DNA encapsulating liposome based rolling circle amplification immunoassay as a versatile platform for ultrasensitive detection of protein.

A novel rolling circle amplification (RCA) immunoassay based on DNA-encapsulating liposomes, liposome-RCA immunoassay, was developed for ultrasensitive protein detection. This technique utilized antibody-modified liposomes with DNA prime probes encapsulated as the detection reagent in the sandwiched immunoassays. The DNA prime probes were released from liposomes and then initiated a linear RCA reaction, generating a long tandem repeated sequences that could be selectively and sensitively detected by a microbead-based fluorescence assay. The developed technique offered very high sensitivity due to primary amplification via releasing numerous DNA primers from a liposome followed by a secondary RCA amplification. A biobarcode design was incorporated in the technique, which allowed the strategy to be directly implemented for multiplex assay of multiple proteins. Also, the technique allowed easy preparation of the DNA-carrying antibody reagent and the implementation with simple instrumentation. The technique was demonstrated for the determination of prostate-specific antigen (PSA), a highly selective biomarker associated with prostate cancer. The results revealed that the technique exhibited a dynamic response to PSA over a 6-decade concentration range from 0.1 fg mL(-1) to 0.1 ng mL(-1) with a limit of detection as low as 0.08 fg mL(-1) and a high dose-response sensitivity. The liposome-RCA immunoassay holds great promise as a versatile, sensitive, and robust platform to combine the nucleic acid amplification with immunoassay for ultrasensitive protein detection.

Surface-enhanced Raman spectroscopic detection of a bacteria biomarker using gold nanoparticle immobilized substrates.

The development of ultrasensitive and rapid methods for the detection of dipicolinic acid (DPA), a biomarker for bacterial spores including Bacillus anthracis, is increasingly important. This paper reports the results of an investigation of surface enhanced Raman spectroscopy (SERS) based ultrasensitive detection of DPA using a gold nanoparticle/polyvinylpyrrolidone/gold substrate (AuNPs/PVP/Au). The strong SERS effect of this substrate exploits the particle-particle and particle-substrate plasmonic coupling, which is optimized by manipulating the diameter of the nanoparticles (50-70 nm). The correlation between the SERS intensity of the diagnostic band and the DPA concentration (0.1 ppb to 100 ppm) was shown to exhibit two linear regions, i.e., the low- (<0.01 ppm) and high-concentration (>1 ppm) regions, with an intermediate region in between. The presence of a linear relationship in the low-concentration region was observed for the first time in SERS detection of DPA. A detection limit of 0.1 ppb was obtained from the substrates with 60 nm sized Au NPs, which is, to our knowledge, the lowest detection limit reported for DPA using this type of SERS substrate. This finding was also supported by the estimated enhancement factor (approximately 10(6)) and a large adsorption equilibrium constant for the low-concentration region (1.7 x 10(7) M(-1)). The adsorption characteristics of DPA on the SERS substrates were analyzed in terms of monolayer and multilayer adsorption isotherms to gain insights into the correlation between the SERS intensity and the DPA concentration. The observed transition from the low- to high-concentration linear regions was found to correspond to the transition from a monolayer to multilayer adsorption isotherm, which was in agreement with the estimated minimum DPA concentration for a monolayer coverage (approximately 0.01 ppm).

A novel piezoelectric immunoagglutination assay technique with antibody-modified liposome.

A simple rapid piezoelectric immunoagglutination assay (PEIA) technique with antibody-modified liposome has been developed for direct quantitative detection of human immunoglobulin G (hIgG). This technique is based on specific agglutination of antibody-coated liposome particles in the presence of the corresponding antigen, which can be monitored by the frequency shift of a piezoelectric device. Compared with conventional piezoelectric assays, this liposome-based PEIA does not require the immobilization of antigen or antibody on the quartz crystal surface, making the developed technique especially useful for rapid and renewable immunochemical determination. To alleviate non-specific adsorption of serum proteins, modification of the quartz crystal surface by different protocols and the composition of the assay medium have been investigated. The results indicate that the background interference can be substantially minimized through modifying the quartz crystal surface with a bovine serum albumin (BSA) layer and introducing an appropriate amount of BSA in the assay medium. The effects of the liposome composition, the liposome concentration and the concentration of poly(ethylene glycol) (PEG) in the assay medium, have also been investigated. The frequency responses of the liposome-based PEIA are linearly correlated to hIgG concentration in the range of 0.05-6 microg mL(-1) with a detection limit of 50 ng mL(-1).

An optode sensor for Cu2+ with high selectivity based on porphyrin derivative appended with bipyridine.

