Zhaoyang Wu

A novel approach of antibody immobilization based on n-butyl amine plasma-polymerized films for immunosensors

A novel method based on plasma-polymerized films (PPFs) is proposed for immobilizing antibodies (antigens) through a polyelectrolyte-mediated layer. The immobilization of goat-anti-IgG antibody, as an example, is investigated. The n-butyl amine PPFs are deposited on the surfaces of quartz crystal microbalance (QCM) with a radio frequency plasma method using n-butyl amine as the precursor, the IR spectrum indicating the existence of amino-groups in the film. After self-assembling a polystyrenesulfonate (PSS) layer on the PPF, the goat-anti-IgG antibody is immobilized in a 0.2 mg ml 1 of antibody immobilizing solution at pH 5.0. The QCM immunosensor can quantitatively determine NH IgG in the range of 0.7‐126m gm l 1 . Moreover, the PSS and protein layers can easily be removed simply by shifting the pH, making the immunosensor regenerable.

Acetylcholinesterase Liquid Crystal Biosensor Based on Modulated Growth of Gold Nanoparticles for Amplified Detection of Acetylcholine and Inhibitor

A novel acetylcholinesterase (AChE) liquid crystal (LC) biosensor based on enzymatic growth of gold nanoparticles (Au NPs) has been developed for amplified detection of acetylcholine (ACh) and AChE inhibitor. In this method, AChE mediates the hydrolysis of acetylthiocholine (ATCl) to form thiocholine, and the latter further reduces AuCl(4)(-) to Au NPs without Au nanoseeds. This process, termed biometallization, leads to a great enhancement in the optical signal of the LC biosensor due to the large size of Au NPs, which can greatly disrupt the orientational arrangement of LCs. On the other hand, the hydrolysis of ATCl is inhibited in the presence of ACh or organophosphate pesticides (OPs, a AChE inhibitor), which will decrease the catalytic growth of Au NPs and, as a result, reduce the orientational response of LCs. On the basis of such an inhibition mechanism, the AChE LC biosensor can be used as an effective way to realize the detection of ACh and AChE inhibitors. The results showed that the AChE LC biosensor was highly sensitive to ACh with a detection limit of 15 μmol/L and OPs with a detection limit of 0.3 nmol/L. This study provides a simple and sensitive AChE LC biosensing approach and offers effective signal enhanced strategies for the development of enzyme LC biosensors.

Label-Free Liquid Crystal Biosensor Based on Specific Oligonucleotide Probes for Heavy Metal Ions

In this study, to enhance the capability of metal ions disturbing the orientation of liquid crystals (LCs), we designed a new label-free LC biosensor for the highly selective and sensitive detection of heavy metal ions. This strategy makes use of the target-induced DNA conformational change to enhance the disruption of target molecules for the orientation of LC leading to an amplified optical signal. The Hg(2+) ion, which possesses a unique property to bind specifically to two DNA thymine (T) bases, is used as a model heavy metal ion. In the presence of Hg(2+), the specific oligonucleotide probes form a conformational reorganization of the oligonucleotide probes from hairpin structure to duplex-like complexes. The duplex-like complexes are then bound on the triethoxysilylbutyraldehyde/N,N-dimethyl-N-octadecyl (3-aminopropyl) trimethoxysilyl chloride (TEA/DMOAP)-coated substrate modified with capture probes, which can greatly distort the orientational profile of LC, making the optical image of LC cell birefringent as a result. The optical signal of LC sensor has a visible change at the Hg(2+) concentration of low to 0.1 nM, showing good detection sensitivity. The cost-effective LC sensing method can translate the concentration signal of heavy metal ions in solution into the presence of DNA duplexes and is expected to be a sensitive detection platform for heavy metal ions and other small molecule monitors.

