Prion protein detection via direct immuno-quantitative real-time PCR

We describe a simple and robust assay for the quantitative detection of prions using immuno-quantitative real-time PCR (iQ-RT-PCR) made possible by a direct conjugate of a prion-specific antibody (ICSM35) and a synthetic 99-bp DNA tail. The DNA tail was engineered to include a single ScrFI restriction site, which enabled subsequent quantification of restricted DNA tails using real-time PCR. The assay was tested with scrapie prions bound to polyvinylidene difluoride membranes and to 96-well plates coated with a capturing antibody from a commercially available immuno-based assay (TeSeE). The iQ-RT-PCR assay had a detection limit corresponding to 2.32 × 10² prion epitopes, which represented a 1000-fold increase in detection sensitivity over the commercial assay. Detection of prions from diluted scrapie-positive brain homogenate bound to membranes was linear over a range of 1.06 × 10⁴ to 3.24 × 10² epitopes (R² = 0.92). Given its sensitivity and versatility, the present assay has potential to enable rapid and reliable detection of agents causing transmissible spongiform encephalopathies.     

Electrochemical detection of uric acid via uricase-immobilized graphene oxide

Measurement of the uric acid level in the body can be improved by biosensing with respect to the accuracy, sensitivity and time consumption. This study has reported the immobilization of uricase onto graphene oxide (GO) and its function for electrochemical detection of uric acid. Through chemical modification of GO using 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS) as cross-linking reagents, the enzyme activity of the immobilized uricase was much comparable to the free enzyme with 88% of the activity retained. The modified GO-uricase (GOU) was then subjected to electrocatalytic detection of uric acid (UA) via cyclic voltammetry (CV). For that reason, a glassy carbon electrode (GCE) was modified by adhering the GO along with the immobilized uricase to facilitate the redox reaction between the enzyme and the substrate. The modified GOU/GCE outperformed a bare electrode through the electrocatalytic activity with an amplified electrical signal for the detection of UA. The electrocatalytic response showed a linear dependence on the UA concentration ranging from 0.02 to 0.49 mM with a detection limit of 3.45 μM at 3σ/m. The resulting biosensor also exhibited a high selectivity towards UA in the presence of other interference as well as good reproducibility.     

Identification of inhibitors of ribozyme self-cleavage in mammalian cells via high-throughput screening of chemical libraries

We have recently described an RNA-only gene regulation system for mammalian cells in which inhibition of self-cleavage of an mRNA carrying ribozyme sequences provides the basis for control of gene expression. An important proof of principle for that system was provided by demonstrating the ability of one specific small molecule inhibitor of RNA self-cleavage, toyocamycin, to control gene expression in vitro and vivo. Here, we describe the development of the high-throughput screening (HTS) assay that led to the identification of toyocamycin and other molecules capable of inhibiting RNA self-cleavage in mammalian cells. To identify small molecules that can serve as inhibitors of ribozyme self-cleavage, we established a cell-based assay in which expression of a luciferase (luc) reporter is controlled by ribozyme sequences, and screened 58,076 compounds for their ability to induce luciferase expression. Fifteen compounds able to inhibit ribozyme self-cleavage in cells were identified through this screen. The most potent of the inhibitors identified were toyocamycin and 5-fluorouridine (FUR), nucleoside analogs carrying modifications of the 7-position and 5-position of the purine or pyrimidine bases. Individually, these two compounds were able to induce gene expression of the ribozyme-controlled reporter approximately 365-fold and 110-fold, respectively. Studies of the mechanism of action of the ribozyme inhibitors indicate that the compounds must be incorporated into RNA in order to inhibit RNA self-cleavage.     

Rapid detection of Mycobacterium tuberculosis based on antigen 85B via real-time recombinase polymerase amplification

Tuberculosis (TB), as a common infectious disease, still remains a severe challenge to public health. Due to the unsatisfied clinical needs of currently available diagnostic vehicles, it is desired to establish a new approach for universally detecting Mycobacterium tuberculosis. Herein, we designed a real-time recombinase polymerase amplification (RPA) technology for identifying M. tuberculosis within 20 min at 39°C via custom-designed oligonucleotide primers and probe, which could specifically target antigen 85B (Ag85B). Particularly, the primers F4-R4 produced the fastest fluorescence signal with the probe among four pairs of designed primers in the RPA assays. The optimal primers/probe combination could effectively identify M. tuberculosis with the detection limit of 4·0 copies per μl, as it could not show a positive signal for the genomic DNA from other mycobacteria or pathogens. The Ag85B-based RPA could determine the genomic DNA extracted from M. tuberculosis with high reliability (100%, 22/22). More importantly, when testing clinical sputum samples, the real-time RPA displayed an admirable sensitivity (90%, 95% CI: 80·0-96·0%) and specificity (98%, 95% CI: 89·0-100·0%) compared to traditional smear microscopy, which was similar to the assay of Xpert MTB/RIF. This real-time RPA based Ag85B provides a promising strategy for the rapid and universal diagnosis of TB.     

