Label-free detection of bacterial RNA using polydiacetylene-based biochip

A novel acetylcholinesterase (AChE)/choline oxidase (ChOx) bienzyme amperometric acetylcholine biosensor based on gold nanoparticles (AuNPs) and multi-walled carbon nanotubes (MWCNTs) has been successfully developed by self-assembly process in combination of sol-gel technique. A thiolated aqueous silica sol containing MWCNTs and ChOx was first dropped on the surface of a cleaned Pt electrode, and then AuNPs were assembled with the thiolated sol-gel network. Finally, the alternate deposition of poly (diallyldimethylammonium chloride) (PDDA) and AChE was repeated to assemble different layers of PDDA-AChE on the electrode for optimizing AChE loading. Among the resulting biosensors, the biosensor based on two layers of PDDA-AChE multilayer films showed the best performance. It exhibited a wide linear range, high sensitivity and fast amperometric response, which were 0.005-0.4mM, 3.395 μA/mM, and within 15s, respectively. The biosensor showed long-term stability and acceptable reproducibility. More importantly, this study could provide a simple and effective multienzyme immobilization platform for meeting the demand of the effective immobilization enzyme on the electrode surface.     

Label-free, multiplexed detection of bacterial tmRNA using silicon photonic microring resonators

This work describes the development of an automated flow-based biosensor that employs genetically modified acetylcholinesterase (AChE) enzymes B394, B4 and wild type B131. The biosensor was based on a screen printed carbon electrode (SPE) that was integrated into a flow cell. Enzymes were immobilised on cobalt (II) phthalocyanine (CoPC) modified electrodes by entrapment in a photocrosslinkable polymer (PVA-AWP). The automated flow-based biosensor was successfully used to quantify three organophosphate pesticides (OPs) in milk samples. The OPs used were chlorpyriphos-oxon (CPO), ethyl paraoxon (EPOx) and malaoxon (MOx). The total analysis time for the assay was less than 15 min. Initially, the biosensor performance was tested in phosphate buffer solution (PBS) using B394, B131 and B4 biosensors. The best detection limits were obtained with B394; therefore, this biosensor was used to produce calibration data in milk with three OPs in the concentration range of 5 × 10(-6)M to 5 × 10(-12)M. The limit of detection (LOD) obtained in milk for CPO, EPOx and MOx were 5 × 10(-12)M, 5 × 10(-9)M and 5 × 10(-10)M, respectively, with a correlation coefficient R(2)=0.9910. The automated flow-based biosensor successfully quantified the OPs in different fat-containing milk samples. There were no false positives or false negatives observed for the analytical figures of merit for the constructed biosensors. This method is inexpensive, sensitive, portable, non-invasive and provides real-time results. This analytical system can provide rapid detection of highly toxic OPs in food matrices such as milk.     

Label-free DNA sequencing using Millikan detection

A label-free method for DNA sequencing based on the principle of the Millikan oil drop experiment was developed. This sequencing-by-synthesis approach sensed increases in bead charge as nucleotides were added by a polymerase to DNA templates attached to beads. The balance between an electrical force, which was dependent on the number of nucleotide charges on a bead, and opposing hydrodynamic drag and restoring tether forces resulted in a bead velocity that was a function of the number of nucleotides attached to the bead. The velocity of beads tethered via a polymer to a microfluidic channel and subjected to an oscillating electric field was measured using dark-field microscopy and used to determine how many nucleotides were incorporated during each sequencing-by-synthesis cycle. Increases in bead velocity of approximately 1% were reliably detected during DNA polymerization, allowing for sequencing of short DNA templates. The method could lead to a low-cost, high-throughput sequencing platform that could enable routine sequencing in medical applications.     

Quantitative and simultaneous detection of four foodborne bacterial pathogens with a multi-channel SPR sensor

We report the quantitative and simultaneous detection of four species of bacteria, Escherichia coli O157:H7, Salmonella choleraesuis serotype typhimurium, Listeria monocytogenes, and Campylobacter jejuni, using an eight-channel surface plasmon resonance (SPR) sensor based on wavelength division multiplexing. Detection curves showing SPR response versus analyte concentration were established for each species of bacteria in buffer at pH 7.4, apple juice at native pH 3.7, and apple juice at an adjusted pH of 7.4, as well as for a mixture containing all four species of bacteria in buffer. Control experiments were performed to show the non-fouling characteristics of the sensor surface as well as the specificity of the amplification antibodies used in this study. The limit of detection (LOD) for each of the four species of bacteria in the tested matrices ranges from 3.4 x 10(3) to 1.2 x 10(5) cfu/ml. Detection curves in buffer of an individual species of bacteria in a mixture of all four species of bacteria correlated well with detection curves of the individual species of bacteria alone. SPR responses were higher for bacteria in apple juice at pH 7.4 than in apple juice at pH 3.7. This difference in sensor response could be partly attributed to the pH dependence of antibody-antigen binding.     

