Development of Lateral Flow Fluorescence Assay for the Detection of Trypanosoma

Trypanosoma such as Trypanosoma brucei and Trypanosoma cruzi, the causative agents for African sleeping sickness and Chagas disease, respectively, have important influence on human health. The methods such as microscopic examination, immunological methods, and molecular methods are used for the identification and detection of Trypanosoma, but none of these methods are ideal to mass screening of samples such as onset of outbreak, epidemiological surveys, and blood unit screening. Therefore, there is a need for an assay which can rapidly, sensitively and specifically detect Trypanosoma. In this study, Ru(bpy)32+-doped silica nanoparticles (RuSNP) were used to target nucleic acid sequences in a lateral flow fluorescent assay. This assay was developed to improve the sensitivity and lower the limit of detection as compared to the traditional lateral flow assay. The assay targeted both the spliced leader sequence as well as the polyA tail of the mRNA. The surface of spherical RuSNP was modified by glycidoxypropyl trimethoxysilane (GOPTMS). Amine-terminated oligonucleotides as a bioreceptor were immobilized onto the RuSNP via the interaction between the NH2 and the epoxy group of the GOPTMS. The conjugate complexes formed were immobilized on the conjugate pad, and the capture oligonucleotides used for test and control lines were immobilized on the nitrocellulose membrane. The effects of the amount of RuSNP, GOPTMS, amine-capped oligonucleotides, and capture oligonucleotides on the test line on the performance of the test strips were investigated and optimized. The fluorescence intensity was evaluated by using a fluorescent microplate reader. The experimental results showed that the nucleic acid sequence-based and RuSNP-labeled lateral flow assay was very sensitive compared with the gold-labeled test strips and the chemiluminescent test strips we developed previously, and that the limit of detection (LOD) of the test strips developed is 0.4 fmol. The LOD can further be reduced about one order of magnitude when dipstick format was used

Development of an Electrowetting Valve in Capillary-Driven Microfluidic Biosensor for Nucleic Acid Detection

This article presents the development of microfluidic valves to be used in capillary flow microfluidic device as a platform for nucleic acid detection. The valve used the principle of electrowetting and was able to be actuated at low voltage. The valve consisted of two silver electrodes which were encountered in series within a microfluidic channel. The second electrode was modified with a hydrophobic monolayer resulting in a cessation of capillary flow. A potential of 4V resulted in a 70° reduction in water contact angle within ten seconds which allowed capillary flow to continue. The final device represented a microfluidic valve for capillary flow microfluidics realized on PMMA substrate. In addition to the valve designed for timed fluid delivery, our PMMA microfluidic chip also consists of self-priming microfluidics with sealed conjugate pads of reagent delivery and an absorbent pad for additional fluid draw. We have developed a single-step surface modification method which allows strong capillary flow within a sealed microchannel. Conjugate pads within the device held trapped complex consisting of the magnetic beads and nucleic-acid-probe-conjugated horseradish peroxidase (HRP). Magnetic beads were released when sample entered the chamber and hybridized with the complex. The complex was immobilized over a magnet in the capture zone while a luminol co-reactant stream containing H2O2 was merged with the channel. A photomultiplier tube was used to quantify the chemiluminescence signal. This new format of biosensor will not only allow for pumpless automatically reagent delivery, but also smaller and more sensitive detection, as well as commercial-scale manufacturing and low materials cost, and it would be an ideal device for fast diagnostic in resource-limited settings

Phage based electrochemical detection of Escherichia coli in drinking water using affinity reporter probes

The monitoring of drinking water for indicators of fecal contamination is crucial for ensuring a safe supply. In this study, a novel electrochemical method was developed for the rapid and sensitive detection of Escherichia coli (E. coli) in drinking water. This strategy is based on the use of engineered bacteriophages (phages) to separate and concentrate target E. coli when conjugated with magnetic beads, and to facilitate the detection by expressing gold binding peptides fused alkaline phosphatase (GBPs-ALP). The fusion protein GBPs-ALP has both the enzymatic activity and the ability to directly bind onto a gold surface. This binding-peptide mediated immobilization method provided a novel and simple approach to immobilize proteins on a solid surface, requiring no post-translational modifications. The concentration of E. coli was determined by measuring the activity of the ALP on gold electrodes electrochemically using linear sweep voltammetry (LSV). This approach was successfully applied in the detection of E. coli in drinking water. We were able to detect 105 CFU mL−1 of E. coli within 4 hours. After 9 hours of preincubation, 1 CFU of E. coli in 100 mL of drinking water was detected with a total assay time of 12 hours. This approach compares favorably to the current EPA method and has the potential to be applied to detect different bacteria in other food matrices

