Avraham Rasooly

Microarray Analysis of Microbial Virulence Factors

ABSTRACT Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I,slt-II, fliC, rfbE, andipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella,Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.

Identification of Listeria Species by Microarray-Based Assay

ABSTRACT We have developed a rapid microarray-based assay for the reliable detection and discrimination of six species of the Listeria genus: L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. seeligeri, and L. grayi. The approach used in this study involves one-tube multiplex PCR amplification of six target bacterial virulence factor genes (iap, hly, inlB, plcA, plcB, and clpE), synthesis of fluorescently labeled single-stranded DNA, and hybridization to the multiple individual oligonucleotide probes specific for each Listeria species and immobilized on a glass surface. Results of the microarray analysis of 53 reference and clinical isolates of Listeria spp. demonstrated that this method allowed unambiguous identification of all six Listeria species based on sequence differences in the iap gene. Another virulence factor gene, hly, was used for detection and genotyping all L. monocytogenes, all L. ivanovii, and 8 of 11 L. seeligeri isolates. Other members of the genus Listeria and three L. seeligeri isolates did not contain the hly gene. There was complete agreement between the results of genotyping based on the hly and iap gene sequences. All L. monocytogenes isolates were found to be positive for the inlB, plcA, plcB, and clpE virulence genes specific only to this species. Our data on Listeria species analysis demonstrated that this microarray technique is a simple, rapid, and robust genotyping method that is also a potentially valuable tool for identification and characterization of bacterial pathogens in general.

Simultaneous Analysis of Multiple Staphylococcal Enterotoxin Genes by an Oligonucleotide Microarray Assay

ABSTRACT Staphylococcal enterotoxins (SEs) are a family of 17 major serological types of heat-stable enterotoxins that are one of the leading causes of gastroenteritis resulting from consumption of contaminated food. SEs are considered potential bioweapons. Many Staphylococcus aureus isolates contain multiple SEs. Because of the large number of SEs, serological typing and PCR typing are laborious and time-consuming. Furthermore, serological typing may not always be practical because of antigenic similarities among enterotoxins. We report on a microarray-based one-tube assay for the simultaneous detection and identification (genetic typing) of multiple enterotoxin (ent) genes. The proposed typing method is based on PCR amplification of the target region of the ent genes with degenerate primers, followed by characterization of the PCR products by microchip hybridization with oligonucleotide probes specific for each ent gene. We verified the performance of this method by using several other techniques, including PCR amplification with gene-specific primers, followed by gel electrophoresis or microarray hybridization, and sequencing of the enterotoxin genes. The assay was evaluated by analysis of previously characterized staphylococcal isolates containing 16 ent genes. The microarray assay revealed that some of these isolates contained additional previously undetected ent genes. The use of degenerate primers allows the simultaneous amplification and identification of as many as nine different ent genes in one S. aureus strain. The results of this study demonstrate the usefulness of the oligonucleotide microarray assay for the analysis of multitoxigenic strains, which are common among S. aureus strains, and for the analysis of microbial pathogens in general.

Food Microbial Pathogen Detection and Analysis Using DNA Microarray Technologies

Culture-based methods used for microbial detection and identification are simple to use, relatively inexpensive, and sensitive. However, culture-based methods are too time-consuming for high-throughput testing and too tedious for analysis of samples with multiple organisms and provide little clinical information regarding the pathogen (e.g., antibiotic resistance genes, virulence factors, or strain subtype). DNA-based methods, such as polymerase chain reaction (PCR), overcome some these limitations since they are generally faster and can provide more information than culture-based methods. One limitation of traditional PCR-based methods is that they are normally limited to the analysis of a single pathogen, a small group of related pathogens, or a small number of relevant genes. Microarray technology enables a significant expansion of the capability of DNA-based methods in terms of the number of DNA sequences that can be analyzed simultaneously, enabling molecular identification and characterization of multiple pathogens and many genes in a single array assay. Microarray analysis of microbial pathogens has potential uses in research, food safety, medical, agricultural, regulatory, public health, and industrial settings. In this article, we describe the main technical elements of microarray technology and the application and potential use of DNA microarrays for food microbial analysis.

