A strategy for sensitivity and specificity enhancements in prostate specific antigen-alpha1-antichymotrypsin detection based on surface plasmon resonance

A biochip based on surface plasmon resonance was fabricated to detect prostate specific antigen-alpha(1)-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.     

Feasibility of protein A for the oriented immobilization of immunoglobulin on silicon surface for a biosensor with imaging ellipsometry

The feasibility of using protein A to immobilize antibody on silicon surface for a biosensor with imaging ellipsometry was presented in this study. The amount of human IgG bound with anti-IgG immobilized by the protein A on silicon surface was much more than that bound with anti-IgG immobilized by physical adsorption. The result indicated that the protein A could be used to immobilize antibody molecules in a highly oriented manner and maintain antibody molecular functional configuration on the silicon surface. High reproducibility of the amount of antibody immobilization and homogenous antibody adsorption layer on surfaces could be obtained by this immobilization method. Imaging ellipsometry has been proven to be a fast and reliable detection method and sensitive enough to detect small changes in a molecular monolayer level. The combination of imaging ellipsometry and surface modification with protein A has the potential to be further developed into an efficient immunoassay protein chip.     

Gold nanoparticle-based colorimetric assays for coagulation-related proteins and their inhibition reactions

In this paper, we describe two simple, label-free, homogenous assays using gold nanoparticles (Au NPs)-one to detect coagulation-related proteins and the other to screen inhibition reactions. The first nanosensor functions on the basis of the fact that thrombin catalyzes fibrinogen to form long-chain fibrins, which then induce aggregation of Au NPs. We applied this sensor to study the interactions of thrombin, inhibitors, cofactors, and antidotes. We further used thrombin-conjugated Au NPs (Thr-Au NPs) to analyze the levels of fibrinogen in plasma samples via fibrinogen-induced aggregation of Thr-Au NPs. The limit of detection (LOD; S/N=3) of this sensor for fibrinogen in plasma was 10nM. The Thr-Au NP probe provided quantitative results for fibrinogen in plasma samples that correlated (R(2)=0.97) with those obtained using a clinical von Clauss clotting rate assay. In addition, the Thr-Au NP-based sensor could be used to monitor thrombin concentrations in plasma samples under physiological conditions. Compared with conventional assays, these label-free assays offer several advantages, such as rapid and simple readout by the naked eye or by UV-vis absorption spectroscopy.     

Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium

An immunosensor based on surface plasmon resonance (SPR) using protein G was developed for the detection of Salmonella typhimurium. A protein G layer was fabricated by binding chemically to self-assembly monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) on gold (Au) surface. The formation of protein G layer on Au surface modified with 11-MUA and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The effect of detergent such as Tween-20 on binding efficiency of antibody and antigen was investigated by SPR. The binding efficiency of antigen to the antibody immobilized on Au surface was improved up to about 85% and 100% by using protein G and Tween-20, respectively. The surface morphology analyses of 11-MUA monolayer on Au substrate, protein G layer on 11-MUA monolayer and antibody layer immobilized on protein G layer were performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. typhimurium using protein G was developed with a detection range of 10(2) to 10(9)CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. typhimurium could be applied to construct other immnosensors or protein chips.     

Surface plasmon resonance immunosensor using self-assembled protein G for the detection of Salmonella paratyphi

A surface plasmon resonance (SPR) based immunosensor using self-assembled protein G was developed for the detection of Salmonella paratyphi. In order to endow a solid substrate binding affinity to protein G, the free amine (-NH2) of protein G was substituted into thiol (-SH) using 2-iminothiolane. Thus, self-assembled protein G was fabricated on gold (Au) substrate. The formation of protein G layer on Au surface, and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analysis of the protein G layer on Au surface was performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. paratyphi using self-assembled protein G was developed with a detection range of 10(2)-10(7) CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. paratyphi could be applied to construct other immnosensors or protein chips.     

