Development and characterization of a lux-modified 2,4-dichlorophenol-degrading Burkholderia sp. RASC

lux-marked biosensors for assessing the toxicity and bioremediation potential of polluted environments may complement traditional chemical techniques. luxCDABE genes were introduced into the chromosome of the 2,4-dichlorophenol (2,4-DCP)-mineralizing bacterium, Burkholderia sp. RASC c2, by biparental mating using the Tn4431 system. Experiments revealed that light output was constitutive and related to cell biomass concentration during exponential growth. The transposon insertion was stable and did not interrupt 2,4-DCP-degradative genes, and expression of luxCDABE did not constitute a metabolic burden to the cell. A bioluminescence response was detectable at sublethal 2,4-DCP concentrations: at <10.26 microg ml(-1), bioluminescence was stimulated (e.g. 218% of control), but at concentrations >60 microg ml(-1) it declined to <1%. Investigating the effect of [14C]-2,4-DCP concentration on the evolution of 14CO2 revealed that, for initial concentrations of 2.5-25 microg ml(-1), approximately equals 55% of the added 14C was mineralized after 24 h compared with

Lux-biosensor assessment of pH effects on microbial sorption and toxicity of chlorophenols

Lux-marked bacterial biosensors and a commercial toxicity testing bacterial strain (Microtox) were exposed to 2,4-dichlorophenol (DCP) and the light output response measured. Increasing DCP concentrations caused a decrease in light output in all three biosensors with an order of sensitivity (in terms of luminescence decrease over the DCP concentration range) of Pseudomonas fluorescens <Escherichia coli <Microtox. Adsorption of DCP to E. coli was measured using uniformly ring labelled [14C]DCP and found to be very rapid. The effect of pH on toxicity and adsorption was also investigated. Low pH values increased the amount of DCP adsorbed to the cell and increased the toxicity of DCP.

Signal enhancement of surface plasmon resonance immunoassay using enzyme precipitation-functionalized gold nanoparticles: A femto molar level measurement of anti-glutamic acid decarboxylase antibody

Colloidal gold nanoparticles (AuNPs) and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance (SPR) biosensor. The AuNPs were synthesized and functionalized with HS-OEG(3)-COOH by self assembling technique. Thereafter, the HS-OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti IgG antibody to form an enzyme-immunogold complex. Characterizations were performed by several methods: UV-vis absorption, DLS, HR-TEM and Fr-IR. The Au-anti IgG-HRP complex has been applied in enhancement of SPR immunoassay using a sensor chip constructed by 1:9 molar ratio of HS-OEG(6)-COOH and HS-OEG(3)-OH for detection of anti-GAD antibody. As a result, AuNPs showed their enhancement as being consistent with other previous studies while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. The limit of detection was found as low as 0.03 ng/ml of anti-GAD antibody (or 200 fM) which is much higher than that of previous reports. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

A strategy for sensitivity and specificity enhancements in prostate specific antigen-[alpha] 1-antichymotrypsin detection based on surface plasmon resonance

A biochip based on surface plasmon resonance was fabricated to detect prostate specific antigen-a1-antichymotrypsin (PSA-ACT complex) in both HBS buffer and human serum. To reduce non-specific binding and steric hindrance effect, the chemical surface of the sensor chips was constructed by using various oligo(ethylene glycol) mixtures of different molar ratios of HS(CH2)11(OCH2CH2)6OCH2COOH and HS(CH2)11(OCH2CH2)3OH. The self-assembled monolayers were biotinylated to facilitate the immobilization of streptavidin. Using the chip surfaces, PSA-ACT complex in HBS buffer and human serum was detected at 20.7 and 47.5 ng/ml by primary immunoresponse, respectively. However, the limit of detection could be simply enhanced by a sandwich strategy to improve the sensitivity and specificity of the immunoassay. An intact PSA polyclonal antibody was used as an amplifying agent in the strategy. As a result, PSA-ACT complex concentrations as low as 10.2 and 18.1 ng/ml were found in the HBS buffer and human serum sample, respectively. The result indicates that this approach could satisfy our goal without modifying the secondary interactant.

