Bioconjugation of alkaline phosphatase to mechanically processed, aqueous suspendible electrospun polymer nanofibers for use in chemiluminescent detection assays

Aqueous suspendible polymer nanostructures were prepared by simple microtome processing of electrospun nylon 6 nanofibers and were used to immobilize calf intestinal alkaline phosphatase (ALP) by either covalent or noncovalent bioconjugation chemistries. It was found that noncovalent immobilization of ALP to the mechanically cut nanofibers (mean length approximately 4 microm; mean diameter approximately 80 nm) using a multi-stacked, layer-by-layer (LBL) approach with the cationic polymer Sapphire II resulted in the highest enzyme loading (48.1 +/- 0.4 microg . mg(-1) nanofiber) when compared to other covalent immobilization methods based on glutaraldehyde crosslinking. The biofunctionalized nanofibers were also characterized for their chemiluminescent activity with the dioxetane substrate, CSPD. The results indicate that the kinetic parameters, K(m) and V(max), for the catalytic activity of the nanostructure-bound ALP enzyme were influenced by the particular types of immobilization methods employed. In terms of the overall catalytic performance of the various immobilized ALP systems, a single-stacked LBL assembly approach resulted in the highest level of enzymatic activity per unit mass of nanofiber support. To the best of our knowledge, this study represents the first report examining the preparation of mechanically shortened, aqueous dispersed electrospun polymer nanofibers for potential application as enzyme scaffolds in chemiluminescent-based assay systems.     

Cotinine-conjugated aptamer/anti-cotinine antibody complexes as a novel affinity unit for use in biological assays

Aptamers are synthetic, relatively short (e.g., 20-80 bases) RNA or ssDNA oligonucleotides that can bind targets with high affinity and specificity, similar to antibodies, because they can fold into unique, three-dimensional shapes. For use in various assays and experiments, aptamers have been conjugated with biotin or digoxigenin to form complexes with avidin or anti-digoxigenin antibodies, respectively. In this study, we developed a method to label the 5'' ends of aptamers with cotinine, which allows formation of a stable complex with anti-cotinine antibodies for the purpose of providing another affinity unit for the application in biological assays using aptamers. To demonstrate the functionality of this affinity unit in biological assays, we utilized two well-known aptamers: AS1411, which binds nucleolin, and pegaptanib, which binds vascular endothelial growth factor. Cotinine-conjugated AS1411/anti-cotinine antibody complexes were successfully applied to immunoblot, immunoprecipitation, and flow cytometric analyses, and cotinine-conjugated pegaptanib/anti-cotinine antibody complexes were used successfully in enzyme immunoassays. Our results show that cotinine-conjugated aptamer/anti-cotinine antibody complexes are an effective alternative and complementary technique for aptamer use in multiple assays and experiments.     

Label-free voltammetric detection of single-nucleotide mismatches recognized by the protein MutS

MutS, a protein involved in DNA mismatch repair, recognizes mispaired and unpaired bases in duplex DNA. We have previously used MutS in an electrochemical double-surface technique (DST) for in-vitro detection of point mutations in DNA. The DST involved binding of unlabeled MutS to DNA heteroduplexes at the surface of magnetic beads followed by a highly sensitive electrochemical determination of the protein by measurement of a catalytic protein signal (peak H) at mercury electrodes. Detection of MutS using a peak resulting from oxidation of tyrosine and tryptophan residues of the protein at a carbon-paste electrode (CPE) was also possible but was approximately three orders of magnitude less sensitive. In this work we present an optimized technique for ex-situ voltammetric determination of MutS at a CPE. Choice of optimum experimental conditions (pH of supporting electrolyte, square-wave voltammetry settings, etc.) resulted in substantial improvement of the sensitivity of the assay, enabling detection of approximately 140 pg (1.6 fmol protein monomer) MutS in a 5-μL sample. The sensitivity was increased further by acid hydrolysis of the protein before measurement. The hydrolyzed protein was detectable down to 5 pg (approx. 56 amol) MutS in 5 μL solution. By using the DST combined with determination of the bound unlabeled MutS at the CPE we demonstrated selective interactions of the protein with single-base mismatches and discrimination among different base mispairs in 30-mer or 95-mer DNA duplexes. In agreement with previous studies, binding of the protein to the 30-mer substrates followed the trend G:T>>C:A>A:A>C:T>homoduplex. The electrochemical data were confirmed by use of an independent technique—a quartz-crystal microbalance for real-time monitoring of MutS interactions with DNA duplexes containing different base mispairs. By using the electrochemical DST a G:T mismatch was detectable in up to 1000-fold excess of homoduplex DNA.     

