Immobilization and Detection of Listeria monocytogenes

A procedure was developed for immobilization of Listeria monocytogenes cells on metal hydroxides coupled with detection and enumeration using an automated optical system. The results of the immobilization procedure (<1 h) and detection during overnight incubation agreed with calculated plate counts, and this technique is simple and rapid and provides samples that are ready for confirmation of the presence of the pathogen by rapid methods.     

Impedance-based detection of DNA sequences using a silicon transducer with PNA as the probe layer

Electrochemical impedance measurements were used for the detection of single-strand DNA sequences using a peptide nucleic acid (PNA) probe layer immobilized onto Si/SiO2 chips. An epoxysilane layer is first immobilized onto the Si/SiO2 surface. The immobilization procedure consists of an epoxide/amine coupling reaction between the amino group of the PNA linker and the epoxide group of the silane. A 20-nucleotide sequence of PNA was used. Impedance measurements allow for the detection of the changes in charge distribution at the oxide/solution interface following modifications to the oxide surface. Due to these modifications, there are significant shifts in the semiconductor's flat-band potential after immobilization and hybridization. The results obtained using this direct and rapid approach are supported by fluorescence measurements according to classical methods for the detection of nucleic acid sequences.     

DNA probe attachment on plastic surfaces and microfluidic hybridization array channel devices with sample oscillation

DNA probe immobilization on plastic surfaces and device assembly are both critical to the fabrication of microfluidic hybridization array channel (MHAC) devices. Three oligonucleotide (oligo) probe immobilization procedures were investigated for attaching oligo probes on four different types of plastic surfaces (polystyrene, polycarbonate, poly(methylmethacrylate), and polypropylene). These procedures are the Surmodics procedure, the cetyltrimethylammonium bromide (CTAB) procedure, and the Reacti-Bind procedure. To determine the optimal plastic substrate and attachment chemistry for array fabrication, we investigated plastic hydrophobicity, intrinsic fluorescence, and oligo attachment efficiency. The Reacti-Bind procedure is least effective for attaching oligo probes in the microarray format. The CTAB procedure performs well enough to use in array fabrication, and the concentration of CTAB has a significant effect on oligo immobilization efficiency. We also found that use of amine-modified oligo probes resulted in better immobilization efficiency than use of unmodified oligos with the CTAB procedure. The oligo probe immobilization on plastic surfaces by the Surmodics procedure is the most effective with regard to probe spot quality and hybridization sensitivity. A DNA hybridization assay on such a device results in a limit of detection of 12pM. Utilizing a CO(2) IR laser machining and adhesive layer approach, we have developed an improved procedure for realizing a DNA microarray inside a microfluidic channel. This device fabrication procedure allows for more feasible spot placement in the channel and reduced sample adsorption by adhesive tapes used in the fabrication procedure. We also demonstrated improved hybridization kinetics and increased detection sensitivity in MHAC devices by implementing sample oscillation inside the channel. A limit of detection of 5pM has been achieved in MHAC devices with sample oscillation.     

Multisubstrate-compatible ELISA procedures for rapid and high-sensitivity immunoassays

This protocol describes an improved and optimized approach to develop rapid and high-sensitivity ELISAs by covalently immobilizing antibody on chemically modified polymeric surfaces. The method involves initial surface activation with KOH and an O(2) plasma, and then amine functionalization with 3-aminopropyltriethoxysilane. The second step requires covalent antibody immobilization on the aminated surface, followed by ELISA. The ELISA procedure developed is 16-fold more sensitive than established methods. This protocol could be used generally as a quantitative analytical approach to perform high-sensitivity and rapid assays in clinical situations, and would provide a faster approach to screen phage-displayed libraries in antibody development facilities. The antibody immobilization procedure is of ～3 h duration and facilitates rapid ELISAs. This method can be used to perform assays on a wide range of commercially relevant solid support matrices (including those that are chemically inert) with various biosensor formats.     