Carbon nanotubes with enhanced chemiluminescence immunoassay for CCD-based detection of Staphylococcal enterotoxin B in food.

Enhanced chemiluminescence (ECL) detection can significantly enhance the sensitivity of immunoassays but often requires expensive and complex detectors. The need for these detectors limits broader use of ECL in immunoassay applications. To make ECL more practical for immunoassays, we utilize a simple cooled charge-coupled device (CCD) detector combined with carbon nanotubes (CNTs) for primary antibody immobilization to develop a simple and portable point-of-care immunosensor. This combination of ECL, CNT, and CCD detector technologies is used to improve the detection of Staphylococcal enterotoxin B (SEB) in food. Anti-SEB primary antibodies were immobilized onto the CNT surface, and the antibody-nanotube mixture was immobilized onto a polycarbonate surface. SEB was then detected by an ELISA assay on the CNT-polycarbonate surface with an ECL assay. SEB in buffer, soy milk, apple juice, and meat baby food was assayed with a LOD of 0.01 ng/mL using our CCD detector, a level similar to the detection limit obtained with a fluorometric detector when using the CNTs. This level is far more sensitive than the conventional ELISA, which has a LOD of approximately 1 ng/mL. Our simple, versatile, and inexpensive point-of-care immunosensor combined with the CNT-ECL immunoassay method described in this work can also be used to simplify and increase sensitivity for many other types of diagnostics and detection assays.

Electrochemical Biosensing Platform Using Hydrogel Prepared from Ferrocene Modified Amino Acid as Highly Efficient Immobilization Matrix

To increase the loading of glucose oxidase (GOx) and simplify glucose biosensor fabrication, hydrogel prepared from ferrocene (Fc) modified amino acid phenylalanine (Phe, F) was utilized for the incorporation of GOx. The synthesized hydrogel displays good biocompatibility and contains a significant number of Fc moieties, which can be considered as an ideal matrix to immobilize enzymes for the preparation of mediator-based biosensors. The hydrogel was studied by scanning electron microscopy, which indicated that it was composed of nanofibers with a diameter of around 50-100 nm and length extended to 1 mm. With the addition of GOx into the hydrogel and by directly dropping the resulting biocomposite onto the electrode surface, a glucose biosensor, that displays good performance due to improved enzyme loading and efficient electron transfer, can be simply constructed. The favorable network structure and good biocompatibility of the hydrogel could effectively avoid enzyme leakage and maintain the bioactivity of the enzymes, which resulted in good stability of the biosensor. The biosensor was utilized for the detection of glucose in blood samples with results comparable to those obtained from the hospital. The hydrogel as a functional component of an amperometric biosensor has implications for future development of biosensors and for clinical applications.

A Simple Portable Electroluminescence Illumination-Based CCD Detector

In this chapter we describe a simple and relatively inexpensive Electroluminescence (EL) illumination and charged-coupled device (CCD) camera (EL-CCD) based detector for monitoring fluorescence and colorimetric assays. The portable battery-operated fluorescence detector includes an EL panel for fluoro-genic excitation at 490 nm, a cooled CCD digital camera to monitor emission at 523 nm, filters and a close up lens. The detector system is controlled by a laptop computer for camera operation, image acquisition and analysis. The system was tested using a fluorogenic peptide substrate (SNAP-25) for botulinum neurotoxin serotype A (BoNT-A) labeled with FITC. The level of detection of the system was found to be 1.25 nM of the peptide, similar to the detection level of a commercial photomultipler-based plate fluorometer. The multichannel EL-CCD was used with an assay plate capable of testing nine samples simultaneously in 1 min at this detection level. The portable system is small and is operated by a 12 V source. The modular detector was designed with easily interchangeable ELs, filters and lenses and can be used and adapted for a wide variety of fluorescence and colorimetric assays, fluorescence labels and assay formats.