A graphene oxide based biosensor for microcystins detection by fluorescence resonance energy transfer

Water safety is one of the most pervasive problems afflicting people throughout the world. Microcystin, a hepatotoxin produced by cyanobacteria, poses a growing and serious threat of water safety. According to World Health Organization (WHO), the limit of content of microcystin-LR (MC-LR) in drinking water is as low as 1μg/L; it is thus necessary to explore a sensitive method for the trace detection of microcystins (MCs). Based on the observation of gold nanoparticles (Au NPs) induced graphene oxide (GO) fluorescence quenching, a reliable biosensor was developed here for microcystins detection. MCs could be attached on Au NPs through the interaction with single strand-DNA (ss-DNA) modified on Au NPs, which formed Au-DNA-MCs complexes. These MCs in the complexes could be immunologically recognized by the antibodies adsorbed on GO sheets, as a result, Au NPs were close enough to quench the photoluminescence of GO by the fluorescence resonance energy transfer (FRET). The fluorescence intensity decreased with the increase of MCs as more Au NPs linked onto GO surface. The limit of detection was 0.5 and 0.3μg/L for microcystin-LR and microcystin-RR (MC-RR), respectively, which satisfies the strictest standard of WHO. Well defined results were also obtained in natural lake water and the specificity experiment. The antibody used here could recognize Adda group, the conservative part of MCs, which allowed the biosensor to detect both single toxin and the total content of MCs existing in the water sample.     

An artificial enzyme-based assay: DNA detection using a peroxidase-like copper-creatinine complex

We report an artificial enzyme-based DNA assay using a peroxidase-like copper (Cu)-creatinine complex as a catalyst for 3,3',5,5'-tetramethylbenzidine (TMB) oxidation. The assay employs double signal amplification and a homogeneous catalytic reaction: (i) fast catalytic growth of Cu on a gold (Au) nanoparticle (NP) label forms a thick Cu layer (first amplification); (ii) dissolution of the Cu layer generates many Cu-creatinine complexes per NP (generation of homogeneous catalysts); (iii) peroxidase-like Cu-creatinine complexes rapidly convert TMB into a colored product (second amplification). To investigate the effect of ligand on the catalytic activities of Cu complexes, the kinetics of catalytic TMB oxidation is tested with and without using imidazole ring-containing ligands (creatinine, imidazole, and poly(l-histidine)). Both fast oxidation of TMB and slow further oxidation of the colored product are required for high signal-to-background ratios. Cu-creatinine complex allows relatively fast oxidation and slow further oxidation. Fast seed-mediated Cu growth on Au NP and slow Cu autonucleation (i.e., slow formation of Cu NP in the absence of Au NP) are also required for high signal-to-background ratios. In tris-EDTA (tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid) buffer (pH 7.7) containing high concentrations of Cu(2+) (90 mM), ascorbic acid (50mM), and Mg(2+) (200 mM), Cu growth on Au NP is very fast and autonucleation is significantly suppressed. Fast catalytic oxidation by Cu-creatinine complex along with fast Cu growth on Au NP allows a detection limit of 0.1 pM for DNA in a simple microplate format.     

Amperometric detection of bilirubin from a micro-sensing electrode with a synthetic bilirubin imprinted poly(MAA-co-EGDMA) film

Poly(methacrylic acid-co-ethyl glycol dimethylacrylate) (poly(MAA-co-EGDMA)) imprinted with alpha-bilirubin was shown to be able to bind alpha-bilirubin in our previous work. In this work, the corresponding imprinted polymer thin film was synthesized onto a thiol treated Au electrode by surface grafting polymerization. Bilirubin was able to be detected by an Au electrode, however, the electrode was not be able to discriminate bilirubin from the other matrix components if clinical samples were used. Therefore, the imprinted material was introduced so that the modified Au electrode could specifically detect bilirubin. Optimal potential was found to be 0.55 V and this was set for the rest of experiments. The imprinting factor of 3.16 was confirmed by comparing the signals from the MIP-Au and the NIP (non-imprinted polymer)-Au electrode. Calibration of the bilirubin concentration with respect to the current by the MIP-Au electrode was made within the range of 5mg/dl and a detection sensitivity of 0.644 microA/mg/dl (2.58 microA/cm(2)/mg/dl) was obtained. Furthermore, a linear correlation of the bilirubin concentration within 1.0mg/dl versus detection current was also achieved. Bilirubin was further detected by the MIP-Au electrode in the presence of fetal bovine serum (FBS). Repeated detection of bilirubin with at least three detection batches was performed and the reproducibility of the same piece of MIP-Au electrode was confirmed. The result was compared to those obtained from the serum and the solvent solution. The results indicated the feasibility of using the bilirubin imprinted poly(MAA-co-EGDMA) film as a sensing electrode for the clinical detection of bilirubin in serum.     