QCM-based DNA biosensor for detection of genetically modified organisms (GMOs)

Development of a mass sensitive quartz crystal microbalance (QCM)-based DNA biosensor for the detection of the hybridization of CaMV 35S promoter sequence (P35S) was investigated for the screening of genetically modified organisms (GMOs). Attention was focused on the choice of the coating chemistry that could be used for the immobilization of probe sequences on the gold surface of the quartz crystal. Two immobilization procedures were tested and compared considering the amount of the immobilized P35S probe and the extent of the hybridization reaction with the target oligonucleotide. In wet chemistry procedure, the interaction between the thiol and gold for the immobilization of a thiolated probe was employed. Direct surface functionalization of piezoelectric quartz crystals were achieved in 13.56 MHz plasma polymerization reactor utilising ethylenediamine (EDA) precursors for the immobilization of amined probes. Results indicated that immobilization of a thiolated probe provides better immobilization characteristics and higher sensitivity for the detection of the hybridization reaction. The thiolated probe was used for the detection of P35S sequence in PCR-amplified DNAs and in real samples of pflp (ferrodoxin like protein)-gene inserted tobacco plants. Fragmentation of the genomic DNAs were achieved by digestion with restriction endonucleases and ultrasonication. The results obtained from the fragmented genomic DNAs demonstrated that it is possible to detect the target sequence directly in non-amplified genomic DNAs by using the developed QCM-based DNA biosensor system. The developed QCM-based DNA biosensor represented promising results for a real-time, label-free, direct detection of DNA samples for the screening of GMOs.     

Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection

Background

Rapid diagnosis for time-sensitive illnesses such as stroke, cardiac arrest, and septic shock is essential for successful treatment. Much attention has therefore focused on new strategies for rapid and objective diagnosis, such as Point-of-Care Tests (PoCT) for blood biomarkers. Here we use a biomimicry-based approach to demonstrate a new diagnostic platform, based on enzymes tethered to nanoparticles (NPs). As proof of principle, we use oriented immobilization of pyruvate kinase (PK) and luciferase (Luc) on silica NPs to achieve rapid and sensitive detection of neuron-specific enolase (NSE), a clinically relevant biomarker for multiple diseases ranging from acute brain injuries to lung cancer. We hypothesize that an approach capitalizing on the speed and catalytic nature of enzymatic reactions would enable fast and sensitive biomarker detection, suitable for PoCT devices.

Methods and findings

We performed in-vitro, animal model, and human subject studies. First, the efficiency of coupled enzyme activities when tethered to NPs versus when in solution was tested, demonstrating a highly sensitive and rapid detection of physiological and pathological concentrations of NSE. Next, in rat stroke models the enzyme-based assay was able in minutes to show a statistically significant increase in NSE levels in samples taken 1 hour before and 0, 1, 3 and 6 hours after occlusion of the distal middle cerebral artery. Finally, using the tethered enzyme assay for detection of NSE in samples from 20 geriatric human patients, we show that our data match well (r = 0.815) with the current gold standard for biomarker detection, ELISA—with a major difference being that we achieve detection in 10 minutes as opposed to the several hours required for traditional ELISA.

Conclusions

Oriented enzyme immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes. Together, our findings provide proof of concept for using oriented immobilization of active enzymes on NPs as the basis for a highly rapid and sensitive biomarker detection platform. This addresses a key challenge in developing a PoCT platform for time sensitive and difficult to diagnose pathologies.     

Quantitation of immobilized proteins

A method for the quantitative determination of immobilized proteins based on the binding and subsequent elution of Coomassie Blue R is presented. Also presented is a method for the immobilization of proteins in solution by entrapment in polyacrylamide. These entrapped proteins are then available for use in the assay method presented. Other analytical procedures can also be performed on the entrapped proteins, either alone or in combination with the protein quantitation. The dye binding and elution method presented provides a sensitive and, in most applications, rapid method for the quantitative detection of immobilized proteins. Rather than immobilization being an obstacle to the assay method, this approach utilizes the advantages of immobilization for the removal of excess reagents. Application of this approach to several types of immobilized protein are presented.     

