduced graphene oxide (CRGNO). The electrochemical approach was green and fast, and unlike chemical reduction, does not result in contamination of the reduced material, and at highly negative potential could reduce the oxygen functionalities (–OH, C–O–C and –COOH) of the graphene oxide more efficiently. ERGNO exhibited better electrochemical and electrocatalytic performances than CRGNO. Gold nanoparticles (AuNPs) were electrodeposited on the ERGNO/GCE to amplify the electrochemical signals. The resulting AuNPs/ERGNO composite film was characterized by scanning electron microscopy, energy dispersive spectroscopy and Raman spectroscopy. The electrochemical responses of guanine (G), adenine (A), thymine (T) and cytosine (C) were investigated at AuNPs/ERGNO/GCE, which showed more favorable electron transfer kinetics than at ERGNO/GCE, demonstrating the significantly synergistic electrocatalytic effect of ERGNO and AuNPs. Synthetic sequence-specific DNA oligonucleotides was successfully detected and the established immobilization-free biosensor had the ability to discriminate single- or double-base mismatched DNA.

Highly sensitive electrochemical impedance spectroscopic detection of DNA hybridization based on Aunano–CNT/PANnano films

A polyaniline nanofibers (PAN(nano))/carbon paste electrode (CPE) was prepared via dopping PAN(nano) in the carbon paste. The nanogold (Au(nano)) and carbon nanotubes (CNT) composite nanoparticles were bound on the surface of the PAN(nano)/CPE. The immobilization and hybridization of the DNA probe on the Au(nano)-CNT/PAN(nano) films were investigated with differential pulse voltammetry (DPV) and cyclic voltammetry (CV) using methylene blue (MB) as indicator, and electrochemical impedance spectroscopy (EIS) using [Fe(CN)(6)](3-/4-) as redox probe. The voltammetric peak currents of MB increased dramatically owing to the immobilization of the probe DNA on the Au(nano)-CNT/PAN(nano) films, and then decreased obviously owing to the hybridization of the DNA probe with the complementary single-stranded DNA (cDNA). The electron transfer resistance (R(et)) of the electrode surface increased after the immobilization of the probe DNA on the Au(nano)-CNT/PAN(nano) films and rose further after the hybridization of the probe DNA. The remarkable difference between the R(et) value at the DNA-immobilized electrode and that at the hybridized electrode could be used for the label-free EIS detection of the target DNA. The loading of the DNA probe on Au(nano)-CNT/PAN(nano) films was greatly enhanced and the sensitivity for the target DNA detection was markedly improved. The sequence-specific DNA of phosphinothricin acetyltransferase (PAT) gene and the polymerase chain reaction (PCR) amplification of nopaline synthase (NOS) gene from transgenically modified beans were determined with this label-free EIS DNA detection method. The dynamic range for detecting the PAT gene sequence was from 1.0 x 10(-12)mol/L to 1.0 x 10(-6)mol/L with a detection limit of 5.6 x 10(-13)mol/L.

Fabrication of DNA/graphene/polyaniline nanocomplex for label-free voltammetric detection of DNA hybridization

A novel DNA electrochemical biosensor was described for the detection of specific gene sequences. Electrochemically reduced graphene oxide (ERGNO) was prepared on polyaniline (PAN) nanofibers modified glassy carbon electrode (GCE). Compared with the electrochemical reduction of graphene oxide directly on bare GCE (reduction potential: ca. -1.3V), more positive reduction potential (ca. -1V) for graphene oxide was observed with the PAN membrane existing. The formed ERGNO/PAN nanocomposites were applied to bind ssDNA probe via the non-covalent assembly. The surface density of ssDNA was calculated by voltammetric studies of redox cations ([Ru(NH(3))(6)](3+)), which were bound to the surface via electrostatic interaction with negative charged phosphate backbone of the DNA. After the hybridization of ssDNA probe with complementary DNA, the response of surface-bound [Ru(NH(3))(6)](3+) changed obviously, which could been adopted to recognize the DNA hybridization. Under optimal conditions, the dynamic range of the DNA biosensor for detecting the sequence-specific DNA of cauliflower mosaic virus (CaMV35S) gene was from 1.0×10(-13) to 1.0×10(-7)molL(-1), with a detection limit of 3.2×10(-14)molL(-1). This biosensor also showed a high degree of selectivity.