A porphyrin derivative (fluorophore) appended with bipyridine (ionophore) has been applied for preparation of a Cu2+-sensitive optical chemical sensor, which is based on fluorescence quenching of porphyrin derivative entrapped in a poly(vinyl chloride) membrane by the energy transfer process. The sensor exhibits a linear response toward Cu2+ in the concentration range 2.0 x 10(-8) - 1.0 x 10(-5) M, with a working pH range from 6.0 to 8.0 and a high selectivity. The detection limit is 5 x 10(-9) M. The response time for Cu2+ is less than 5 min with concentrations lower than 5 x 10(-6) M. The optode can be regenerated using 0.3 M EDTA (pH 9) and acetate buffer solution. The effect of the composition of the sensor membrane was studied, and the experimental conditions were optimized. The sensor has been used for direct determination of Cu2+ in water samples with satisfied results.

Quartz crystal microbalance bioaffinity sensor for biotin based on mixed self-assembled monolayers and metastable molecular complex receptor.

A quartz crystal microbalance (QCM) sensor was proposed for the detection of small molecule biotin based on the mixed self-assembled monolayer (SAM) of thiols on gold substrate and the bioaffinity difference between an analyte (biotin) and an analogue compound (HABA) in binding avidin. Avidin formed a metastable complex with 2-[(4-hydroxyphenyl)azo]benzoic acid (HABA) immobilized on the crystal surface. When the sensor contacts a sample solution containing biotin, the avidin was released from the sensor surface to form a more stable complex with biotin in solution. The frequency change recorded is proportional to the desorbed mass of avidin, and there is a clear mathematic relationship between the frequency change and the biotin concentration. The use of mixed SAMs allows the stable attachment of bioreceptor molecules on the QCM, and enhances the amount of the immobilized molecules on the QCM, as a longer "space arm" in the mixed SAMs makes this monolayer membrane more accessible to capture the immobilized molecules. The proposed bioaffinity sensor has nice response to biotin in the range of 0.017-1.67 microg/mL. The sensor could be regenerated under very mild conditions simply by reimmersion of the sensor into a biotin solution to desorb the surplus avidin.

A highly selective potentiometric sensor of molybdate based on a mu-oxo-bridged manganeseporphyrin dimer.

A novel high-selective potentiometric sensor for molybdate was prepared with a PVC membrane combining mu-oxo-bis[5,10,15,20-tetra(p-methylphenyl)porphinatomanganese(III)] [[Mn(p-Me)TPP](2)O] as an electroactive material and 2-nitrophenyl octyl ether (o-NPOE) as a plasticizer in the percentage ratio of 3:65:32, [Mn(p-Me)TPP](2)O:o-NPOE:PVC (w:w). The sensor exhibited a linear response with a Nernstian slope of 30.5 mV per decade within a concentration range of 2.1 x 10(-6) to 1.0 x 10(-1) M MoO4(2-), with a working pH range from 5.0 to 12.5, and a fast response time of less than 15 s. The electrode showed improved selectivity toward molybdate with respect to common coexisting anions compared to monometalloporphyrin counterparts. Several electroactive materials and solvent mediators were compared and the experimental conditions were optimized. The sensor is preliminary applied to the assay of MoO4(2-) in corrosion inhibitor samples with satisfactory results.

Piezoelectric immunoassay for complement C4 based on a Nafion-modified interface for antibody immobilization.

A piezoelectric immunosensor based on a Nafion membrane interface with a simple immobilization procedure has been developed for the determination of complement C4 in human serum. The polyanionic perfluorosulfonated Nafion polymer was used to modify the electrode surface of quartz-crystal microbalance (QCM) as a platform for the immobilization of complement C4 antibodies. The surface morphology of QCM modified with Nafion membrane was investigated using scanning electron microscopy. The system was optimized with regard to the parameters involved in the preparation of the immunosensor and the assay process. The proposed immunosensor responds well to C4 in the range of 0.08-1.6 microg/ml with a relative standard deviation of <5.3%. Moreover, the proposed immunosensor has advantages in terms of the speed and simplicity of the immobilization procedure as well as the simple and advantageous regeneration process. The experimental results obtained with regard to nonspecific adsorption and recovery indicate that the proposed immunosensor offers a promising alternative tool for the clinical diagnosis of complement C4.

An electrochemical stripping metalloimmunoassay based on silver-enhanced gold nanoparticle label.