DNA Generated Electric Current Biosensor

In addition to its primary function as a genetic material, deoxyribonucleic acid (DNA) is also a potential biologic energy source for molecular electronics. For the first time, we demonstrate that DNA can generate a redox electric current. As an example of this new functionality, DNA generated redox current was used for electrochemical detection of human epidermal growth factor receptor 2 (HER2), a clinically important breast cancer biomarker. To induce redox current, the phosphate of the single stranded DNA aptamer backbone was reacted with molybdate to form redox molybdophosphate precipitate and generate an electrochemical current of ∼16.8 μA/μM cm2. This detection of HER2 was performed using a sandwich detection assay. A HER2 specific peptide was immobilized onto a gold electrode surface for capturing HER2 in buffer and serum. The HER2 specific aptamer was used as both ligand to bind the captured HER2 and to generate a redox current signal. When tested for HER2 detection, the electrochemical current generated by the aptasensor was proportional to HER2 concentration in the range of 0.01 to 5 ng/mL, with a current generated in the range of ∼6.37 to 31.8 μA/cm2 in both buffer and serum. This detection level is within the clinically relevant range of HER2 concentrations. This method of electrochemical signal amplification greatly simplifies the signal transduction of aptasensors, broadening their use for HER2 analysis. This novel approach of using the same aptamer as biosensor ligand and as transducer can be universally extended to other aptasensors for a wide array of biodetection applications. Moreover, electric currents generated by DNA or other nucleic acids can be used in molecular electronics or implanted devices for both power generation and measurement of output.

Biosensors and Biodetection

Here is presented a miniaturized, fiber-optic (FO) nanoprobe biosensor based on the localized surface plasmon resonance (LSPR) at the reusable dielectric-metallic hybrid interface with a robust, gold nano-disk array at the fiber end facet. The nanodisk array is directly fabricated using electron beam lithography (EBL) and metal lift-off process. The free prostate-specific antigen (f-PSA) has been detected with a mouse antihuman prostate-specific antigen (PSA) monoclonal antibody (mAb) as a specific receptor linked with a selfassembled monolayer (SAM) at the LSPR-FO facet surfaces. Experimental investigation and data analysis found near field refractive index (RI) sensitivity at ~226 nm/RIU with the LSPR-FO nanoprobe, and demonstrated the lowest limit of detection (LOD) at 100 fg/mL (~3 fM) of f-PSA in PBS solutions. The SAM shows insignificant nonspecific binding to the target biomarkers in the solution. The control experimentation using 5 mg/mL bovine serum albumin in PBS and nonspecific surface test shows the excellent specificity and selectivity in the detection of f-PSA in PBS. These results indicate important progress toward a miniaturized, multifunctional fiber-optic technology that integrates informational communication and sensing function for developing a high-performance, label-free, point-of-care (POC) device.

An ELISA Lab-on-a-Chip (ELISA-LOC).

Laminated object manufacturing (LOM) technology using polymer sheets is an easy and affordable method for rapid prototyping of Lab-on-a-Chip (LOC) systems. It has recently been used to fabricate a miniature 96 sample ELISA lab-on-a-chip (ELISA-LOC) by integrating the washing step directly into an ELISA plate. LOM has been shown to be capable of creating complex 3D microfluidics through the assembly of a stack of polymer sheets with features generated by laser micromachining and by bonding the sheets together with adhesive. A six layer ELISA-LOC was fabricated with an acrylic (poly(methyl methacrylate) (PMMA)) core and five polycarbonate layers micromachined by a CO(2) laser with simple microfluidic features including a miniature 96-well sample plate. Immunological assays can be carried out in several configurations (1 × 96 wells, 2 × 48 wells, or 4 × 24 wells). The system includes three main functional elements: (1) a reagent loading fluidics module, (2) an assay and detection wells plate, and (3) a reagent removal fluidics module. The ELISA-LOC system combines several biosensing elements: (1) carbon nanotube (CNT) technology to enhance primary antibody immobilization, (2) sensitive ECL (electrochemiluminescence) detection, and (3) a charge-coupled device (CCD) detector for measuring the light signal generated by ECL. Using a sandwich ELISA assay, the system detected Staphylococcal enterotoxin B (SEB) at concentrations as low as 0.1 ng/ml, a detection level similar to that reported for conventional ELISA. ELISA-LOC can be operated by a syringe and does not require power for operation. This simple point-of-care (POC) system is useful for carrying out various immunological assays and other complex medical assays without the laboratory required for conventional ELISA, and therefore may be more useful for global healthcare delivery.