A piezoelectric immunoagglutination assay for Toxoplasma gondii antibodies using gold nanoparticles

The serologic detection of anti-Toxoplasma gondii immunoglobulins plays a key role in the clinical diagnosis of Toxoplasmosis. In this paper, a simple, rapid and highly sensitive agglutination-based piezoelectric immunoassay has been firstly developed for directly detecting anti-T. gondii immunoglobulins in infected rabbit serum (IRS) and infected rabbit blood (IRB). The proposed technique is based on that the specific agglutination of antigen-coated gold nanoparticles, averaging 10nm in diameter, in the presence of the corresponding antibody causes a frequency change that is monitored by a piezoelectric device. In contrast to the commonly used piezoelectric assays, it possesses an attractive advantage in that the immobilization of antibody or antigen on the crystal is unnecessary. Use of a newly prepared sensing probe which was modified by a plasma-polymerized film (PPF) of n-butyl amine and further by a heparin layer resulted in a response-enhanced immunoagglutination and a high compatibility of the probe with biological samples. An appropriate reagent consisting of 1% normal rabbit serum (NRS) and 0.1% bovine serum albumin (BSA) for diluting the analytes were verified in counteracting the background interference of assay. Moreover, an optimization of assay medium composition with the addition of poly(ethylene glycol) (PEG) serving as immunoagglutination rate and sensitivity enhancer was investigated in detail. It is found that the developed immunoagglutination assay system is sensitive to dilution ratio of anti-T. gondii antibody as low as 1:5500. Analytical results of several specimens obtained using the developed technique are in satisfactory agreement with those given by the ELISA method, implying a promising alternative approach for detecting anti-T. gondii antibodies in the clinical diagnosis.

Oligonucleotide probes applied for sensitive enzyme-amplified electrochemical assay of mercury(II) ions

We developed a novel electrochemical sensor for Hg(2+) detection using two mercury-specific oligonucleotide probes and streptavidin-horseradish peroxidase (HRP) enzymatic signal amplification. The two mercury-specific oligonucleotide probes comprised a thiolated capture probe and a biotinated signal probe. The thiolated capture probe was immobilized on a gold electrode. In the presence of Hg(2+), the thymine-Hg(2+)-thymine (T-Hg(2+)-T) interaction between the mismatched T-T base pairs directed the biotinated signal probe hybridizing to the capture probe and yielded a biotin-functioned electrode surface. HRP was then immobilized on the biotin-modified substrate via biotin-streptavidin interaction. The immobilized HRP catalyzed the oxidation of hydroquinone (H(2)Q) to benzoquinone (BQ) by hydrogen peroxide (H(2)O(2)) and the generated BQ was further electrochemically reduced at the modified gold electrode, producing a readout signal for quantitative detection of Hg(2+). The results showed that the enzyme-amplified electrochemical sensor system was highly sensitive to Hg(2+) in the concentration of 0.5 nM to 1 μM with a detection limit of 0.3 nM, and it also demonstrated excellent selectivity against other interferential metal ions.

Modulated Dye Retention for the Signal-On Fluorometric Determination of Acetylcholinesterase Inhibitor

A novel fluorometric assay method based on target-induced signal on was developed for acetylcholinesterase (AChE) inhibitor with obviously improved detection sensitivity. In this method, the AChE molecules catalyzed the hydrolysis of acetylthiocholine (ATCl) to form thiocholine, which in turn can specifically react with fluorescent squaraine derivative, a specific chemodosimeter for thiol-containing compounds, resulting in fluorescence quenching and offering a low fluorometric background for the further detection of AChE inhibitor. In the presence of AChE inhibitor, the catalytic hydrolysis of ATCl is blocked, and then the squaraine derivative remains intact and shows signal-on fluorescence. The amount of the remaining fluorescent squaraine derivative is positively correlated with that of the AChE inhibitor in solution. This new designed sensing system shows an obviously improved sensitivity toward target with a detection limit of 5 pg mL(-1) (0.018 nM) for the AChE inhibitor, comparing favorably with previously reported fluorometric methods. To our best knowledge, this new method is the first example of fluorometric enzymatic assay for AChE inhibitors based on such a signal-on principle and using a specific reaction, which has potential to offer an effective strategy for the detection of AChE inhibitors.