A novel, sensitive electrochemical immunoassay has been developed based on the precipitation of silver on colloidal gold labels which, after silver metal dissolution in an acidic solution, was indirectly determined by anodic stripping voltammetry (ASV) at a glassy-carbon electrode. The method was evaluated for a noncompetitive heterogeneous immunoassay of an immunoglobulin G (IgG) as a model. The influence of relevant experimental variables, including the reaction time of antigen with antibody, the dilution ratio of the colloidal gold-labeled antibody and the parameters of the anodic stripping operation, upon the peak current was examined and optimized. The anodic stripping peak current depended linearly on the IgG concentration over the range of 1.66 ng ml(-1) to 27.25 microg ml(-1) in a logarithmic plot. A detection limit as low as 1 ng ml(-1) (i.e., 6 x 10(-12) M) human IgG was achieved, which is competitive with colorimetric enzyme linked immuno-sorbent assay (ELISA) or with immunoassays based on fluorescent europium chelate labels. The high performance of the method is attributed to the sensitive ASV determination of silver (I) at a glassy-carbon electrode (detection limit of 5 x 10(-9) M) and to the catalytic precipitation of a large number of silver on the colloidal gold-labeled antibody.

Amperometric hydrogen peroxide biosensor based on horseradish peroxidase-labeled nano-Au colloids immobilized on poly(2,6-pyridinedicarboxylic acid) layer by cysteamine.

A sensitive hydrogen peroxidase (H2O2) amperometric sensor based on horseradish peroxidase (HRP)-labeled nano-Au colloids has been proposed. Nano-Au colloids were immobilized by the thiol group of cysteamine, which was associated with the carboxyl groups of poly(2,6-pyridinedicarboxylic acid) (PPDA). With the aid of the hydroquinone, the sensor displayed excellent electrocatalytical response to the reduction of H2O2. Compared with the non-Au-colloid modified electrode, i.e., PPDA/HRP, the Au-colloid modified electrode exhibited better performance characteristics, including stability, reproducibility, sensitivity and accuracy. The biosensor shows a linear response to H2O2 in the range of 3.0 x 10(-7) - 2 x 10(-3) M. The detection limit was 1.0 x 10(-7) M.

Glycosylated metalloporphyrins as neutral carriers for PVC membrane electrodes.

In this paper, the synthesis of a novel ionophore, chloro[5,10,15,20-tetrakis[2-(2,3,4,6-tetraacetyl-beta-D-glucopyranosyl)-1-O-phenyl]porphinato]manganese (MnT(o-glu)PPCl), and its application as a neutral carrier for a PVC membrane electrode are described. The MnT(o-glu)PPCl-based PVC membrane electrode shows a potentiometric responses to SCN- over a concentration range of 3.4 x 10(-7) - 1.0 x 10(-1) mol L(-1) with a Nernstian slope and a response time of 20 s. The electrode exhibits an anti-Hofmeister selectivity toward SCN- with respect to common coexisting anions. As active materials, MnT(o-glu)PPCl shows better selectivity toward SCN- than chloro(tetraphenylporphinato)manganese (MnTPPCl). The effect of the electrode membrane compositions has been studied and the experimental conditions were optimized. The electrode was applied to the determination of SCN- in body fluids with satisfactory results.

A rhodamine-appended water-soluble conjugated polymer: an efficient ratiometric fluorescence sensing platform for intracellular metal-ion probing.

Thewater-soluble CP was conjugatedwith a rhodamine spirolactam for the first time to develop a new FRET-based ratiometric fluorescence sensing platform(CP 1) for intracellular metal-ion probing. CP 1 exhibits excellent water-solubility with twowell-resolved emission peaks, which benefit ratiometric intracellular imaging applications.

An efficient fluorescent sensing platform for biomolecules based on fenton reaction triggered molecular beacon cleavage strategy.

A universal sensing platform for fluorescence turn-on detection of biomolecules is developed based on Fenton reaction triggered molecular beacon cleavage. Due to its high quenching efficiency, molecular beacons (MBs)-based sensing systems usually show low background fluorescence and large signal-to-background ratio. Glucose is chosen as a model biomolecule for constructing an MB-based fluorescence sensing system. In the presence of glucose, the glucose oxidase will bind with it and catalyze the oxidation to generate H(2)O(2), which is further decomposed to produce (·)OH through the Fe(2+)-catalyzed Fenton reaction. Then, in-situ-generated OH can trigger the cleavage of the MB, and its fluorescence intensity will be dramatically increased because of the complete separation of the fluorophore from the quencher. By employing molecular beacon as both recognition and reporter probes to low background signal, the proposed biosensors showed high sensitivity to targets. It also exhibited high selectivity owing to the high specificity of the enzymatic oxidation, which make it valuable for the detection of target biomolecule in complex biological samples.

[Extracellular polymeric substances (EPS) of white-rot fungus and their effects on Pb2+ adsorption by biomass].