胶体金免疫层析方法快速检测腹泻性贝毒软海绵酸的初步研究

建立了赤潮毒素腹泻性贝毒软海绵酸的快速胶体金免疫层析检测方法。通过细胞融合,制备抗软海绵酸单克隆抗体,胶体金标记抗体,建立快速检测软海绵酸的免疫层析试纸条方法。检出限500 ng/mL(50 ng/条),探讨了影响测试方法的因素和提高灵敏度的可能手段。     

Label-free detection of DNA mutations by SPR: application to the early detection of inherited breast cancer

A screening analysis of DNA hybridization and the presence of DNA mutations using an surface plasmon resonance (SPR) biosensor is shown. The influence of lateral and vertical spacers, as well as several hybridization conditions, was studied to optimize the differentiation between fully complementary and mismatched DNA strands. Our results demonstrated that SPR biosensors were able to detect mismatch sequences related to inherited breast cancer, with high specificity and sensitivity. Using PCR synthetic sequences as targets, mutant sequences were clearly discriminated from fully complementary ones, and detection limits below 50 nM were achieved.     

Label-free colorimetric detection of specific sequences in genomic DNA amplified by the polymerase chain reaction

We document the surprising result that single-stranded DNA adsorbs on negatively charged gold nanoparticles (Au-nps) with a rate that depends on sequence length and temperature. After ss-DNA adsorbs on Au-nps, we find that the particles are stabilized against salt-induced aggregation. These observations can be rationalized on the basis of electrostatics and form the basis for a colorimetric assay to identify specific sequences and single nucleotide polymorphisms on polymerase chain reaction (PCR)-amplified DNA. The assay is label-free, requires no covalent modification of the DNA or Au-np surfaces, and takes on the sensitivity of PCR. Most important, binding of target and probe takes place in solution where hybridization occurs in less than 1 min. As an example, we test PCR-amplified genomic DNA from clinical samples for single nucleotide polymorphisms (SNPs) associated with a fatal arrhythmia known as long QT syndrome.     

Two molecular assays for the rapid and inexpensive detection of GJB2 and GJB6 mutations

The hypoacusia can be classified in two clinical forms: Syndromic (SHL) and Nonsyndromic (NSHL). In particular, the NSHL describes the 70–80% of hypoacusia cases and it is mainly due to genetic factors, which are causative of the deafness at the birth. The genetic hypoacusia presents different inheritance patterns: autosomal dominant (20%), autosomal recessive (80%), X‐linked (1%), and mitochondrial (1%), respectively. To date, about 35 deafness‐causative genes have been identified and most of them codify for connexin transmembrane proteins. Approximately 1:2500 children with NSHL carries mutations in the GJB2 and GJB6 (13q12) genes, which code for connexin 26 (Cx26) and connexin 30 (Cx30), respectively. In the Caucasian population, the most common mutations are 35delG, M34T and 167delT, and D13S1830. Given the frequency distribution of the four mutations in the Caucasian population and the pathogenic connection with NSHL, the development of accurate, rapid, and “low‐cost” molecular assays should be strongly encouraged. To this purpose, we set up two different molecular assays (namely the Cx26 and Cx26‐30 molecular assays) for the fast and inexpensive detection of 35delG, M34T, 167delT, and D13S1830 mutations. Both the molecular approaches showed to be accurate, sensitive, reproducible, and “low‐cost” alternatives for the proper evaluation of the GJB2 and GJB6 genes, which are causative of NSHL. In conclusion, the Cx26 and Cx26‐30 molecular assays can be applied to individual, preconception, prenatal, or postnatal screening for the causative‐mutations of NSHL.     

Fluorescence‐based lateral flow assays for rapid oral fluid roadside detection of cannabis use

With the recent worldwide changes in the legalization of marijuana, there is a significant need for rapid, roadside screening test for driving under the influence of drugs. A robust, sensitive, lateral flow assay has been developed to detect recent use via oral‐fluid testing for Δ⁹‐tetrahydrocannabinol (THC). This proof‐of‐concept assay uses a fluorescent‐based immunoassay detection of polymeric beads, conjugated to antibodies against native THC. The fluorescent technique allows for significantly lower limits of detection and higher precision determination of recent marijuana use without the use of urine or blood sampling—thus allowing for roadside identification. Detection levels of 0.01 ng/mL were distinguished from background and the lower limit of quantification was determined to approach 1 ng/mL.     