Non-surgical catheterization of the jugular vein in young pigs

Current methods utilized for serial blood collection in the young pig are limited due to the stress and/or discomfort to which the pig is exposed. Thus, we have developed a non-surgical, minimally invasive cannulation technique which allows jugular vein catheter placement in the young pig without causing extended discomfort or stress. The procedure described is rapid (approximately 8 min/pig) and relatively simple, requiring only minimal anaesthesia for immobilization of the pig during the procedure. Routinely, 2-week-old piglets are standing in their pens within 15-20 min from initiation of the procedure. Piglets recover rapidly from the procedure and display no clinical indications of pain or discomfort. Serum concentrations of cortisol, a standard indicator of stress and/or discomfort, are asymptomatic within 2 h of completing the procedure (k = 26.14+/-3.03 ng/ml). Stress is limited to the initial immobilization of the piglets. With this technique of cannulation, we routinely maintain catheter patency for 2 days, and often for as long as 5 days.     

Detecting minimal traces of DNA using DNA covalently attached to superparamagnetic nanoparticles and direct PCR-ELISA

A single bond covalent immobilization of aminated DNA probes on magnetic particles suitable for selective molecular hybridization of traces of DNA samples has been developed. Commercial superparamagnetic nanoparticles containing amino groups were activated by coating with a hetero-functional polymer (aldehyde-aspartic-dextran). This new immobilization procedure provides many practical advantages: (a) DNA probes are immobilized far from the support surface preventing steric hindrances; (b) the surface of the nanoparticles cannot adsorb DNA ionically; (c) DNA probes are bound via a very strong covalent bond (a secondary amine) providing very stable immobilized probes (at 100 degrees C, or in 70% formamide, or 0.1N NaOH). Due to the extreme sensitivity of this purification procedure based on DNA hybridization, the detection of hybridized products could be coupled to a PCR-ELISA direct amplification of the DNA bond to the magnetic nanoparticles. As a model system, an aminated DNA probe specific for detecting Hepatitis C Virus cDNA was immobilized according to the optimised procedure described herein. Superparamagnetic nanoparticles containing the immobilized HCV probe were able to give a positive result after PCR-ELISA detection when hybridized with 1 mL of solution containing 10(-18) g/mL of HCV cDNA (two molecules of HCV cDNA). In addition, the detection of HCV cDNA was not impaired by the addition to the sample solution of 2.5 million-fold excess of non-complementary DNA. The experimental data supports the use of magnetic nanoparticles containing DNA probes immobilized by the procedure here described as a convenient and extremely sensitive procedure for purification/detection DNA/RNA from biological samples. The concentration/purification potential of the magnetic nanoparticles, its stability under a wide range of conditions, coupled to the possibility of using the particles directly in amplification by PCR greatly reinforces this methodology as a molecular diagnostic tool.     

Rapid detection of salmonellae by immunoassays with titanous hydroxide as the solid phase

Radioimmunometric and enzyme-immunometric assays were developed for the detection of salmonellae in pure and mixed cultures as well as in 59 food samples. The performances of titanous hydroxide suspension and microtiter plates as the solid phase for the immobilization of microorganisms were compared in these immunoassays. Detection of populations of salmonella cells in pure culture, diluted with saline, was 4- to 10-fold more sensitive with the microtiter plates. However, with mixed culture of salmonella and other enterobacterial species, the detection sensitivity with titanous hydroxide was 100- to 160-fold more sensitive than with microtiter plates. Good correlation existed between results of a standard cultural method for the detection of salmonellae in foods and those obtained from radioimmunometric and enzyme-immunometric assays utilizing titanous hydroxide. However, a high incidence of false-positive and false-negative results with food samples occurred with the enzyme-immunometric assay utilizing microtiter plates. The results provided strong evidence for the merits of substituting titanous hydroxide for microtiter plates as the solid phase for the immobilization of salmonellae for their detection by immunoassays. The immunoassays were rapid and enabled the analysis of a large number of selective enrichment cultures of food samples for salmonellae within 8 h.     

Rapid detection of salmonellae by immunoassays with titanous hydroxide as the solid phase.