A phage-based assay for the rapid, quantitative, and single CFU visualization of E. coli (ECOR #13) in drinking water

Drinking water standards in the United States mandate a zero tolerance of generic E. coli in 100mL of water. The presence of E. coli in drinking water indicates that favorable environmental conditions exist that could have resulted in pathogen contamination. Therefore, the rapid and specifc enumeration of E. coli in contaminated drinking water is critical to mitigate signifcant risks to public health. To meet this challenge, we developed a bacteriophage-based membrane fltration assay that employs novel fusion reporter enzymes to fully quantify E. coli in less than half the time required for traditional enrichment assays. A luciferase and an alkaline phosphatase, both specifcally engineered for increased enzymatic activity, were selected as reporter probes due to their strong signal, small size, and low background. The genes for the reporter enzymes were fused to genes for carbohydrate binding modules specifc to cellulose. These constructs were then inserted into the E. coli-specifc phage T7 which were used to infect E. coli trapped on a cellulose flter. During the infection, the reporters were expressed and released from the bacterial cells following the lytic infection cycle. The binding modules facilitated the immobilization of the reporter probes on the cellulose flter in proximity to the lysed cells. Following substrate addition, the location and quantifcation of E. coli cells could then be determined visually or using bioluminescence imaging for the alkaline phosphatase and luciferase reporters, respectively. As a result, a detection assay capable of quantitatively detecting E. coli in drinking water with similar results to established methods, but less than half the assay time was developed

A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples

A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (101 to 106) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortuitum, M. scrofulaceum, M. intracellulare, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR

Application of a unique server-based oligonucleotide probe selection tool toward a novel biosensor for the detection of Streptococcus pyogenes

We developed a software program for the rapid selection of detection probes to be used in nucleic acid-based assays. In comparison to commercially available software packages, our program allows the addition of oligotags as required by nucleic acid sequence-based amplification (NASBA) as well as automatic BLAST searches for all probe/primer pairs. We then demonstrated the usefulness of the program by designing a novel lateral flow biosensor for Streptococcus pyogenes that does not rely on amplification methods such as the polymerase chain reaction (PCR) or NASBA to obtain low limits of detection, but instead uses multiple reporter and capture probes per target sequence and an instantaneous amplification via dye-encapsulating liposomes. These assays will decrease the detection time to just a 20 min hybridization reaction and avoid costly enzymatic gene amplification reactions. The lateral flow assay was developed quantifying the 16S rRNA from S. pyogenes by designing reporter and capture probes that specifically hybridize with the RNA and form a sandwich. DNA reporter probes were tagged with dye-encapsulating liposomes, biotinylated DNA oligonucleotides were used as capture probes. From the initial number of capture and reporter probes chosen, a combination of two capture and three reporter probes were found to provide optimal signal generation and significant enhancement over single capture/reporter probe combinations. The selectivity of the biosensor was proven by analyzing organisms closely related to S. pyogenes, such as other Streptococcus and Enterococcus species. All probes had been selected by the software program within minutes and no iterative optimization and re-design of the oligonucleotides was required which enabled a very rapid biosensor prototyping. While the sensitivity obtained with the biosensor was only 135 ng, future experiments will decrease this significantly by the addition of more reporter and capture probes for either the same rRNA or a different nucleic acid target molecule. This will lead to the possibility of detecting S. pyogenes with a rugged assay that does not require a cell culturing or gene amplification step and will therefore enable rapid, specific and sensitive onsite testing

“Design and fabrication of a microfluidic device for near-single cell mRNA isolation using a copper hot embossing master

