Chengxiao Zhang

Label-free and sensitive faradic impedance aptasensor for the determination of lysozyme based on target-induced aptamer displacement

A label-free and sensitive faradic impedance spectroscopy (FIS) aptasensor based on target-induced aptamer displacement was developed for the determination of lysozyme as a model system. The aptasensor was fabricated by self-assembling the partial complementary single strand DNA (pcDNA)-lysozyme binding aptamer (LBA) duplex on the surface of a gold electrode. To measure lysozyme, the change in interfacial electron transfer resistance of the aptasensor using a redox couple of [Fe(CN)(6)](3-/4-) as the probe was monitored. The introduction of target lysozyme induced the displacement of the LBA from the pcDNA-LBA duplex on the electrode into the solution, decreasing the electron transfer resistance of the aptasensor. The decrease in the FIS signal is linear with the concentration of lysozyme in the range from 0.2 nM to 4.0 nM, with a detection limit of 0.07 nM. The fabricated aptasensor shows a high sensitivity, good selectivity and satisfactory regeneration. This work demonstrates that a high sensitivity of the fabricated aptasensor can be obtained using a relatively short pcDNA. This work also demonstrates that the target-induced aptamer displacement strategy is promising in the design of an electrochemical aptasensor for the determination of lysozyme with good selectivity and high sensitivity.

Electrochemical detection of DNA hybridization based on polypyrrole/ss-DNA/multi-wall carbon nanotubes paste electrode

A sensitive electrochemical detection of DNA hybridization using a paste electrode assembled by multi-wall carbon nanotubes (MWNT) and immobilizing DNA probe within electropolymerized polypyrrole (ppy) was developed. The detection approach relied on entrapping of DNA probe within electropolymerized ppy film on the MWNT paste electrode and monitoring the current change generated from an electroactive intercalator of ethidium bromide (EB) after DNA hybridization. As a consequence of DNA hybridization, significant changes in the current of EB intercalated with double-stranded DNA (ds-DNA) on the MWNT paste electrode were observed. Based on the response of EB, only the complementary DNA sequence gave an obvious current signal compared with the five-point mismatched and non-complementary sequences. The oxidation peak current was linearly related to the logarithm of the concentration of the complementary DNA sequence from 1.0x10(-10) to 1.0x10(-8)M with a detection limit of 8.5x10(-11)M. This work demonstrates that the incorporation of MWNT paste electrode with electropolymerization is a promising strategy of functional interfaces for the immobilization of biological recognition elements.

Ultrasensitive electrogenerated chemiluminescence detection of DNA hybridization using carbon-nanotubes loaded with tris(2,2′-bipyridyl) ruthenium derivative tags

An ultrasensitive electrogenerated chemiluminescence (ECL) detection method of DNA hybridization based on single-walled carbon-nanotubes (SWNT) carrying a large number of ruthenium complex tags was developed. The probe single strand DNA (ss-DNA) and ruthenium complex were loaded at SWNT, which was taken as an ECL probe. When the capture ss-DNA with a thiol group was self-assembled onto the surface of gold electrode, and then hybridized with target ss-DNA and further hybridized with the ECL probe to form DNA sandwich conjugate, a strong ECL response was electrochemically generated. The ECL intensity was linearly related to the concentration of perfect-matched target ss-DNA in the range from 2.4x10(-14) to 1.7x10(-12)M with a detection limit of 9.0x10(-15)M. The ECL signal difference permitted to discriminate the perfect-matched target ss-DNA and two-base-mismatched ss-DNA. This work demonstrates that SWNT can provide an amplification platform for carrying a large number of ECL probe and thus resulting in an ultrasensitive ECL detection of DNA hybridization.

Label-free and sensitive electrogenerated chemiluminescence aptasensor for the determination of lysozyme.