Electrochemical aptasensor for tetracycline detection

An electrochemical aptasensor was developed for the detection of tetracycline using ssDNA aptamer that selectively binds to tetracycline as recognition element. The aptamer was highly selective for tetracycline which distinguishes minor structural changes on other tetracycline derivatives. The biotinylated ssDNA aptamer was immobilized on a streptavidin-modified screen-printed gold electrode, and the binding of tetracycline to aptamer was analyzed by cyclic voltammetry and square wave voltammetry. Our results showed that the minimum detection limit of this sensor was 10 nM to micromolar range. The aptasensor showed high selectivity for tetracycline over the other structurally related tetracycline derivatives (oxytetracycline and doxycycline) in a mixture. The aptasensor developed in this study can potentially be used for detection of tetracycline in pharmaceutical preparations, contaminated food products, and drinking water.     

Electrochemical detection of lesions in DNA

Electrochemical DNA-based sensors that exploit the inherent sensitivity of DNA-mediated charge transport (CT) to base pair stacking perturbations are capable of detecting base pair mismatches and some common base damage products. Here, using DNA-modified gold electrodes, monitoring the electrocatalytic reduction of DNA-bound methylene blue, we examine a wide range of base analogues and DNA damage products. Among those detected are base damage products O4-methyl-thymine, O6-methyl-guanine, 8-oxo-guanine, and 5-hydroxy-cytosine, as well as a therapeutic base, nebularine. The efficiency of DNA-mediated CT is found not to depend on the thermodynamic stability of the helix. However, general trends in how base modifications affect CT efficiency are apparent. Modifications to the hydrogen bonding interface in Watson-Crick base pairs yields a substantial loss in CT efficiency, as does added steric bulk. Base structure modifications that may induce base conformational changes also appear to attenuate CT in DNA as do those that bury hydrophilic groups within the DNA helix. Addition and subtraction of methyl groups that do not disrupt hydrogen bonding interactions do not have a large effect on CT efficiency. This sensitive detection methodology based upon DNA-mediated CT may have utility in diagnostic applications and implicates DNA-mediated CT as a possible damage detection mechanism for DNA repair enzymes.     

Electrochemical detection and characterization of proteins

On-line detection of serum proteins is of clinical relevance, in detecting leaks and biofouling in hemofiltration equipment, biofilm growth on prosthetic devices, or hemolysis within a prosthetic or therapeutic device. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were employed to detect and analyze micromolar concentrations of four globular proteins of clinical importance. CV testing showed that identification and quantification of each of these proteins was possible through analysis of current changes at specific potentials. Preliminary CV studies into the contamination of Bovine Serum Albumin with a microgram amount of one of the other three proteins illustrated that direct detection of the contaminant protein was possible. The analysis of the EIS data demonstrated that with increase in relative concentration of proteins, the amount of electroactive proteins adsorption at the interface increases, leading to increase in surface charge density and capacitance, especially for lower molecular weight proteins. The impedance data was used to determine the values of Gibbs adsorption energy, adsorption coefficients for the four proteins, and develop an equivalent circuit model for the protein-containing solutions.     

Electrochemical biosensor based on functional composite nanofibers for detection of K-ras gene via multiple signal amplification strategy

An electrochemical biosensor based on functional composite nanofibers for hybridization detection of specific K-ras gene that is highly associated with colorectal cancer via multiple signal amplification strategy has been developed. The carboxylated multiwalled carbon nanotubes (MWCNTs) doped nylon 6 (PA6) composite nanofibers (MWCNTs–PA6) was prepared using electrospinning, which served as the nanosized backbone for thionine (TH) electropolymerization. The functional composite nanofibers [MWCNTs–PA6–PTH, where PTH is poly(thionine)] used as supporting scaffolds for single-stranded DNA1 (ssDNA1) immobilization can dramatically increase the amount of DNA attachment and the hybridization sensitivity. Through the hybridization reaction, a sandwich format of ssDNA1/K-ras gene/gold nanoparticle-labeled ssDNA2 (AuNPs–ssDNA2) was fabricated, and the AuNPs offered excellent electrochemical signal transduction. The signal amplification was further implemented by forming network-like thiocyanuric acid/gold nanoparticles (TA/AuNPs). A significant sensitivity enhancement was obtained; the detection limit was down to 30 fM, and the discriminations were up to 54.3 and 51.9% between the K-ras gene and the one-base mismatched sequences including G/C and A/T mismatched bases, respectively. The amenability of this method to the analyses of K-ras gene from the SW480 colorectal cancer cell lysates was demonstrated. The results are basically consistent with those of the K-ras Kit (HRM: high-resolution melt). The method holds promise for the diagnosis and management of cancer.     