Label-free detection of proteins using SERS-based immuno-nanosensors

This paper describes the development and optimization of a novel class of SERS-based immuno-nanosensors for the label-free detection of specific proteins in complex environments (e.g., cell culture matrices and intracellular environments). These SERS-based nanosphere sensors are fabricated by depositing multiple layers of silver on silica nanospheres, followed by binding of the antibody of interest to the silver surface via a short rigid crosslinker. In these studies, several different crosslinkers were characterized and evaluated for optimal nanosensor activity. The crosslinkers evaluated contained either thiol or isothiocyanate functionalities, which bind to the silver surface on one end, while the other end of the crosslinker contained either a carboxylic or primary amine group, which reacts readily with the antibodies. These SERS-based nanosensors were also optimized for underlying silica sphere diameters, silica sphere coating conditions during silver deposition, number of silver layers applied, and silver surface coverage with crosslinkers. Upon optimization, the nanosensors were evaluated by monitoring their response to various antigens (e.g., human insulin or interleukin II) in complex environments.     

Direct detection of bacterial pathogens in representative dairy products using a combined bacterial concentration-PCR approach

AIMS: To develop a simple, rapid method to concentrate and purify bacteria and their nucleic acids from complex dairy food matrices in preparation for direct pathogen detection using polymerase chain reaction (PCR). METHODS AND RESULTS: Plain non-fat yogurt and cheddar cheese were each seeded with Listeria monocytogenes or Salmonella enterica serovar. Enteritidis in the range of 10(1)-10(6) CFU per 11-g sample. Samples were then processed for bacterial concentration using high-speed centrifugation (9700 g) followed by DNA extraction, PCR amplification, and amplicon confirmation by hybridization. Bacterial recoveries after centrifugation ranged from 53 to >100% and 71 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the non-fat yogurt samples; and from 77 to >100% and 69 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the cheddar cheese samples. There were no significant differences in recovery efficiency at different inocula levels, and losses to discarded supernatants were always <5%, regardless of dairy product or pathogen. CONCLUSIONS: When followed by pathogen detection using PCR and confirmation by amplicon hybridization, detection limits of 10(3) and 10(1) CFU per 11-g sample were achieved for L. monocytogenes and serovar. Enteritidis, respectively, in both product types and without prior cultural enrichment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents progress toward the rapid and efficient direct detection of pathogens from complex food matrices at detection limits approaching those that might be anticipated in naturally contaminated products.     

Label-free virus detection using silicon photonic microring resonators

Viruses represent a continual threat to humans through a number of mechanisms, which include disease, bioterrorism, and destruction of both plant and animal food resources. Many contemporary techniques used for the detection of viruses and viral infections suffer from limitations such as the need for extensive sample preparation or the lengthy window between infection and measurable immune response, for serological methods. In order to develop a method that is fast, cost-effective, and features reduced sample preparation compared to many other virus detection methods, we report the application of silicon photonic microring resonators for the direct, label-free detection of intact viruses in both purified samples as well as in a complex, real-world analytical matrix. As a model system, we demonstrate the quantitative detection of Bean pod mottle virus, a pathogen of great agricultural importance, with a limit of detection of 10 ng/mL. By simply grinding a small amount of leaf sample in buffer with a mortar and pestle, infected leaves can be identified over a healthy control with a total analysis time of less than 45 min. Given the inherent scalability and multiplexing capability of the semiconductor-based technology, we feel that silicon photonic microring resonators are well-positioned as a promising analytical tool for a number of viral detection applications.     

Label-free amplified bioaffinity detection using terahertz wave technology

A new affinity biosensor based on pulsed terahertz (THz) wave technology has been used to monitor binding between biotin and avidin molecules. Amplified detection of avidin-biotin binding is obtained on supported membranes composed of biotin layers on quartz surface, which is modified with octadecanol. Agarose particles are conjugated with avidin and then applied to biotin, which is already bound to the octadecanol quartz surface, the biotin binds to the conjugate rapidly and causes an enhancement of the THz difference signal between biotin and biotin-avidin complexes by a factor greater than eight fold when compared to the same sample without agarose beads. The technique was able to detect less than 10.3 ng/cm2 avidin, thus, giving the THz system a detection capability of sub-thin solid films better than ellipsometry and reflectometry techniques. Further improvement is underway using highly refractive beads together with appropriate surface chemistry. This newly developed method is being saliently optimized for future application, including the detection of DNA hybridization and ligand-analyte affinity binding.     

