Highly sensitive electrochemical aptasensor for immunoglobulin E detection based on sandwich assay using enzyme-linked aptamer

Here, we describe the fabrication of an electrochemical immunoglobulin E (IgE) aptasensor using enzyme-linked aptamer in the sandwich assay method and thionine as redox probe. In this protocol, 5′-amine-terminated IgE aptamer and thionine were covalently attached on glassy carbon electrode modified with carbon nanotubes/ionic liquid/chitosan nanocomposite. Furthermore, another IgE aptamer was modified with biotin and enzyme horseradish peroxidase (HRP), which attached to the aptamer via biotin–streptavidin interaction. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry were performed at each stage of the chemical modification process to confirm the resulting surface changes. The presence of IgE induces the formation of a double aptamer sandwich structure on the electrode, and the electrocatalytic reduction current of thionine in the presence of hydrogen peroxide was measured as the sensor response. Under optimized conditions and using differential pulse voltammetry as the measuring technique, the proposed aptasensor showed a low detection limit (6 pM) and high sensitivity (1.88 μA nM⁻¹). This aptasensor also exhibited good stability and high selectivity for IgE detection without an interfering effect of some other proteins such as bovine serum albumin (BSA) and lysozyme. The application of the aptasensor for IgE detection in human serum sample was also investigated. The proposed protocol is quite promising as an alternative sandwich approach for various protein assays.     

Highly sensitive oligonucleotide-based fluorometric detection of mercury(II) in aqueous media

This paper describes a highly sensitive and selective Hg(2+) sensor using a label free Hg(2+) specific probe (5'-18T-3') and an intercalation dye SYBR Green I (SG). The Hg(2+) specific probe is composed of thymines (T) and readily forms T-Hg(2+)-T complexes in the presence of Hg(2+). This specific T-Hg(2+)-T formation affects the hybridization of the Hg(2+) specific probe and the intercalation of SG. Upon treatment of 1 nM 5'-18T-3' with different amount of Hg(2+) (0.1-10nM), which is followed by hybridization with 1 nM 5'-18T-3' and incubation with 1 microL of SG, the solution fluorescence gave a linear response (R=0.996) to the concentration of Hg(2+). The detection limit for Hg(2+) was 0.5 nM (0.1 ppb). The overall test only takes few minutes and very little interference is observed from non-specific metal ions. This approach may find potential applications in monitoring the Hg(2+) concentration in drinking water.     

Highly sensitive enzyme-linked assay based on monoclonal antibodies for detection of Brucella antigens

Mice monoclonal antibodies against lypopolysaccharides (LPS) of Brucella abortus has been obtained and characterized. The antibodies detected LPS of B. abortus, B. melitensis and B. suis with high sensivity and specificity and did not react with LPS of Yersinia enterocolitica O:3, Y. enterocolitica O:9, Salmonella typhimurium, and Francisella tularensis. It has been shown that interaction of monoclonal antibodies and LPS of Brucella species can be critically dependent from buffer system. Obtained monoclonal antibodies allowed to develop highly sensitive assay which was able to detect antigens of Brucella species in concentrations 0.05 - 0.1 ng/ml. The assay can be used for detection and identification of Brucella species.     

Highly sensitive detection of cocaine using a piezoelectric immunosensor

This paper describes the development of a highly sensitive competitive immunoassay with the piezoelectric sensor. The immobilized derivative of cocaine was benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO). For the immobilization of BZE-DADOO, the conjugate BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) was synthesized via 2-(5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TNTU), followed by the creation of the conjugate monolayer on the piezosensor electrodes. For the optimization of the competitive assay we used electrodes with rough or smooth gold areas and for the interaction with immobilized antigen different anti-cocaine sheep polyclonal (pAb, either whole IgG or Fab fragment) and mouse monoclonal (mAb, whole IgG) antibodies. The assay of cocaine developed achieved a detection limit (LOD) of 100 pmol/l (34 ng/l) using the sheep antibody (IgG) and piezoelectric sensors with a smooth gold surface. The total time of one analysis was 15 min and the measuring area of the sensor could be used more than 40 times without losing its sensitivity.     

Highly sensitive amperometric detection of genomic DNA in animal tissues

A simple and highly sensitive method for the detection of genomic DNA in tissue samples is described. It is based on amperometric detection of target DNA by forming an analyte/polymeric activator bilayer on a gold electrode. The biotinylated target DNA is hybridized to oligonucleotide capture probes immobilized on the gold electrode, forming the first layer. A subsequent binding of glucose oxidase– avidin conjugate to the target DNA and the introduction of a second layer of a redox polymer to the electrode, via layer-by-layer electrostatic self-assembly, allow for electrochemical detection of the catalytic oxidation current of glucose in a PBS solution. Less than 2.0 fg of rat genomic DNA, for both regulated and house-keeping genes, can be easily detected in 2.5 µl droplets. The proposed procedure shows very high specificity for genomic DNA in a RT–PCR mixture.     