Enhancement of relaxivity rates of Gd-DTPA complexes by intercalation into layered double hydroxide nanoparticles

In this paper we report the preparation and characterization of [Gd(dtpa)](2-) intercalated layered double hydroxide (LDH) nanomaterials. [Gd(dtpa)](2-) (gadolinium(III) diethylene triamine pentaacetate) was transferred into LDH by anionic exchange. The intercalation of [Gd(dtpa)](2-) into LDH was confirmed by X-ray diffraction for the new phase with the interlayer spacing of 3.5-4.0 nm and by FTIR for the characteristic vibration peaks of [Gd(dtpa)](2-). The morphology of the nanoparticles was influenced by the extent of [Gd(dtpa)](2-) loading, in which the poly-dispersity quality decreased as the [Gd(dtpa)](2-) loading was increased. Compared with the morphology of the original Mg(2)Al-Cl-LDH nanoparticles (hexagonal plate-like sheets of 50-200 nm), the modified LDH-Gd(dtpa) nanoparticles are bar-like with a width of 30-60 nm and a length of 50-150 nm. LDH-Gd(dtpa) was expected to have an increased water proton magnetic resonance relaxivity due to the intercalation of [Gd(dtpa)](2-) into the LDH interlayer that led to slower molecular anisotropic tumbling compared with free [Gd(dtpa)](2-) in solution. Indeed, LDH-nanoparticle suspension containing approximately 1.6 mM [Gd(dtpa)](2-) exhibits a longitudinal proton relaxivity r(1) of approximately 16 mM(-1) s(-1) and a transverse proton relaxivity r(2) of approximately 50 mM(-1) s(-1) at room temperature and a magnetic field of 190 MHz, which represents an enhancement four times (r(1)) and 12 times (r(2)) that of free [Gd(dtpa)](2-) in solution under the same reaction conditions. We have thus tailored LDH-nanoparticles into a novel contrast agent with strong relaxivity, promising for great potential applications in magnetic resonance imaging.     

Label-free electrochemical aptasensor constructed by layer-by-layer technology for sensitive and selective detection of cancer cells

Here, a cytosensor was constructed with ferrocene-appended poly(allylamine hydrochloride) (Fc-PAH) functionalized graphene (Fc-PAH-G), poly(sodium-p-styrenesulfonate) (PSS) and aptamer (AS1411) by layer-by-layer assembly technology. The hybrid nanocomposite Fc-PAH-G not only brings probes on the electrode and also promotes electron transfer between the probes and the substrate electrode. Meanwhile, LBL technology provides more effective probes to enhance amplified signal for improving the sensitivity of the detection. While AS1411 forming G-quardruplex structure and binding cancer cells, the current response of the sensing electrode decreased due to the insulating properties of cellular membrane. Differential pulse voltammetry (DPV) was performed to investigate the electrochemical detection of HeLa cells attributing to its sensitivity of the current signal change. The as-prepared aptasensor showed a high sensitivity and good stability, a widely detection range from 10 to 10⁶ cells/mL with a detection limit as low as 10 cells/mL for the detection of cancer cells.     

Label-free detection of DNA molecules on the dendron based self-assembled monolayer by electrochemical impedance spectroscopy

We report preparation of a novel platform for effective DNA hybridization and its application to the detection of single mismatched DNA. Cone-shaped dendrimer molecules have been immobilized on the gold surface at equidistance, 3.1 nm, from each other with a probe DNA molecule attached to the top of each dendrimer so that enough space would be secured for effective hybridization. This arrangement allows each probe DNA molecule to form a natural DNA double helix upon hybridization with a target DNA molecule. The single nucleotide polymorphism at either the central or end position of the 25-mer target DNA has been shown to be effectively discriminated against on this platform from each other as well as from a complementary DNA by electrochemical impedance measurements. We also report adverse effects exerted by probe ions, Fe(CN)₆^3−/4−, on DNA hybridization reactions. The significance of the results for the use in DNA analysis is discussed.     