Enhancement of RNA self-cleavage by micellar catalysis.

It has been reported recently that naturally occurring catalytic RNAs like hammerhead and hairpin ribozyme do not require metal ions for efficient catalysis. It seems that the folded tertiary structure of the RNA contributes more to the catalytic function than was initially recognized. We found that a highly specific self-cleavage reaction can occur within a small bulge loop of four nucleotides in a mini-substrate derived from Arabidopsis thaliana intron-containing pre-tRNA(Tyr) in the absence of metal ions. NH(4)(+) cations and non-ionic or zwitter-ionic detergents at or above their critical micelle concentration are sufficient to catalyze this reaction. The dependence on micelles for the reaction leads to the assumption that physical properties, i.e. the hydrophobic interior of a micelle, are essential for this self-cleavage reaction. We suggest that NH(4)(+)-ions play a crucial role for the entry of the negatively charged RNA into the hydrophobic interior of a detergent micelle. A change of the pattern of hydration or hydrogen bonds caused by the hydrophobic surrounding enhances the reaction by a factor of 100. These findings suggest that highly structured RNAs may shift pK(a) values towards neutrality via the local environment and thereby enhance their ability to perform general acid-base catalysis without the participation of metal ions.     

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.¹ PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.^2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.⁵DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.^6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 10¹⁶ individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).^9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.^6-8Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.^17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.More recently, we developed a method of isolating RFDs for bacterial detection.⁵ These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).⁵ This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.     

Multiplex real-time PCR detection of Vibrio cholerae

Cholera is an important enteric disease, which is endemic to different regions of the world and has historically been the cause of severe pandemics. Vibrio cholerae is a natural inhabitant of the aquatic environment and the toxigenic strains are causative agents of potentially life-threatening diarrhoea. A multiplex, real-time detection assay was developed targeting four genes characteristic of potentially toxigenic strains of V. cholerae, encoding: repeat in toxin (rtxA), extracellular secretory protein (epsM), mannose-sensitive pili (mshA) and the toxin coregulated pilus (tcpA). The assay was developed on the Cepheid Smart Cycler using SYBR Green I for detection and the products were differentiated based on melting temperature (Tm) analysis. Validation of the assay was achieved by testing against a range of Vibrio and non-Vibrio species. The detection limit of the assay was determined to be 10(3) CFU using cells from pure culture. This assay was also successful at detecting V. cholerae directly from spiked environmental water samples in the order of 10(4) CFU, except from sea water which inhibited the assay. The incorporation of a simple DNA purification step prior to the addition to the PCR increased the sensitivity 10 fold to 10(3) CFU. This multiplex real-time PCR assay allows for a more reliable, rapid detection and identification of V. cholerae which is considerably faster than current conventional detection assays.     

Near real-time biosensor-based detection of 2,4-dinitrophenol

A fluorescent biosensor assay has been developed for near real-time detection of 2,4-dinitrophenol (DNP). The assay was based on fluorescent detection principles that allow for the analysis of antibody/antigen interactions in solution using the KinExA immunoassay instrument. Our KinExA consisted of a capillary flow observation cell containing a microporous screen that maintains a compact capture antigen-coated bead bed. The bead bed was comprised of polymethylmethacrylate (PMMA) beads coated with dinitrophenol-human serum albumin (DNP-HSA) conjugate. Phosphate buffered saline (PBS) solutions, containing various concentrations of free DNP, were incubated for 30 min with mouse anti-DNP monoclonal antibody to equilibrium. Solutions containing the DNP-monoclonal antibody complex and possible excess free antibodies were then passed over DNP-HSA labeled beads. The free monoclonal anti-DNP antibody, if available, was then bound to the DNP-HSA fixed on the beads. The system was then flushed with excess PBS to remove unbound reactants in the bead bed. The beads were then subjected to brief contact with PBS solutions containing goat anti-mouse fluorescein isothiocyanate (FITC)-labeled secondary antibody, once again, followed by a short PBS flush. The fluorescence was recorded during the addition of the FITC labeled secondary antibody to the bead bed through the final PBS flushing with the KinExA. The amount of DNP detected could then be determined from the fluorescent slopes that were generated or by the remaining fluorescence that was retained on the beads after final PBS flushing of the system. This assay has been able to detect a minimum of 5 ng/ml of DNP in solution and can be adapted for other analytes of interest simply by changing the capture antigen and antibody pairs.     