Detection of pathogen based on the catalytic growth of gold nanocrystals

A homogenous detection of pathogen (Giardia lamblia cysts) based on the catalytic growth of gold nanoparticles (AuNPs) has been studied. In this study, centrifugal filters were employed as tools to concentrate and separate the pathogen cells, and moreover amplify the detection signal. The catalytic growth of gold nanoparticles was verified to be positively related to gold seeds concentration. On this basis, homogenous detection of the pathogenic bacteria in liquid phase was established by means of conjugating antibody to gold seeds. Under the given experimental condition, detection limit of G. lamblia cysts was determined as low as 1.088 × 103 cells ml-1. The additional nonspecific binding tests were also conducted to verify the detection specificity. This sensing platform has been proved to be a sensitive, reliable and simple method for large-scale pathogen detection, and provide valuable insight for the development of gold nanocrystals based colorimetric biosensors.

Homogenous growth of gold nanocrystals for quantification of PSA protein biomarker

We report on another alternative sensing platform for the detection of protein biomarker (PSA–ACT complex) based on homogenous growth of Au nanocrystals in solution phase. The immuno-recognition event is translated into the gold nanoparticle growth signal which can be intuitively recognized by an unaided eye, or quantitatively measured by an UV–vis spectrophotometric analysis. Surface plasmonic signature and kinetics of the Au nanogrowth in the homogenous phase containing of HAuCl4, AA, and CTAB have also been studied to provide suitable parameters for the immunoassay. As a result, detection limit of PSA–ACT complex was determined to be 10 fM. The result indicated that this is a very sensitive, robust, simple, and economic strategy to detect protein biomarkers, and it has great potential to detect other biological interactions.

Gold-based optical biosensor for single-mismatched DNA detection using salt-induced hybridization

In this study, a gold nanoparticle (Au-NP)-based detection method for sensitive and specific DNA-based diagnostic applications is described. A sandwich format consisting of Au-NPs/DNA/PMP (Streptavidin-coated MagnetSphere Para-Magnetic Particles) was fabricated. PMPs captured and separated target DNA while Au-NPs modified with oligonucleotide detection sequences played a role in recognition and signal production. Due to the much lower stability of mismatched DNA strands caused by unstable duplex structures in solutions of relatively low salt concentration, hybridization efficiency in the presence of different buffers was well investigated, and thus, the optimized salt concentration allowed for discrimination of single-mismatched DNA (MMT) from perfectly matched DNA (PMT). Therefore, quantitative information concerning the target analyte was translated into a colorimetric signal, which could easily and quantitatively measured by low-cost UV–vis spectrophotometric analysis. The results indicated this to be a very simple and economic strategy for detection of single-mismatched DNA strands.

Quantitative detection of DNA by autocatalytic enlargement of hybridized gold nanoprobes

Quantitative detection of specific viral DNA has become a pressing issue for the earlier clinical diagnosis of viral infectious diseases. Therefore, in this paper, we report a simple, sensitive, and inexpensive quantitative approach for DNA detection based on the autocatalytic Au deposition of gold nanoprobes via the surface reduction of AuCl4- to Au0 on their surface in the presence of ascorbic acid (AA) and cetyltrimethylammonium bromide (CTAB). On this basis, signal enhancements in the absorbance intensity and kinetic behavior of gold enlargement in the aqueous phase have been well investigated and explained for the selection of analytical parameters. To achieve high sensitivity, magnetic particles conjugated with capture probes (PMPs) were employed for the collection of gold nanoprobes. After denaturated by ion a pH 11 solution, the amplified signals of gold nanoprobes, which is proportional to the concentration of the target DNA, could easily be confirmed by a UV-vis scanning spectrophotometer. Limit of detection could be obtained as low as 1.0 fM by this simple method.