Highly sensitive method for the determination of melatonin by normal-phase high-performance liquid chromatography with fluorometric detection

In the present study a new chromatographic method was developed to quantify melatonin in rat pineal that can be extended to other tissues. Melatonin was extracted from an acid homogenate with ethyl acetate to avoid amine interference. HPLC was performed with silica normal-phase column and fluorescence detection. This method is sensitive enough for detecting melatonin in a single pineal gland with a detection limit of 3 pg/mg tissue.     

Highly sensitive time-resolved fluorometric determination of estrogens by high-performance liquid chromatography using a beta-diketonate europium chelate

A new fluorescent europium chelate labeling reagent, 5-(4"-chlorosulfo-1',1"-diphenyl-4'-yl)-1,1,1,2,2-pentafluoro-3,5-pentanedione (CDPP), was synthesized for the time-resolved fluorometric detection of HPLC. The label can be directly bound to amino or phenolic hydroxyl groups of analytes with its chlorosulfonyl group, and the labeled analytes are separated on a HPLC column. After separation, EuCl(3), TOPO (tri-n-octylphosphine oxide), and Triton X-100 were added by post-column introduction to the eluent, and the fluorescence of the europium chelate was measured with the time-resolved fluorometric detector. Estrone (E1), 17beta-estradiol (E2), ethynylestradiol (EE2) and estriol (E3) were measured with the detection limits of 0.65, 0.65, 0.65 and 0.60 ng/ml, respectively. The recovery for river water samples was in the range of 86.0-105.1% with the RSD of 1.9-5.8%. The method was applied to the analysis of a river water sample and estrone (E1) was determined to be 2.1 ng/l. The results and processing have been compared with those of a GC-MS method and a high degree of correlation (r> or =0.98) was observed.     

A sensitive fluorometric assay for the determination of DNA

A sensitive fluorometric assay for DNA determination using m-diaminobenzoic acid dihydrochloride for the reaction with deoxyribose liberated by perchloric acid is described. Fluorescence is proportional to the amount of DNA over the range 0.05–5 μg of DNA when the reaction is conducted in a volume of 400 μl and over the range 0.01–0.5 μg when the reaction is conducted in a volume of 40 μl. The assay is suitable for estimating DNA both in perchloric acid extracts and in complex mixtures with protein and RNA. The determination of DNA in cells grown as a monolayer by the described method is simple and rapid.     

Highly sensitive and selective detection of thiol-containing biomolecules using DNA-templated silver deposition

In this work, we report a new fluorescent method for the label-free assay of thiol-containing amino acids and peptide by use of silver deposited DNA duplex and the intercalating dye. The sensing approach is based on the specific interactions between thiols and DNA-templated silver deposition via robust Ag-S bonds. In the presence of thiols, the intercalating dye gives a dramatic increase in fluorescence as a result of the strong interaction between the intercalator and the released DNA from silver surfaces. The detection limit of this method is lower than or at least comparable to previous fluorescence-based methods, and the turn-on sensing mode offers additional advantage to efficiently reduce background noise. Moreover, the detection and discrimination process can be observed by the naked eye with the aid of an UV transilluminator. This method also exhibits excellent selectivity for these thiol-containing biomolecules over various other amino acids. As far as we know, our method is the first example using the specific interaction between thiols and silver-coated DNA to fabricate a turn-on fluorescent sensor for biological thiols with high sensitivity and selectivity.     

Colorimetric silver detection of DNA microarrays

Development of microarrays has revolutionized gene expression analysis and molecular diagnosis through miniaturization and the multiparametric features. Critical factors affecting detection efficiency of targets hybridization on microarray are the design of capture probes, the way they are attached to the support, and the sensitivity of the detection method. Microarrays are currently detected in fluorescence using a sophisticated confocal laser-based scanner. In this work, we present a new colorimetric detection method which is intented to make the use of microarray a powerful procedure and a low-cost tool in research and clinical settings. The signal generated with this method results from the precipitation of silver onto nanogold particles bound to streptavidin, the latter being used for detecting biotinylated DNA. This colorimetric method has been compared to the Cy-3 fluorescence method. The detection limit of both methods was equivalent and corresponds to 1 amol of biotinylated DNA attached on an array. Scanning and data analysis of the array were obtained with a colorimetric-based workstation.     