A Protein L -Based Immunodiagnostic Approach Utilizing Time-Resolved F?rster Resonance Energy Transfer

Chelated lanthanides such as europium (Eu) have uniquely long fluorescence emission half-lives permitting their use in time-resolved fluorescence (TRF) assays. In Förster resonance energy transfer (FRET) a donor fluorophore transfers its emission energy to an acceptor fluorophore if in sufficiently close proximity. The use of time-resolved (TR) FRET minimizes the autofluorescence of molecules present in biological samples. In this report, we describe a homogenous immunoassay prototype utilizing TR-FRET for detection of antibodies in solution. The assay is based on labeled protein L, a bacterial protein that binds to immunoglobulin (Ig) light chain, and labeled antigen, which upon association with the same Ig molecule produce a TR-FRET active complex. We show that the approach is functional and can be utilized for both mono- and polyvalent antigens. We also compare the assay performance to that of another homogenous TR-FRET immunoassay reported earlier. This novel assay may have wide utility in infectious disease point-of-care diagnostics.     

Determination and analysis of antigenic epitopes of prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2) using synthetic peptides and computer modeling.

Prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2), produced essentially by the prostate gland, are 237-amino acid monomeric proteins, with 79% identity in primary structure. Twenty-five anti-PSA monoclonal antibodies (Mabs) were studied for binding to a large array of synthetic linear peptides selected from computer models of PSA and hK2, as well as to biotinylated peptides covering the entire PSA sequence. Sixteen of the Mabs were bound to linear peptides forming four independent binding regions (I-IV). Binding region I was localized to amino acid residues 1-13 (identical sequence for PSA and hK2), II (a and b) was localized to residues 53-64, III (a and b) was localized to residues 80-91 (= kallikrein loop), and IV was localized to residues 151-164. Mabs binding to regions I and IIa were reactive with free PSA, PSA-ACT complex, and with hK2; Mabs binding to regions IIb, IIIa, and IV were reactive with free PSA and PSA-ACT complex, but unreactive with hK2; Mabs binding to region IIIb detected free PSA only. All Mabs tested (n = 7) specific for free PSA reacted with kallikrein loop (binding region IIIb). The presence of Mabs interacting with binding region I did not inhibit the catalytic activity of PSA, whereas Mabs interacting with other binding regions inhibited the catalysis. Theoretical model structures of PSA, hK2, and the PSA-ACT complex were combined with the presented data to suggest an overall orientation of PSA with regard to ACT.     

A novel ultrahigh-resolution surface plasmon resonance biosensor with an Au nanocluster-embedded dielectric film

The detection performance of conventional surface plasmon resonance (SPR) biosensors is limited to a 1 pg/mm(2) surface coverage of biomolecules, and consequently, such sensors struggle to detect the interaction of small molecules in low concentrations. The present study is attempted to propose the use of a novel SPR biosensor with Au nanoclusters embedded in a dielectric film to achieve a 10-fold improvement in the resolution performance. A co-sputtering method utilizing a multi-target sputtering system is used to fabricate the present dielectric films (SiO(2)) with embedded Au nanoclusters. It is shown that the sensitivity of the developed SPR biosensor can be improved by adjusting the size and volume fraction of the embedded Au nanoclusters in order to control the surface plasmon effect. The present gas detection and DNA hybridization experimental results confirm that the proposed Au nanocluster-enhanced SPR biosensor provides the potential to achieve an ultrahigh-resolution detection performance of approximately 0.1 pg/mm(2) surface coverage of biomolecules.     