Gold nanoparticle based signal enhancement liquid crystal biosensors for DNA hybridization assays

A novel signal enhanced liquid crystal biosensor based on using AuNPs for highly sensitive DNA detection has been developed. This biosensor not only significantly decreases the detection limit, but also offers a simple detection process and shows a good selectivity to distinguish perfectly matched target DNA from two-base mismatched DNA.

Electrocatalytic assay of mercury(II) ions using a bifunctional oligonucleotide signal probe

Engineered nucleic acid probes containing recognition and signaling functions find growing interest in biosensor design. In this paper, we developed a novel electrochemical biosensor for sensitive and selective detecting of Hg(2+) based on a bifunctional oligonucleotide signal probe combining a mercury-specific sequence and a G-quadruplex (G4) sequence. For constructing the electrochemical Hg(2+) biosensor, a thiolated, mercury-specific oligonucleotide capture probe was first immobilized on gold electrode surface. In the presence of Hg(2+), a bifunctional oligonucleotide signal probe was hybridized with the immobilized capture probe through thymine-mercury(II)-thymine interaction-mediated surface hybridization. The further interaction between G4 sequence of the signal probe and hemin generated a G4-hemin complex, which catalyzed the electrochemical reduction of hydrogen peroxide, producing amplified readout signals for Hg(2+) interaction events. This electrochemical Hg(2+) biosensor was highly sensitive and selective to Hg(2+) in the concentration of 1.0 nM to 1 μM with a detection limit of 0.5 nM. The new design of bifunctional oligonucleotide signal probes also provides a potential alternative for developing simple and effective electrochemical biosensors capable of detecting other metal ions specific to natural or artificial bases.

Microwave plasma treated carbon nanotubes and their electrochemical biosensing application.

A convenient microwave plasma treatment method with ammonia precursor was proposed to enhance the solubility of carbon nanotubes (CNTs). The SEM, XRD and FTIR spectra clearly demonstrated that the carbon skeleton structure of the resultant ammonia plasma-treated CNTs (ammonia PT-CNTs) was not destroyed and amine groups of different forms were successfully coupled to CNTs in the MWP treatment process. The ammonia PT-CNTs have excellent solubility in water and are insoluble in nonpolar tetrahydrofuran, and the cyclic voltammograms suggest that the enhanced wetting properties clearly favor faster electron transfer kinetics on the ammonia PT-CNT electrodes. By choosing glucose oxidase as a model enzyme, the application of the ammonia PT-CNTs in construction of biosensors was further investigated. Due to the biocompatibility and electron transfer capability of the ammonia PT-CNTs, the resultant GOD biosensor displayed a good sensing performance. The biosensor has a fast response of less than 10s, and the response current linearly increases with the glucose concentration in the range of 1.2x10(-4) to 7.5x10(-3)M with a detection limit of 1.0x10(-5)M.

A reusable piezo-immunosensor with amplified sensitivity for ceruloplasmin based on plasma-polymerized film

A reusable piezoelectric immunosensor with amplified sensitivity has been developed for the detection of ceruloplasmin (CP) in human serum. The quartz crystal microbalance (QCM) was deposited with plasma-polymerized n-butyl amine film with the surface topology further characterized by using atomic force microscopy (AFM). Anti-ceruloplasmin antibody (CP-Ab) was electrostatically adsorbed on the PPF-modified crystal via an oppositely charged polyelectrolyte layer of alginate. It was found that the alginate-mediated immobilization interface could allow for antibodies to be largely immobilized with well-retained immunoactivity. In particular, a simple regeneration process for the sensor produced, i.e. by shifting the pH, can also be realized. Moreover, an optimized assay medium containing polyethylene glycol (PEG) was tested with enhanced immunosensing response (sensitivity). A dynamic concentration range of two orders of magnitude and a detection limit of 0.15mugml(-1) of CP were observed. Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assay (ELISA) method. However, it presents some superior advantages over the traditional sandwich format in that the analyzing performances are direct, rapid and simple without multiple separation and labeling steps.