We describe investigations toward a disposable polymer-based chip for the isolation of eukaryotic mRNA. This work focuses here on the improvement of the fabrication methods for rapid prototyping and the actual application at lowest RNA concentrations with total channel volumes of 3.5 μL. Messenger RNA isolation was achieved using paramagnetic oligo (dT)25 beads within a microfluidic channel which incorporated a sawtooth microstructured design to aid in mixing. The structures were shown to facilitate mixing beteen two fluids in parallel flow when compared to a channel without structures. The chip was fabricated by means of hot embossing poly(methyl methacrylate) (PMMA) using a copper master. Copper was used as the master material due to its excellent thermal, mechanical, and electroplating properties. Fabrication of the master consisted of the structuring of a polished copper plate using KMPR 1050 as an electroplating mold for forming the microchannel structures. The copper master was found to be much more robust than traditional silicon masters used for prototyping. The use of KMPR enabled the generation of high straight walls in contrast to SU-8 masters. In addition, embossing times were able to be decreased by a factor of 3 due to improved heat conduction and avoidance of a lengthy and delicate de-embossing step

Monomeric streptavidin phage display allows efficient immobilization of bacteriophages on magnetic particles for the capture, separation, and detection of bacteria

Abstract Immobilization of bacteriophages onto solid supports such as magnetic particles has demonstrated ultralow detection limits as biosensors for the separation and detection of their host bacteria. While the potential impact of magnetized phages is high, the current methods of immobilization are either weak, costly, inefficient, or laborious making them less viable for commercialization. In order to bridge this gap, we have developed a highly efficient, site-specific, and low-cost method to immobilize bacteriophages onto solid supports. While streptavidin–biotin represents an ideal conjugation method, the functionalization of magnetic particles with streptavidin requires square meters of coverage and therefore is not amenable to a low-cost assay. Here, we genetically engineered bacteriophages to allow synthesis of a monomeric streptavidin during infection of the bacterial host. The monomeric streptavidin was fused to a capsid protein (Hoc) to allow site-specific self-assembly of up to 155 fusion proteins per capsid. Biotin coated magnetic nanoparticles were functionalized with mSA-Hoc T4 phage demonstrated in an E. coli detection assay with a limit of detection of < 10 CFU in 100 mLs of water. This work highlights the creation of genetically modified bacteriophages with a novel capsid modification, expanding the potential for bacteriophage functionalized biotechnologies

Water-Soluble Electrospun Nanofibers as a Method for On-Chip Reagent Storage

This work demonstrates the ability to electrospin reagents into water-soluble nanofibers resulting in a stable on-chip enzyme storage format. Polyvinylpyrrolidone (PVP) nanofibers were spun with incorporation of the enzyme horseradish peroxidase (HRP). Scanning electron microscopy (SEM) of the spun nanofibers was used to confirm the non-woven structure which had an average diameter of 155 ± 34 nm. The HRP containing fibers were tested for their change in activity following electrospinning and during storage. A colorimetric assay was used to characterize the activity of HRP by reaction with the nanofiber mats in a microtiter plate and monitoring the change in absorption over time. Immediately following electrospinning, the activity peak for the HRP decreased by approximately 20%. After a storage study over 280 days, 40% of the activity remained. In addition to activity, the fibers were observed to solubilize in the microfluidic chamber. The chromogenic 3,3′,5,5′-tetramethylbenzidine solution reacted immediately with the fibers as they passed through a microfluidic channel. The ability to store enzymes and other reagents on-chip in a rapidly dispersible format could reduce the assay steps required of an operator to perform

An Engineered Reporter Phage for the Fluorometric Detection of Escherichia coli in Ground Beef

Despite enhanced sanitation implementations, foodborne bacterial pathogens still remain a major threat to public health and generate high costs for the food industry. Reporter bacteriophage (phage) systems have been regarded as a powerful technology for diagnostic assays for their extraordinary specificity to target cells and cost-effectiveness. Our study introduced an enzyme-based fluorescent assay for detecting the presence of E. coli using the T7 phage engineered with the lacZ operon which encodes beta-galactosidase (β-gal). Both endogenous and overexpressed β-gal expression was monitored using a fluorescent-based method with 4-methylumbelliferyl β-d-galactopyranoside (MUG) as the substrate. The infection of E. coli with engineered phages resulted in a detection limit of 10 CFU/mL in ground beef juice after 7 h of incubation. In this study, we demonstrated that the overexpression of β-gal coupled with a fluorogenic substrate can provide a straightforward and sensitive approach to detect the potential biological contamination in food samples. The results also suggested that this system can be applied to detect E. coli strains isolated from environmental samples, indicating a broader range of bacterial detection