Fabrication and characteristics of MOSFET protein chip for detection of ribosomal protein

A metal oxide silicon field effect transistor (MOSFET) protein chip for the easy detection of protein was fabricated and its characteristics were investigated. Generally, the drain current of the MOSFET is varied by the gate potential. It is expected that the formation of an antibody-antigen complex on the gate of MOSFET would lead to a detectable change in the charge distribution and thus, directly modulate the drain current of MOSFET. As such, the drain current of the MOSFET protein chip can be varied by ribosomal proteins absorbed by the self-assembled monolayer (SAM) immobilized on the gate (Au) surface, as ribosomal protein has positive charge, and these current variations then used as the response of the protein chip. The gate of MOSFET protein chip is not directly biased by an external voltage source, so called open gate or floating gate MOSFET, but rather chemically modified by immobilized molecular receptors called self-assembled monolayer (SAM). In our experiments, the current variation in the proposed protein chip was about 8% with a protein concentration of 0.7 mM. As the protein concentration increased, the drain current also gradually increased. In addition, there were some drift of the drain current in the device. It is considered that these drift might be caused by the drift from the MOSFET itself or protein absorption procedures that are relied on the facile attachment of thiol (-S) ligands to the gate (Au) surface. We verified the formation of SAM on the gold surface and the absorption of protein through the surface plasmon resonance (SPR) measurement.     

Surface plasmon resonance immunosensor for detection of<Emphasis Type="Italic">Legionella pneumophila</Emphasis>

An immunosensor based on surface plasmon resonance (SPR) onto a protein G layer by self-assembly technique was developed for detection ofLegionella pneumophila. The protein G layer by self-assembly technique was fabricated on a gold (Au) surface by adsorbing the 11-mercaptoundecanoic acid (MUA) and an activation process for the chemical binding of the free amine (-NH₂) of protein G and 11-(MUA) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) in series. The formation of the protein G layer by self-assembly technique on the Au substrate and the binding of the antibody and antigen in series were confirmed by SPR spectroscopy. The surface topographies of the fabricated thin films on an Au substrate were also analyzed by using an atomic force microscope (AFM). Consequently, an immunosensor for the detection ofL. pneumophila using SPR was developed with a detection limit of up to 10² CFU per mL.     

Enhanced surface plasmon resonance with the modified catalytic growth of Au nanoparticles

The catalytic growth of Au nanoparticles (AuNPs) has been employed in several analytical methods for improving the detection sensitivity, or integrated with the enzyme reactions for the quantitative detection of the respective substrates. However, the catalytic growth of Au nanoparticles do not work in some situations, such as surface plasmon resonance (SPR), electrochemistry, where metal matrices were used, because metal matrices used in these techniques, e.g. Au, are susceptible to metal deposition, which increased the background seriously. In this work, a SiO(2) layer was vapor-deposited on the gold film. The inhibition of metal deposition by this SiO(2) layer was investigated by SPR sensor. The results showed that the SiO(2) layer could avoid the deposition of metal on Au film. With the low background achieved by SiO(2)-coated Au films, sensitive detection of DNA hybridization using the catalytic growth of Au nanoparticles enhanced SPR was demonstrated. The work described here maybe helpful for the development of sensitive bioanalytical methods.     

Allosteric aptamers controlling a signal amplification cascade allow visual detection of molecules at picomolar concentrations

A broadly applicable homogeneous detection system has been developed. It utilizes components of the blood coagulation cascade in the presence of polystyrene microspheres (MS) as a signal amplifier. Russell's viper venom factor X activator (RVV-X) triggers the cascade, which results in an eye-visible phase transition (precipitation) of MS bound to clotted fibrin. An allosteric RNA aptamer, RNA132, with affinity for RVV-X and human vascular endothelial growth factor (VEGF(165)) was created. RNA132 inhibits enzymatic activity of RVV-X. The effector molecule, VEGF(165), reverses the inhibitory activity of RNA132 on RVV-X and restores its enzymatic activity, thus, triggering the cascade and enabling the phase transition. As few as 5 fmol of VEGF(165) could be detected by the naked eye within an hour. Similar results were obtained for another allosteric aptamer modulated by a protein tyrosine phosphatase. The assay is instrumentation-free for both processing and readout and can be modified to detect molecules to which aptamers can be obtained.     