The extracellular polymeric substances (EPS) of P. chrysosporium and their effects on Pb2+ biosorption were studied. The product, composition of EPS and the effects on Pb2+ biosorption capacity were investigated in lab via flask experiments. The surface changes of mycelium before and after EPS extraction, before and after Pb2+ adsorption were researched by environment scanning electron microscope with energy-dispersive X-ray analysis (ESEM-EDX). Results showed that at 113 h, the maximum yield of EPS was 125.5 mg/L, which contained 46.6% - 54.3% of sugar and 31.2% - 35.1% of protein. The results of control test after EPS extraction displayed a decrease of biosorption capacity of Pb2+ among 2.12 mg/g (113 h) - 7.73 mg/g (41 h). The results of environment scanning electron microscope (ESEM) showed that the EPS extraction affected the cell wall of white-rot fungus and the Pb-contained globular particle after Pb2+ uptake, which was very useful for further study on heavy metal biosorption mechanism.

Construction of supported lipid membrane modified piezoelectric biosensor for sensitive assay of cholera toxin based on surface-agglutination of ganglioside-bearing liposomes.

A novel piezoelelctric biosensor has been developed for cholera toxin (CT) detection based on the analyte-mediated surface-agglutination of ganglioside (GM1)-functionalized liposomes. To achieve a CT-specific agglutination at the surface, the gold electrode is modified by a GM1-functionalized supported lipid membrane via spontaneous spread of the liposomes on a self-assembled monolayer of a long-chain alkanethiol. In the presence of CT, the GM1-incorporated liposomes in assay medium will rapidly specifically agglutinate at the electrode surface through the binding of CT to GM1 on the electrode surface and the liposome interface. This results in an enormous mass loading on the piezoelelctric crystal as well as a significant increase of density and viscosity at the interface, thereby generating a decrease in frequency of the piezoelelctric crystal. The combination of mass loading with interfacial change in the surface-agglutination reaction allows the developed piezoelelctric biosensor to show substantial signal amplification in response to the analyte CT. The detection limit can be achieved as low as 25 ng mL(-1) CT. This is the first demonstration on CT detection based on specific surface-agglutination of GM1-modified liposomes. The supported lipid layer based sensing interface can be prepared readily and renewably, making the developed technique especially useful for simple, reusable and sensitive determination of proteins.

An electrochemical immunosensor based on enzyme-encapsulated liposomes and biocatalytic metal deposition.

A novel electrochemical immunosensor based on double signal amplification of enzyme-encapsulated liposomes and biocatalytic metal deposition was developed for the detection of human prostate specific antigen (PSA). Alkaline phosphatase (ALP)-encapsulated and detection antibody-functionalized liposomes were first prepared and used as the detection reagent. In the sandwich immunoassay, the model analyte PSA was first captured by anti-PSA capture antibody immobilized on the electrode and then sandwiched with the functionalized liposomes. The bound liposomes were then lysed with surfactant to release the encapsulated ALP, which served as secondary signal amplification means. ALP on the electrode surface initiated the hydrolysis of ascorbic acid 2-phosphate (AA-p) to produce ascorbic acid. The latter, in turn, reduced silver ions on the electrode surface, leading to deposition of the metal silver on the electrode surface. Linear sweep voltammetry (LSV) was chosen to detect the amount of the deposited silver. The results showed that the anodic stripping peak current was linearly dependent on the PSA concentration in the range of 0.01-100 ng mL(-1), and a detection limit as low as 0.007 ng mL(-1) can be obtained. Since the cut-off value of human PSA is 4 ng mL(-1), the proposed electrochemical immunosensor would be expected to gain widespread applications for the detection of PSA in clinical diagnosis.

A reusable capacitive immunosensor based on a CuS ultrathin film constructed by using a surface sol-gel technique.

A capacitive sensing method based on a CuS ultrathin film modified electrode prepared by a surface sol-gel technique has been developed for the direct detection of human IgA. The resulting CuS film was investigated with cyclic voltammetry (CV), impedance spectroscopy, and quartz crystal microbalance (QCM). CV and impedance examinations showed that the CuS film formed on the gold electrode surface was insulated, and was applicable to form an insulating layer of a capacitive immunosensor. With QCM measurements, the thickness of the CuS film was evaluated to be 5.8 nm. The capacitance change was greatly increased by a CuS nanofilm-based immunosensor, which was initiated by the recognition of an immobilized antibody and the target antigen. The capacitance of the immunosensor corresponding to the concentration of human IgA was investigated by potentiostatic-step measurements. A linear calibration curve was obtained in the range of 1.81 - 90.5 ng ml(-1) with a detection limit of 1.81 ng ml(-1). There were no obvious interferences from the nonspecific adsorption of other proteins. With nice reproducibility and regeneration capacity, the CuS ultrathin film modified immunosensor could be used for the detection of human IgA in serum samples with a recovery of 96.1 - 104.4%, showing its promising applicability and reliability.