A novel label-free electrogenerated chemiluminescence (ECL) aptasensor for the determination of lysozyme is designed employing lysozyme binding aptamer (LBA) as molecular recognition element for lysozyme as a model analyte and Ru(bpy)(3)(2+) as an ECL signal compound. This ECL aptasensor was fabricated by self-assembling the thiolated LBA onto the surface of a gold electrode. Using this aptasensor, sensitive quantitative detection of lysozyme is realized on basis of the competition of lysozyme with Ru(bpy)(3)(2+) cation for the binding sites of LBA. In the presence of lysozyme, the aptamer sequence prefers to form the LBA-lysozyme complex, the less negative environment allows Ru(bpy)(3)(2+) cations to be less bound electrostatically to the LBAs on the electrode surface, in conjunction with the generation of a decreased ECL signal. The integrated ECL intensity versus the concentration of lysozyme was linear in the range from 6.4×10(-10) M to 6.4×10(-7) M. The detection limit was 1.2×10(-10) M. This work demonstrates that using the competition of target protein with an ECL signal compound Ru(bpy)(3)(2+) for binding sites of special aptamer confined on the electrode is promising approach for the design of label-free ECL aptasensors for the determination of proteins.

Sensitive detection of endonuclease activity and inhibition using gold nanorods.

It is important to develop reliable and sensitive methods for assay of nuclease activity. With this goal in mind, we report a new strategy for nuclease assay by taking advantage of efficient fluorescence resonance energy transfer (FRET) between gold nanorods (GNRs) and fluorescein-tagged single-stranded DNA (FDNA). Upon mixing with GNRs, the FRET between positively charged GNRs and negatively charged FDNA caused a decrease in fluorescence of FDNA. The formation of FDNA/cDNA duplex further improved the FRET efficiency, leading to a significant decrease in fluorescence intensity. However, fluorescence is restored when FDNA1/cDNA1 hybrid was cleaved into small fragments by EcoRI endonucleases, resulting in a decrease in FRET efficiency because of weakened electrostatic interaction between GNRs and the shortened DNA fragments. Activity of EcoRI endonuclease has been real-time studied by monitoring fluorescence change with the prolonging of interaction time. Under optimized conditions, the cleaved fraction is linear with EcoRI concentration over the range of 1.0×10(-3) to 1.0×10(-1) U μL(-1), with a limit of detection of 6.5×10(-4) U μL(-1) which is much better or at least comparable to previous reports. Site-specific DNA cleavage by EcoRI endonuclease has also been verified by gel electrophoresis, fluorescence anisotropy and TEM analysis, which indicated that this method is a feasible and reasonable approach to study sequence-specific protein-DNA interactions. Assay of BamHI activity demonstrated that it is a more universally applied method for studying the activity of endonuclease. Furthermore, this fluorescence assay has been also used for studying the inhibition of EcoRI endonuclease activity. Importantly, experimental results suggested that endonuclease inhibitors can be screened by monitoring the change of fluorescence change. Therefore, this FRET assay is a simple, sensitive and effective approach to study endonuclease activity and inhibition, and as such, it promises to provide a feasible method to screen nuclease inhibitors.

Flow injection analytical system for glucose with screen-printed enzyme biosensor incorporating Os-complex mediator

A novel flow injection analytical system with a screen-printed enzyme sensor incorporating Os-complex mediator has been developed. A newly created glucose biosensor was fabricated using thick-film technique and co-immobilization of glucose oxidase and Os-complex on a permeable membrane. The composition of the enzyme and mediator in the membrane and the analytical conditions were optimized. The flow injection electrochemical system showed that glucose was detected linearly in the concentration range from 0.1 to 10 mM with a detection limit of 0.03 mM by 50 μl of sample injection, giving a throughout of about 40 samples per hour. The glucose biosensors retained their constant response after more than 100 injections and storage over a month. The designed electrochemical flow system with a disposable biosensor is suitable for automatic and rapid determination of glucose.

Single-labeled hairpin probe for highly specific and sensitive detection of lead(II) based on the fluorescence quenching of deoxyguanosine and G-quartet