Highly sensitive detection of protein phosphorylation by using improved Phos-tag Biotin

We have previously shown that the dinuclear zinc(II) complex Phos-tag and its derivatives act as phosphate-capture molecules in aqueous solution under conditions of neutral pH. In this study, our aim was to develop more-advanced applications for the detection of phosphopeptides and phosphoproteins by using several newly synthesized Phos-tag derivatives, including a bisbiotinylated Phos-tag (BTL-108), a tetrakisbiotinylated Phos-tag (BTL-109), and a monobiotinylated Phos-tag with a dodeca(ethylene glycol) spacer (BTL-111), as well as the commercially available product BTL-104. Among these complexes, BTL-111 showed the best performance in Western blotting by an ECL system using HRP conjugated streptavidin. In addition, in a quartz-crystal microbalance analysis of a phosphoprotein, the presence of the long hydrophilic dodeca(ethylene glycol) spacer in a novel Phos-tag sensor chip coated with BTL-111 resulted in a greater sensitivity than was achieved with a similar chip coated with BTL-104. Moreover, a peptide microarray technique using the ECL system and BTL-111 permitted high-throughput assays for the specific and highly sensitive detection of protein kinase activities in cell lysates.     

Nanoparticle assembly for sensitive DNA detection using SERRS

SERRS (surface-enhanced resonance Raman scattering) is a vibrational technique, whereby a relatively weak Raman scattering effect is enhanced through the use of a visible chromophore and a roughened metal surface. The direct analysis of DNA by SERRS requires the modification of a nucleic acid sequence to incorporate a chromophore, and adsorption of the modified sequence on to a roughened metal surface. Aggregated metallic nanoparticles are commonly used in the analysis of dye-labelled DNA by SERRS, allowing for detection levels that rival those gained from standard fluorescence-based techniques. In the present paper, we report on how SERRS can be exploited for the analysis of clinically relevant DNA samples. We also report on the ability of nanoparticles to aggregate as the result of a biologically significant event, as opposed to the use of an external charge-modifying agent. The self-assembly of metallic nanoparticles is shown to be a promising new technique in the move towards extremely sensitive methods of DNA analysis by SERRS.     

Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a

Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify B. pseudomallei DNA from clinical isolates. Through in silico search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to B. pseudomallei, named crBP34. The crBP34-based detection assay can detect as few as 40 copies of B. pseudomallei genomic DNA while discriminating against other tested common pathogens. When coupled with a lateral flow dipstick, the assay readout can be simply performed without the loss of sensitivity and does not require expensive equipment. This crBP34-based detection assay provides high sensitivity, specificity and simple detection method for B. pseudomallei DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA.     

Highly sensitive detection of organophosphorus insecticides using magnetic microbeads and genetically engineered acetylcholinesterase

This work presents a biosensor for organophosphorus pesticides based on immobilisation of a highly sensitive genetically engineered acetylcholinesterase (B394) by affinity interactions on metal chelate-functionalised magnetic microbeads. The developed sensor has been compared with those based on the widely used Electric eel cholinesterase and a classical entrapment procedure in a polyvinylalcohol-based matrix. The use of the B394 enzyme allowed lowering both IC50 and LOD by a factor of 100 when compared with Electric eel enzyme sensor. The oriented and site-specific immobilisation combined with the high specificity of the B349 mutant allows a more sensitive detection of insecticides, concentrations as low as 1.31(-11)M (IC10) being detected for both pesticides chlorpyriphos-oxon and chlorfenvinphos.     

Highly sensitive detection of cytotoxicity using a modified HSP70B' promoter

We have previously found that the DNA fragment from nucleotides (nts) -287 to +110 in the HSP70B' gene is a functional promoter responding to Cadmium Chloride-induced cytotoxicity (Wada et al., Biotechnol Bioeng, 92, 410-415, 2005). In order to increase the cytotoxic response of this promoter, we first determined the location of the cytotoxic responding element (CRE) and then constructed tandem repeats of the CRE in front of the HSP70B' promoter. 5'- and 3'-deletion analysis revealed that the DNA fragment from nts -192 to -56 in the HSP70B' gene induces a significant response to cytotoxicity. When the AP-1 binding site in this region was mutated, the basal activity of HSP70B' gene promoter decreased but the cytotoxic response was unchanged. Thus, the CRE is located in nts -192 to -56 in the HSP70B' promoter, and the AP-1 binding site is not essential for the cytotoxic response. In addition, cells transfected with a luciferase construct carrying three tandem repeats of the CRE upstream of the HSP70B' promoter and containing AP-1 binding site mutation, showed a 2.28-fold higher response than that of no repeats. Moreover, the detection limit of Cadmium Chloride in the cells was 382 pmol/mL. Thus, highly sensitive sensor cells for Cadmium Chloride can be constructed using a HSP70B' promoter construct containing upstream tandem repeats of the CRE and mutation of the AP-1 binding site.     

Highly sensitive detection of target gene by electrochemical method.

A highly sensitive DNA sensing method was developed using electrochemically active ligand. This method is based on the detection of electric current generated by electrochemically active ligand concentrated on the electrode. Electrochemically active, intercalating ligand can bind to the double stranded DNA of target gene sequence on the electrode, where the complementary single strand is immobilized as a probe. We succeeded in the detection of 0.1 amol target gene. The technique was applicable to the detection of 0.1-10 amol gene.