Label-free multiplexed virus detection using spectral reflectance imaging

We demonstrate detection of whole viruses and viral proteins with a new label-free platform based on spectral reflectance imaging. The Interferometric Reflectance Imaging Sensor (IRIS) has been shown to be capable of sensitive protein and DNA detection in a real time and high-throughput format. Vesicular stomatitis virus (VSV) was used as the target for detection as it is well-characterized for protein composition and can be modified to express viral coat proteins from other dangerous, highly pathogenic agents for surrogate detection while remaining a biosafety level 2 agent. We demonstrate specific detection of intact VSV virions achieved with surface-immobilized antibodies acting as capture probes which is confirmed using fluorescence imaging. The limit of detection is confirmed down to 3.5 × 10(5)plaque-forming units/mL (PFUs/mL). To increase specificity in a clinical scenario, both the external glycoprotein and internal viral proteins were simultaneously detected with the same antibody arrays with detergent-disrupted purified VSV and infected cell lysate solutions. Our results show sensitive and specific virus detection with a simple surface chemistry and minimal sample preparation on a quantitative label-free interferometric platform.     

Photonic detection of bacterial pathogens in living hosts

The study of pathogenic is often limited to ex vivo assays and cell-culture correlates. A greater understanding of infectious diseases would be facilitated by in vivo analyses. Therefore, we have developed a method for detecting bacterial pathogens in a living host and used this method to evaluate disease processes for strains of Salmonella typhimurium that differ in their virulence for mice. Three strains of Salmonella were marked with bioluminescence through transformation with a plasmid conferring constitutive expression of bacterial luciferase. Detection of photons transmitted through tissues of animals infected with bioluminescent Salmonella allowed localization of the bacteria to specific tissues. In this manner progressive infections were distinguished from those that were persistent or abortive. We observed patterns of bio-luminescence that suggested the caecum may play a pivotal role in Salmonella pathogenesis. In vivo efficacy of an antibiotic was monitored using this optical method. This study demonstrates that the real time non-invasive analyses of pathogenic events and pharmacological monitoring can be performed in vivo .     

The specific and sensitive detection of bacterial pathogens within 4 h using bacteriophage amplification

This paper describes a novel approach, termed the 'phage amplification assay', for the rapid detection and identification of specific bacteria. The technique is based on the phage lytic cycle with plaque formation as the assay end-point. It is highly sensitive, quantitative and gives results typically within 4 h. The assay comprises four main stages : (1) phage infection of target bacterium ; (2) destruction of exogenous phage ; (3) amplification of phage within infected host and (4) plaque formation from infected host with the aid of helper bacteria. A key component of this assay is a potent virucidal agent derived from natural plant extracts, pomegranate rind extract (PRE). In combination with ferrous sulphate PRE can bring about an 11 log-cycle reduction in phage titre within 3 min. This is achieved without any injury to the infected target bacteria. Subsequently, any resulting plaques are derived only from infected target organisms. Data are presented for a range of bacterial hosts including Pseudomonas aeruginosa, Salmonella typhimurium and Staphylococcus aureus. The detection limit for Ps. aeruginosa was 40 bacteria ml⁻¹ in a time of 4 h and 600 bacteria m⁻¹ for Salm. typhimurium. Application of the principles of this technology to other bacterial genera is discussed.     

Quantitation of yeast ceramides using high-performance liquid chromatography-evaporative light-scattering detection

A high-performance liquid chromatograph equipped with an evaporative light scattering detector (ELSD) (HPLC-ELSD) was used to assay the ceramides in yeast cells. The HPLC-ELSD method employed a cyanopropyl bonded column (CN column) that effectively separated the main interfering substance ergosterol without any derivatization process; most other interfering substances were also removed. The method can be applied for routine assay of ceramide content in yeast.     

Polymerase chain reaction detection of nonviable bacterial pathogens.

Polymerase chain reaction (PCR) methodologies for detection of pathogens in environmental samples are currently available. However, positive amplification products for any set of primers only signal that the appropriate target nucleic acid sequences were present in the sample. The presence of the amplification products does not imply that the target organisms were viable. Here we show that PCR will detect nonviable cells, as long as intact target nucleic acid sequences are available. In an environmental water sample, nucleic acids degraded quickly and were not detectable by PCR after 3 weeks even when stored at 4 degrees C. However, these data show that there is a window of opportunity for PCR analyses to result in false positives with respect to viable cells. We further show that care must be taken in the way samples are stored for future PCR amplifications and that filter sterilization of media is not acceptable for long-term preservation of samples for PCR.     