Assessment of a multiplexing high throughput immunochemical SPR biosensor in measuring multiple proteins on a single biosensor chip

Multiplexed immunochemical detection platforms offer the potential to decrease labour demands, increase sample throughput and decrease overall time to result. A prototype four channel multiplexed high throughput surface plasmon resonance biosensor was previously developed, for the detection of food related contaminants. A study focused on determining the instruments performance characteristics was undertaken. This was followed by the development of a multiplexed assay for four high molecular weight proteins. The protein levels were simultaneously evaluated in serum samples of 10-week-old veal calves (n = 24) using multiple sample preparation methods. Each of the biosensor's four channels were shown to be independent of one another and produced multiplexed within run repeatability (n = 6) ranging from 2.0 to 6.7%CV, for the four tested proteins, whilst between run reproducibility (n = 4) ranged from 1.5 to 8.9%CV. Four calibration curves were successfully constructed before serum sample preparation was optimised for each protein. Multiplexed concentration analysis was successfully performed on four channels revealing that each proteins concentration was consistent across the twenty-four tested animals. Signal reproducibility (n > 19) on a further long term study revealed coefficient of variation ranging from 1.1% to 7.3% and showed that the multiplexed assay was stable for at least 480 cycles. These findings indicate that the performance characteristics fall within the range of previously published data for singleplex optical biosensors and that the multiplexing biosensor is fit-for-purpose for simultaneous concentration analysis in many different types of applications such as the multiplexed detection of markers of growth-promoter abuse and multiplexed detection of residues of concern in food safety. © 2013 Elsevier B.V.

Metamaterials Nanosensor for Label-free Analysis of Conformation and Molecular Interaction of Biomolecules

Analysis of molecular interaction and conformational dynamics of biomolecules is of paramount importance in understanding of their vital functions in complex biological systems, disease detection, and new drug development. Plasmonic biosensors based upon surface plasmon resonance and localized surface plasmon resonance have become the predominant workhorse for detecting accumulated biomass caused by molecular binding events. However, unlike surface-enhanced Raman spectroscopy (SERS), the plasmonic biosensors indeed are not suitable tools to interrogate vibrational signatures of conformational transitions required for biomolecules to interact. Here, we show that plasmonic metamaterials can offer two transducing channels for parallel acquisition of optical transmission and sensitive SERS spectra at the biointerface, simultaneously probing the conformational states and binding affinity of biomolecules, e.g. G-quadruplexes, in different environments (Fig. 1). We further demonstrate the use of the metamaterials for fingerprinting and detection of arginine-glycine-glycine domain of nucleolin, a cancer biomarker which specifically binds to a G-quadruplex, with the picomolar sensitivity. The dual-mode nanosensor will significantly contribute to unraveling the complexes of the conformational dynamics of biomolecules as well as to improving specificity of biodetection assays.

Effects of Gold Nanoparticle and Enzyme Precipitation on Enhancement of Surface Plasmon Resonance Immunoassay: A Super Sensitive Measurement of Anti- Glutamic Acid Decarboxylase Antibody

Introduction: In this study, colloidal gold nanoparticle and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance biosensor.

Methods: The colloidal gold nanoparticle was synthesized as described by Turkevitch et al., and their surface was firstly functionalized with HS(CH2)11(OCH2CH2)3COOH (OEG3¬-COOH) by self assembling technique. Thereafter, those OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti-IgG antibody (specific to the Fc portion of all human IgG subclasses) to form an enzyme-immunogold complex. Characterization was performed by several methods: UV-Vis absorption, dynamic light scattering (DLS), transmission electron microscopy (TEM) and FTIR. The as-prepared enzyme-immunogold complex has been applied in enhancement of SPR immunoassay. A sensor chip used in the experiment was constructed by using 1:10 molar ratio of HS(CH2)11(OCH2CH2)6COOH and HS(CH2)11(OCH2CH2)3OH. The capture protein, GAD65 (autoantigen) which is recognized by anti-GAD antibody (autoantibody) in the sera of insulin-dependent diabetes mellitus patients, was immobilized onto the 1:10 surface via biotin-streptavidin interaction.

Results and conclusions: In the research, we reported the influences of gold nanoparticle and enzyme precipitation on the enhancement of SPR signal. Gold nanoparticle showed its enhancement as being consistent with other previous studies, while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. As the results, anti-GAD antibody could be detected at pg/ml level which is far higher than that of commercial ELISA detection kit. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

Growth of Gold Nanocrystals Incorporated with Magnetic Microbead-based Separation as a Tool for Quantification of Biological Interactions