A label-free nanoparticle aggregation assay for protein complex/aggregate detection and study

The detection, analysis, and understanding of protein complexes/aggregates and their formation process are extremely important for biomolecular research, diagnosis, and biopharmaceutical development. Unfortunately, techniques that can be used conveniently for protein complex/aggregate detection and analysis are very limited. Using gold nanoparticle immunoprobes coupled with dynamic light scattering (DLS), we developed a label-free nanoparticle aggregation immunoassay (NanoDLSay) for protein aggregate detection and study. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a protein target used routinely in Western blot as a loading control, is demonstrated here as an example. Through this study, we discovered that GAPDH has a strong tendency to form large aggregates in certain buffer solutions at a concentration range of 10-25 μg/ml. The strong light scattering property of gold nanoparticles immunoprobes greatly enhanced the sensitivity of the dynamic light scattering for protein complex/aggregate detection. In contrast to fluorescence techniques for protein complex and aggregation study, the protein targets do not need to be labeled with fluorescent probe molecules in NanoDLSay. NanoDLSay is a very convenient and sensitive tool for protein complex/aggregate detection and study.     

A three-dimensional gold nanodendrite network porous structure and its application for an electrochemical sensing

A three dimensional (3D) gold (Au) nanodendrite network porous structure constructed by a simple electrochemical synthetic method has been presented, and its utility for sensitive electrochemical measurement was demonstrated in this study. The 3D nanodendrite network porous structure was constructed on a platinum surface through electrodeposition of Au under the presence of hydrogen bubbles generated from the same surface. Iodide, used as a co-reagent, played an important role in the construction of the nanodendrite network by preventing continual growth of Au into larger agglomerates as well as inhibiting coalescence of neighboring nanodendrites. An electrochemical sensor incorporating the structure was built and used to detect As(III) in ultra low concentration range. For the purpose of comparison, bare gold and gold nanoparticle-incorporated electrodes were also prepared. With the use of 3D nanodendrite network porous structure, a much more sensitive detection of As(III) was possible due to its large surface area.     

Interface-Induced Ag Monolayer Film for Surface-Enhanced Raman Scattering Detection of Water-Insoluble Enrofloxacin

Water-insoluble molecules usually show poor surface-enhanced Raman scattering (SERS) signals, because they are hardly adsorbed on the surface of most commonly used SERS substrates, such as aqueous Ag or Au colloids. In this work, a highly sensitive and reproducible Ag monolayer film (Ag MLF) SERS substrate prepared by self-assembly of Ag nanoparticles (Ag NPs) on water/oil interface can realize the trace SERS detection of water-insoluble enrofloxacin. The positively charged phase transfer catalyst can transfer the negatively charged Ag nanoparticles in aqueous solution to the water/oil interface. At the same time, the water-insoluble enrofloxacin can also be attracted to the interface because of its lipophilic group. The type/volume of the oil phase and phase transfer catalyst and the vortex mixing time were all optimized to maximize the SERS effect of Ag MLF. Results showed that trace water-insoluble enrofloxacin can be identified by Ag MLF and its detection sensitivity was significantly improved. The proposed novel Ag MLF can be further applied to detect other water-insoluble molecules in SERS.

     

Immunosensor for detection of Legionella pneumophila using surface plasmon resonance

Immunosensor using surface plasmon resonance (SPR) onto self-assembled protein G layer was developed for the detection of Legionella pneumophila. A self-assembled protein G layer on gold (Au) surface was fabricated by adsorbing a mixture of 11-mercaptoundecanoic acid (MUA) and hexanethiol (molar ratio of 1:2) and the activation process for chemical binding between free amine (-NH(2)) of protein G and 11-(MUA) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) in series. The formation of self-assembled protein G layer on Au substrate and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analyses of self-assembled protein G layer on Au substrate and monoclonal antibody against L. pneumophila immobilized on protein G were performed by atomic force microscope (AFM). The immunosensor for detection of L. pneumophila using SPR was developed and its detection limit could find up to 10(5) cells/ml.