Label-free capacitive immunosensor based on quartz crystal Au electrode for rapid and sensitive detection of Escherichia coli O157:H7

The capability of ICP-MS equipped with the high matrix introduction system (HMI) for accurate analysis of lanthanoids in environmental samples was investigated. Compared to the conventional operation, the amounts of oxide and hydroxide molecular species formed in the plasma were reduced by up to 5 times. The relative yields of oxides did not exceed 0.02% for BaO⁺ species and were as low as 0.3% for lanthanoids with the highest oxide-formation rates (LaO⁺, CeO⁺, PrO⁺ and NdO⁺). Hydroxide formation was less than 0.02% when HMI system was used. In addition, two digestion procedures were evaluated by the analysis of standard reference materials (SRMs) of different matrices. The digestion efficiency and limits of detection (LOD) were improved when samples were digested with a mixture of HNO₃/H₂O₂/HCl/HF, and HF was removed by evaporation in presence of concentrated HCl. Using this procedure and HMI-ICP-MS analysis, LOD ranged from 0.1 μg kg⁻¹ to 6 μg kg⁻¹. Recoveries ranged from 85 to 115% for La to Ho and from 75 to 85% for the other lanthanoids. Relative standard deviations for replicate analysis of SRMs were less than 10%.     

Label-free detection of amino acids using gold nanoparticles in electrokinetic chromatography-thermal lens microscopy

To achieve label-free detection of amino acids in capillary-based electrokinetic chromatography-thermal lens microscopy (EKC-TLM), gold nanoparticles (GNPs), which possess the absorption around 500nm attributed to surface plasmon resonance (SPR), were added to the background solution (BGS). Since the SPR absorption of the GNPs exhibits a sensitive response toward environmental changes and the degree of aggregation, the sensitive detection of non-absorbing species is expected by using the GNPs in EKC-TLM (GNP-EKC-TLM). In the GNP-EKC-TLM analysis of glutamic acid (Glu), a sharp peak was observed when the GNPs were added to the BGS. The plot of the peak area of Glu against its concentration gave a good linear relationship and the limit of detection was estimated to be 25mug/mL. Furthermore, a baseline separation of lysine and Glu was successfully achieved. Thus, the EKC separation and label-free TLM detection of the amino acids can be realized only by adding the GNPs into the BGSs.     

Evaluation of binding affinity of protein-mutant dna complexes in solution by laser spray mass spectrometry

We have applied laser spray mass spectrometry developed by Hiraoka et al. to investigate the binding affinity of protein-mutant DNA complexes. The results were compared with our previous data of collision-induced dissociation (CID) experiments using electrospray ionization mass spectrometry (ESI-MS). Systematic experiments were carried out on the complexes of the c-Myb DNA binding domain (c-Myb DBD) bound to eight kinds of 16- or 22-mer point mutant double-stranded DNA (dsDNA), whose solution K(d) values are different in the range from 10(-9) M to 10(-7) M. The dissociation curve as a function of laser power was plotted for each complex, and the laser power where 50% of complex was dissociated (E(50%)) in population was obtained. The correlation coefficient between E(50%) and the relative binding free-energy change (DeltaDeltaG) of each complex formation in solutions was 0.9808, which is much better than the coefficient obtained by the previous ESI-CID experiments that was 0.859. In addition, complexes of the c-Myb DBD with five other mutant dsDNA were also examined to confirm that laser spray can be used to estimate the K(d) values of a DNA-protein complex in solutions if an appropriate calibration curve is available. In the process of laser spray, dissociations of these noncovalent complexes occur in solutions, but not in the gas phase. This differs greatly from ESI-CID. Laser spray mass spectrometry has been found to be better than ESI-CID in evaluating binding affinity of a protein to various mutant DNA.     

Development and validation of rapid disequilibrium enzyme-linked immunosorbent assays for the detection of Methyl Yellow and Rhodamine B dyes in foods

Sensitive and specific enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of two illegal synthetic dyes: Methyl Yellow (MY) and Rhodamine B (RB) in food. Polyclonal antibodies were raised against synthesised immunogens and employed in unique direct disequilibrium ELISAs. The time of the assays was only twenty minutes (five minutes for each incubation step with sample and enzyme conjugate and ten minutes with enzyme substrate). The IC(50) for MY was in the range 1.4-4.2 ng mL(-1) and for RB 0.1-0.5 ng mL(-1). A simple sample preparation method was developed for the analysis of a range of sauces. In the case of spices a dispersive solid phase extraction was applied to purify the extracts. The testing of twenty samples took approximately one and a half hours (including sample preparation and analysis). Both assays were validated according to the Commission Decision 2002/657/EC criteria for use in sauces and spices. The detection capability for MY in sauces and spices was determined to be less than 15 ng g(-1) and 50 ng g(-1), respectively and for RB, 10 ng g(-1) for both types of food samples. The precision of the developed assays was determined in a repeatability study. The intra- and inter-assay coefficients of variation were less than 25% for both tests and matrix types. The simplicity and performance of both assays indicate that they will be very reliable screening methods for the detection of the illegal dyes MY and RB in a range of food products.