Au nanoparticles (AuNPs) have been widely used not only as optical labels or ‘weight” labels for the detections of biorecognition events but also an amplifier of surface plasmon resonance biosensors. The intrinsic property of gold nuclei composing of a group of Au atoms to catalyze the reduction of metal ions on the NPs and thereby to enlarge the metallic nanoparticles is employed in different biosensing paths. In a solution containing Au+ ions (e.g. HAuCl4) and the Au clusters, hydrated electrons which are reduced from oxidation of reducers (H2O2, sodium citrate, ascorbic acid, or NaBH4) will be used to reduce the Au+ ion leading to the deposition of Au+ to the Au0 (Au clusters). The reaction will be catalyzed continuously by the Au0 until the Au+ ions and hydrated electrons are exhausted. As a result, the AuNPs will be grown and their optical properties are also changed. If the AuNP nanoclusters are used as probes, the color change will be dependent on amount of analytes, thus give a quantitative monitoring of the analytes.

In this study, we incorporate the use of magnetic beads with the nanocrystalline growth to quantify a target protein based on immunoreactions. Prostate specific antigen (PSA) is chosen as the target analyte because of its values in diagnosis of prostate cancer. A double-sandwiched immunoassay is performed by gold-tagged monoclonal PSA antibody-PSA antigen – magnetic bead-tagged polyclonal PSA antibody interactions. After the immunoreactions, the target analytes are preconcentrated and separated by the magnetic beads while the nanogrowth plays a role of colorimetric signal developer.

The result shows that this is a very sensitive, robust and excellent strategy to detect biological interactions. PSA antigen is detected at femtomolar level with very high specificity under the presence of undesired proteins of crude samples. Furthermore, the method also shows great potential to detect other biological interactions. More details will be described in our presentation.

Label-free Analysis of Conformational Dynamics and Molecular Interaction of Biomolecules by Vis-NIR Metasensor

Analysis of binding recognition and conformation of biomolecules is of paramount important in understanding of their vital functions in complex biological systems. By enabling sub-wavelength light localization and strong local field enhancement, plasmonic biosensors have become dominant tools used for such analysis owing to their label-free and real-time attributes1,2. However, the plasmonic biosensors are not well-suited to provide information regarding conformation or chemical fingerprint of biomolecules. Here, we show that plasmonic metamaterials, consisting of periodic arrays of artificial split-ring resonators (SRRs)3, can enable capabilities of both sensing and fingerprinting of biomolecules. We demonstrate that by engineering geometry of individual SRRs, localized surface plasmon resonance (LSPR) frequency of the metamaterials could be tuned to visible-near infrared regimes (Vis-NIR) such that they possess high local field enhancement for surface-enhanced Raman scattering spectroscopy (SERS). This will provide the basis for the development of a dual mode label-free conformational-resolving and quantitative detection platform. We present here the ability of each sensing mode to independently detect binding adsorption and to identify different conformational states of Guanine (G)-rich DNA monolayers in different environment milieu. Also shown is the use of the nanosensor for fingerprinting and detection of Arginine-Glycine-Glycine (RGG) peptide binding to the G-quadruplex aptamer. The dual-mode nanosensor will significantly contribute to unraveling the complexes of the conformational dynamics of biomolecules as well as to improving specificity of biodetection assays that the conventional, population-averaged plasmonic biosensors cannot achieve.

Metamaterials-Based Label-Free Nanosensor for Conformation and Affinity Biosensing

Analysis of molecular interaction and conformational dynamics of biomolecules is of paramount importance in understanding of their vital functions in complex biological systems, disease detection, and new drug development. Plasmonic biosensors based upon surface plasmon resonance and localized surface plasmon resonance have become the predominant workhorse for detecting accumulated biomass caused by molecular binding events. However, unlike surface-enhanced Raman spectroscopy (SERS), the plasmonic biosensors indeed are not suitable tools to interrogate vibrational signatures of conformational transitions required for biomolecules to interact. Here, we show that highly tunable plasmonic metamaterials can offer two transducing channels for parallel acquisition of optical transmission and sensitive SERS spectra at the biointerface, simultaneously probing the conformational states and binding affinity of biomolecules, e.g. G-quadruplexes, in different environments. We further demonstrate the use of the metamaterials for fingerprinting and detection of arginine-glycine-glycine domain of nucleolin, a cancer biomarker which specifically binds to a G-quadruplex, with the picomolar sensitivity.