Freely switchable impedimetric detection of target gene sequence based on synergistic effect of ERGNO/PANInanocomposites

An impedimetric and freely switchable DNA sensor based on electrochemically reduced graphene oxide (ERGNO) and polyaniline (PANI) film was presented, where ERGNO was prepared on PANI modified glassy carbon electrode (GCE). When the probe DNA was noncovalently assembled on the surface of electrode through π-π* stacking between the ring of nucleobases and the rich-conjugated structure of the nanocomposite, the electron transfer resistance value of [Fe(CN)₆]³⁻/⁴⁻ increased. The negative ssDNA and the steric hindrance blocked the effective electron transfer channel of the [Fe(CN)₆]³⁻/⁴⁻. After hybridization with the complementary DNA, the formation of helix induced dsDNA to release from the surface of conjugated nanocomposite, accompanied with the curtailment of the impedimetric value. The selectivity and sensitivity of this DNA sensing platform were characterized using electrochemical impedance spectroscopy in detail. The fabricated biosensor exhibited excellent performance for the detection of specific DNA sequence with a wide linear range (1.0×10⁻¹⁵ to 1.0×10⁻⁸ mol/L) and a low detection limit of 2.5×10⁻¹⁶ mol/L due to the synergistic effect of ERGNO/PANI nanocomposites. The hosphinothricin acetyltransferase gene (PAT) was also detected to show the switchable ability of ERGNO/PANI.

Ionic liquid-functionalized graphene as modifier for electrochemical and electrocatalytic improvement: Comparison of different carbon electrodes

Electrochemical activities of typically electrochemical targets at three kinds of modified carbon electrodes, i.e. carbon ionic liquid electrode (CILE), graphene/carbon paste electrode (CPE), and ionic liquid-functionalized graphene (IL-graphene)/CPE, were compared in detail. The redox processes of the probes at IL-graphene/CPE were faster than those at CILE and graphene/CPE from cyclic voltammetry. An electrochemical method for the simultaneous determination of guanine and adenine was described with detection limits of 6.5×10(-8) mol L(-1) (guanine) and 3.2×10(-8) mol L(-1) (adenine). Single A→G mutation of sequence-specific DNA could be discriminated by the IL-graphene/CPE.

Electrochemical synthesis of Fe2O3 on graphene matrix for indicator-free impedimetric aptasensing.

Herein, an electrochemical platform was employed for the detection of protein. Fe2O3 was electrochemically deposited on graphene modified glassy carbon electrode surface. Electrodeposition conditions, such as temperature, and time, were optimized for controlling morphologies and electrochemical activities of Fe2O3. Negatively charged lysozyme-binding aptamer (LBA) was immobilized on positively charged Fe2O3 (isoelectric point ≈ 7.0) via electrostatic interaction. Electrochemical impedance spectroscopy was adopted for indicator-free detection of lysozyme. The LBA on the outermost layer would catch lysozyme in solution by physical affinity, which induced the increase of impedimetric signals. In this strategy, a wide detection range (0.5 ng mL(-1)-5 μg mL(-1)) and low detection limit (0.16 ng mL(-1)) for model target lysozyme was obtained. The results showed that indicator-free impedimetric aptasensing strategy had good sensitivity and selectivity.

Rapid DNA electrochemical biosensing platform for label-free potentiometric detection of DNA hybridization

This paper described a novel electrochemical DNA biosensor for rapid specific detection of nucleic acids based on the sulfonated polyaniline (SPAN) nanofibre and cysteamine-capped gold nanoparticle (CA-G(NP)) layer-by-layer films. A precursor film of 3-mercaptopropionic acid (MPA) was firstly self-assembled on the Au electrode surface. CA-G(NP) was covalently deposited on the Au/MPA electrode to obtain a stable substrate. SPAN nanofibre and CA-G(NP) were alternately layer-by-layer assembled on the stable substrate by electrostatic force. Cyclic voltammetry was used to monitor the consecutive growth of the multilayer films by utilizing [Fe(CN)(6)](3-/4-) as the redox indicator. The (CA-G(NP)/SPAN)(n) films showed satisfactory ability of electron transfer and excellent redox activity in neutral media. Negatively charged probe ssDNA was immobilized on the outer layer of the multilayer film (CA-G(NP)) through electrostatic affinity. Chronopotentiometry and electrochemical impedance spectroscopy were employed to obtain the direct electrochemical readout for probe ssDNA immobilization and hybridization using [Fe(CN)(6)](3-/4-) in solution as the mediator. While electrochemical impedance spectroscopy led to the characterization of the electron-transfer resistance at the electrode, chronopotentiometry provided the total resistance at the interfaces of the modified electrodes. A good correlation between the total electrode resistances and the electron-transfer resistances at the conducting supports was found. Chronopotentiometry was suggested as a rapid transduction means (a few seconds). Based on the (CA-G(NP)/SPAN)(n) films, the target DNA with 20-base could be detected up to 2.13x10(-13)mol/L, and the feasibility for the detection of base-mismatched DNA was also demonstrated.

Al 3+ /graphene composites for electrochemical detection of DNA cleavage

Herein, we present the electrochemical co-deposition of Al3+/graphene composites directly from an aqueous mixture containing graphene oxide (GO) and Al3+. The obtained Al3+/graphene composites with good electrochemical activity were regarded as an appropriate immobilization platform for double-stranded DNA (dsDNA). The nontoxic redox probe xanthurenic acid (XA) was successfully applied to recognize single-stranded DNA and dsDNA. We illustrated that the scission of dsDNA caused by GO combining with some metal ions could be detected by monitoring the electrochemical signals of XA.