Quantitative detection of periodontopathogens by real-time PCR

Specific bacteria are believed to play an important role in chronic periodontitis, yet the significance of their relative numbers in initiation and progress of the disease is still unclear. We report here the development of a sensitive, quantitative PCR technique for enumerating Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Dialister pneumosintes (Dp) and Micromonas micros (Mm) as well as total eubacteria in subgingival plaque samples from subjects with periodontitis. Quantification was performed with specific 16S rRNA target sequences with double fluorescence labeled probes and serial dilutions of plasmid standard by real-time PCR. This method showed a broad quantification range from 10(2) to 10(8) and accurate sensitivity and specificity. Fifty subgingival plaque samples from periodontitis patients and 33 from periodontally healthy subjects were subsequently examined. Higher levels of total bacteria numbers, Aa, Pg, Dp and Mm were found in samples from periodontitis subjects in comparison to samples from periodontally healthy subjects. Quantitative real-time PCR thus provides a reliable and valuable method for quantification of periodontopathogens in subgingival plaque samples.     

Electrochemical detection of dopamine using porphyrin-functionalized graphene

A new type of porphyrin-functionalized graphene was synthesized and used for highly selective and sensitive detection of dopamine (DA). The aromatic π-π stacking and electrostatic attraction between positively-charged dopamine and negatively-charged porphyrin-modified graphene can accelerate the electron transfer whereas weakening ascorbic acid (AA) and uric acid (UA) oxidation on the porphyrin-functionalized graphene-modified electrode. Differential pulse voltammetry was used for electrochemical detection, the separation of the oxidation peak potentials for AA-DA, DA-UA and UA-AA is about 188 mV, 144 mV and 332 mV, which allows selectively determining DA. The detection limit of DA can be as low as 0.01 μM. More importantly, the sensor we presented can detect DA in the presence of large excess of ascorbic acid and uric acid. With good sensitivity and selectivity, the present method was applied to the determination of DA in real hydrochloride injection sample, human urine and serum samples, respectively, and the results was satisfactory.     

Electrochemical immunosensor for the detection of atrazine

An amperometric immunosensor for the detection of the herbicide atrazine has been developed. A redox polymer PVPOs(bpy)2Cl was co-immobilized with the specific antibody on the surface of the electrode by crosslinking with PEGDGE to form an electron-conducting hydrogel. In a competitive assay the occurrence of the antibody-antigen reaction on the surface of the sensing film was detected through the 'electrical wiring' of the redox centres of antigen-labelled horseradish peroxidase and the electrode surface in the presence of H2O2 at 0.1 V (vsAg/AgCl).     

Nested real-time PCR for hepatitis A detection

Aims:  The hepatitis A virus (HAV) is one of the most important human foodborne pathogens causing a number of worldwide outbreaks each year. The detection of HAV in food samples remains a complex issue, because commonly used detection tools, such as conventional or even real-time PCR assays, are often unable to detect HAV with sufficient sensitivity. The aims of this study were to develop highly sensitive and specific nested real-time PCR (NRT-PCR)-based method for HAV detection in food and to compare it with currently available methods.
Methods and Results:  By combining conventional PCR, nested PCR and real-time PCR techniques, we have developed a specific NRT-PCR assay for the detection of HAV. The procedure involves two consecutive PCRs, the first of which is performed as a conventional RT-PCR using primers specific for HAV 5' noncoding region. The second reaction involves a real-time PCR using a nested primer pair specific for the first PCR product and a TaqMan probe.
Conclusions:  We have developed a novel NRT-PCR method capable of detecting as little as 0·2 PFU of HAV, which is significantly more sensitive than any other PCR technique tested in our system.
Significance and Impact of the Study:  NRT-PCR provides a potentially useful method for detecting HAV at extremely low levels, as frequently found in food samples, and can be potentially adopted as a regulatory method to ensure food safety.