Specific and homogeneous detection of heavy metal ion is of great importance for both human health care and environmental protection. We reported a highly specific and sensitive assay for fluorescent detection of Pb(2+) based on the difference in quenching ability between deoxyguanosines and G-quartet by using carboxyfluorescein-labeled hairpin DNA (F-hpDNA) as a recognition probe. In the absence of target, the fluorescence of F-hpDNA can be quenched through photoinduced electron transfer from the dye to deoxyguanosines because the formation of hairpin brings deoxyguanosines close to the FAM. In the presence of Pb(2+), the formation of G-quadruplex DNA leads to a significant decrease in fluorescence due to the effective stack of dye on the G-quartet, which obviously intensified the quenching of fluorophore. In comparison with linear DNA probe, hairpin DNA probe greatly improved the specificity, and Pb(2+) can be highly selective detected even when coexisted with other metal ions. The quenching efficiency is linear with the concentration of lead(II) over the range of 0.5-500 nM, with a limit of detection of 0.4 nM. Conformational switch from hairpin to G-quadruplex was verified by CD measurements. Moreover, the application for detection of real samples further demonstrated its reliability. Therefore, it is a selective, simple and sensitive approach for detection of lead ion, as such, it promises to provide a solid foundation for developing universal analytical method for heavy metal ions.

A biosensor prepared by co-entrapment of a glucose oxidase and a carbon nanotube within an electrochemically deposited redox polymer multilayer.

A glucose biosensor based on a nanocomposite made by layer-by-layer electrodeposition of the redox polymer into a multilayer containing glucose oxidase (GOx) and single-walled carbon nanotubes (SWCNT) on a screen-printed carbon electrode (SPCE) surface was developed. The objectives of the electrodeposition of redox polymer are to stabilize further the multilayer using a coordinative cross-linked redox polymer and to wire the GOx. The electrochemistry of the layer-by-layer assembly of the GOx/SWCNT/redox polymer nanocomposite was followed by cyclic voltammetry. The resultant biosensor provided stable and reproducible electrocatalytic responses to glucose, and the electrocatalytic current for glucose oxidation was enhanced with an increase in the number of layers. The biosensor displayed a linear range from 0.5 to 6.0mM, a sensitivity of 16.4μA/(mMcm(2)), and a response time of about 5s. It shows no response to 0.05mM of ascorbic acid, 0.32mM of uric acid and 0.20mM of acetaminophen using a Nafion membrane covering the nanocomposite-modified electrode surface.

Sensitive and antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample

A highly sensitive and attractive antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample was developed. The aptasensor was fabricated by co-assembling thiol-modified anti-thrombin binding aptamer, dithiothreitol and mercaptohexanol on the surface of gold electrode. The performance of aptasensor was characterized by atomic force microscopy, contact angle and electrochemical impedance spectroscopy. In the measurement of thrombin, the change in interfacial electron transfer resistance of aptasensor was monitored using a redox couple of Fe(CN)(6)(3-/4-). The increase in the electron transfer resistance was linearly proportional to the concentration of thrombin in the range from 1.0 to 20ng/mL and a detection limit of 0.3ng/mL thrombin was achieved. The fabricated aptasensor displayed attractive antifouling properties and allowed direct quantification of extrinsic thrombin down to 0.08ng/mL in undiluted serum sample. This work provides a promising strategy for clinical application with impressive sensitivity and antifouling characteristics.

Homogenous electrogenerated chemiluminescence immunoassay for human immunoglobulin G using N-(aminobutyl)-N-ethylisoluminol as luminescence label at gold nanoparticles modified paraffin-impregnated graphite electrode.

A homogeneous electrogenerated chemiluminescence (ECL) immunoassay for human immunoglobulin G (hIgG) has been developed using a N-(aminobutyl)-N-ethylisoluminol (ABEI) as luminescence label at gold nanoparticles modified paraffin-impregnated graphite electrode (PIGE). ECL emission was electrochemically generated from the ABEI-labeled anti-hIgG antibody and markedly increased in the presence of hIgG antigen due to forming a more rigid structure of the ABEI moiety. The concentration of hIgG antigen was determined by the increase of ECL intensity at a gold nanoparticles modified PIGE. It was found that the ECL intensity of ABEI in presence of hydrogen peroxide was dramatically enhanced at gold nanoparticles modified PIGE in neutral aqueous solution and the detection limit of ABEI was 2 x 10(-14)mol/L (S/N=3). The integral ECL intensity was linearly related to the concentration of hIgG antigen from 3.0 x 10(-11) to 1.0 x 10(-9)g/mL with a detection limit of 1 x 10(-11)g/mL (S/N=3). The relative standard deviation was 3.1% at 1.0 x 10(-10)g/mL (n=11). This work demonstrates that the enhancement of the sensitivity of ECL and ECL immunoassay at a nanoparticles modified electrode is a promising strategy.