Label-free antibody-antigen binding detection by optical sensor array based on surface-synthesized gold nanoparticles

Gold nanoparticles grown in situ from printed seed particles on a glass substrate have been fabricated into a biosensor array. The light-scattering properties of the resulting surfaces show sensitivity to changes in the local refractive index. Each array spot is functionalized with fibrinogen or bovine serum albumin and scattered radiation is used to monitor the refractive index change on label-free binding of the antibodies to their antigens from whole blood antiserum. Data were collected real-time and the association rate constants for the specific antibody-antigen binding were derived from a kinetic analysis. The minimum antibody concentration detection sensitivity is of 100 nM.     

Label-free protein and pathogen detection using the atomic force microscope

The atomic force microscope (AFM) uses a sharp micron-scale tip to scan and amplify surface features, providing exceptionally detailed topographical information with magnification on the order of x10(6). This instrument is used extensively for quality control in the computer and semiconductor industries and is becoming a progressively more important tool in the biological sciences. Advantages of the AFM for biological application include the ability to obtain information in a direct, label-free manner and the ability to image in solution, providing real-time data acquisition under physiologically relevant conditions. A novel application of the AFM currently under development combines its surface profiling capabilities with fixed immuno-capture using antibodies immobilized in a nanoarray format. This provides a distinctive platform for direct, label-free detection and characterization of viral particles and other pathogens.     

Label-free detection of protein interactions using deep UV fluorescence lifetime microscopy

We present a label-free detection of protein interaction between beta-galactosidase from Escherichia coli (Ecbeta-Gal) and monoclonal anti-Ecbeta-Gal using deep UV laser-based fluorescence lifetime microscopy. The native fluorescence from intrinsic tryptophan emission was observed after one-photon excitation at 266 nm. Applying the time-correlated single-photon counting (TCSPC) method, we investigated the mean fluorescence lifetime and lifetime distributions from tryptophan residues in Ecbeta-Gal protein, monoclonal anti-Ecbeta-Gal, and corresponding complex. The results demonstrate that deep UV laser-based fluorescence lifetime microscopy is useful for sensitive identification of biological macromolecules interaction using intrinsic fluorescence.     

Label-free detection of cupric ions and histidine-tagged proteins using single poly(pyrrole)-NTA chelator conducting polymer nanotube chemiresistive sensor

Novel chemical and biological sensors based on a single poly(pyrrole)-NTA chelator nanotube for sensitive, selective, rapid and real-time detection of histidine-tagged protein and cupric ions are reported. NTA groups on the nanotube surface provided a simple mechanism for metal ion sensing via the high-affinity interaction between NTA and the subsequent detection of histidine-tagged protein through the coordination with metal chelated nanotube. Poly(pyrrole)-NTA chelator nanotubes of 190 nm outside diameter, 35 nm wall thickness and 30 microm long were synthesized by electrochemical polymerization of pyrrole-NTA inside a 200 nm diameter alumina template and assembled as a chemoresistive device by bottom-up contact geometry on a pair of parallel gold electrodes with a gap distance of 3 microm. The chemoresistive sensors based on single poly(pyrrole)-NTA chelator nanotube exhibited detection as low as one-hundredth attomolar (0.6 ppt) cupric ions and 1 ng/ml of penta-histidine tagged syntaxin protein.     

Direct detection and quantification of Alternaria alternata lycopersici toxins using high-performance liquid chromatography-evaporative light-scattering detection

Alternaria alternata lycopersici toxins are produced by the tomato pathogenic fungus A. alternata f. sp. lycopersici. They are primary determinants for stem canker disease of tomato and induce apoptosis in animals and plants. Due to the lack of a UV chromophore, their detection has relied on derivatizations or immunoassays, which require tedious pre-assay processes. Here, we describe a method for direct and quantitative detection of the toxins. The method employs an evaporative light-scattering detector (ELSD) coupled to HPLC. It gives a linear response in the range of 400-4000 ng toxin, with a detection limit of about 6 ng/mul. The detection of all major toxin congeners by HPLC-ESLD was confirmed by liquid chromatography-mass spectrometry. Thus, the method provides a simple and sensitive tool for the studies of this group of host-specific mycotoxins.