Rapid detection of picloram in agricultural field samples using a disposable immunomembrane-based electrochemical sensor.

Picloram, a widely used chlorinated herbicide, is quite persistent and mobile in soil and water with adverse health and environmental effects. It is essential to establish a rapid and sensitive method for accurate detection of trace picloram in agricultural samples. We employed a disposable, nontoxic, and conductive chitosan/gold nanoparticles composite membrane on electrochemical sensor for the sensitive detection of picloram in several agricultural field samples. A self-synthesized picloram antibody was encapsulated in the immunomembrane to form an immunoconjugate by a competitive immunoreaction in sample solution, followed by the immobilization of horseradish peroxidase (HRP)-labeled secondary antibody. The immunomembrane possessed good reproducibility for fabrication in batch, providing a congenial microenvironment for the immune molecules. The diffused colloidal Au nanoparticles shuttled the electron transfer between the immobilized HRP and the electrode surface. To demonstrate the suitability of the immunosensor for on-site detection, rice, lettuce, and paddy field water were spiked with picloram and assayed without preconcentration. Under optimal conditions, picloram could be detected in the range from 0.005 to 10 microg/mL with the correlation coefficient of 0.9937, and the detection limit is 5 ng/ mL. The proposed immunosensor exhibited good precision, sensitivity, selectivity, and storage stability.

An ultrasensitive chemiluminescence biosensor for cholera toxin based on ganglioside-functionalized supported lipid membrane and liposome.

A novel chemiluminescence biosensor based on a supported lipid layer incorporated with ganglioside GM1 was developed for the detection of cholera toxin. The planar supported lipid membrane was prepared as biosensing interface via spontaneous spread of ganglioside-incorporated phospholipid vesicles on the octadecanethiol-coated gold surface. The specific interaction of multivalent CT by ganglioside GM1 molecules enables the biosensor to be implemented via a sandwiched format using a liposome probe functionalized with GM1 and horseradish peroxidase (HRP). Then, the presence of the target CT could be determined via the HRP-catalyzed enhanced chemiluminescence reaction. The developed strategy offers several unique advantages over conventional biosensors in that it allows for an easy construction and renewal of the sensing interface, a small background signal due to low non-specific adsorption of serum constituents on the lipid membrane, and effective immobilization of multiple biocatalytic amplifiers and recognition components via common phospholipid reagents. The developed biosensor was shown to give chemiluminescence signal in linear correlation to CT concentration within the range from 1pgmL(-1) to 1ngmL(-1) with readily achievable detection limit of 0.8pgmL(-1).

A sensitive immunoassay based on electropolymerized films by capacitance measurements for direct detection of immunospecies.

Fabrication of a capacitive immunosensor based on electropolymerized polytyramine (Pty) film for the direct detection of human serum albumin (HSA) without any labeling is described. The capacitance change of the heterostructures, Pty films/covalently bonded antibodies/buffered medium, is utilized for monitoring the specific antibody-antigen interaction. The Pty films are ultrathin and the HSA assay is nearly specific. Experimental parameters affecting antibody immobilization and the sensing of HSA are investigated in detail and optimized. This capacitive sensor prepared with the present method can provide high sensitivity. Under the optimized experimental conditions, a linear calibration curve in the concentration range 1.84-368.6 ng/ml when plotted vs the logarithm of the antigen concentration is obtained and the detection limit (S/N=3) is 1.60 ng/ml. After an acidic washing the present system can be used again. The applicability and reliability of the sensor are also demonstrated.

A coumarin derivative covalently immobilized on sensing membrane as a fluorescent carrier for nitrofurazone.

A coumarin derivative 4-methyl-8-methylacrylamide-2H, 5H-pyrano [3, 2-C] benzpyran 2, 5-dione (MMPBD) has been synthesized as a fluorescent carrier for preparing an optical chemical sensor. The carrier is immobilized on a quartz glass plate surface treated with a silanizing agent to prevent the leakage of the dye. This MMPBD sensor can be utilized for a nitrofurazone (NF) assay based on fluorescence quenching. The sensor shows good repeatability, a long lifetime and a fast response of less then 50 s. NF can be determined in the range between 1.0x10(-6)-1.0x10(-3 )mol L(-1) with a detection limit of 8.0x10(-7) mol L(-1 )at pH 7.0.