Radioimmunometric and enzyme-immunometric assays were developed for the detection of salmonellae in pure and mixed cultures as well as in 59 food samples. The performances of titanous hydroxide suspension and microtiter plates as the solid phase for the immobilization of microorganisms were compared in these immunoassays. Detection of populations of salmonella cells in pure culture, diluted with saline, was 4- to 10-fold more sensitive with the microtiter plates. However, with mixed culture of salmonella and other enterobacterial species, the detection sensitivity with titanous hydroxide was 100- to 160-fold more sensitive than with microtiter plates. Good correlation existed between results of a standard cultural method for the detection of salmonellae in foods and those obtained from radioimmunometric and enzyme-immunometric assays utilizing titanous hydroxide. However, a high incidence of false-positive and false-negative results with food samples occurred with the enzyme-immunometric assay utilizing microtiter plates. The results provided strong evidence for the merits of substituting titanous hydroxide for microtiter plates as the solid phase for the immobilization of salmonellae for their detection by immunoassays. The immunoassays were rapid and enabled the analysis of a large number of selective enrichment cultures of food samples for salmonellae within 8 h.     

Electrochemical biosensing of pesticide residues based on affinity biocomposite platforms

A novel and very sensitive electrochemical immunosensing strategy for the detection of atrazine based on affinity biocomposite transducers is presented. Firstly, the graphite-epoxy composite transducer was bulk-modified with different universal affinity biomolecules, such as avidin and Protein A. Two strategies for the immobilization of the anti-atrazine antibodies on both biocomposite transducers were evaluated: 'wet-affinity' and 'dry-assisted affinity' immobilization. Finally, the performance of a novel anti-atrazine immunocomposite bulk-modified with anti-atrazine antibodies was also evaluated. The better immobilization performance of the anti-atrazine antibodies was achieved by 'dry-assisted affinity' immobilization on Protein A (2%) graphite-epoxy biocomposite (ProtA(2%)-GEB) as a transducer. The immunological reaction for the detection of atrazine performed on the ProtA(2%)-GEB biosensors is based on a direct competitive assay using atrazine-HRP tracer as the enzymatic label. The electrochemical detection is thus achieved through a suitable substrate and a mediator for the enzyme HRP. This novel strategy was successfully evaluated using spiked orange juice samples. The detection limit for atrazine in orange juices using the competitive electrochemical immunosensing assay was found to be 6 x 10(-3) microgL-1 (0.03 nmolL-1) thus this biosensing method accomplishes by far the LODs required for the European Community directives for potable water and food samples (0.1 microgL-1). This strategy offers great promise for rapid, simple, cost effective, and on-site biosensing of biological, food, and environmental samples.     

Detection of immobilized proteins on nitrocellulose membranes using a biotinylation-dependent system.

Proteins are biotinylated after immobilization on nitrocellulose sheets by reaction with a biotinyl-succinimide ester. The biotinyl residues are visualized by streptavidin-peroxidase-based detection systems either by deposition of a colored formazan dye or by enhanced chemiluminescence (ECL), the latter being 10-fold more sensitive. The sensitivity of the staining procedure is dramatically improved by the inclusion of the reporter deposit technique into the staining procedure: the initially bound peroxidase generates phenolic radicals from biotinyltyramide, enhancing the number of biotinyl residues in the vicinity of the first biotinylation site. Thus the detection limit is lowered to 1 pg of protein with the ECL detection. The new method is compared with silver stain and immunochemical staining in Western blots and furthermore its suitability is demonstrated for 2-D gel electrophoresis.     

Human cytomegalovirus detection by a quartz crystal microbalance immunosensor

A piezoelectric affinity sensor has been developed to detect distinctive antigens of the human cytomegalovirus. Either the specific antibodies or the antigen were immobilized on the gold electrode. To develop a rapid immunoassay, various assay formats were tested in relation with the different antigen composition. First, a direct assay was carried out immobilizing the specific antibody on the crystal surface by passive adsorption. Next, Protein A, thiol/poly L-lysine mixed self-assembled monolayers were tested as methods of gold modification. A competitive format was exploited by immobilization of the antigen onto the crystal activated by SAM and poly L-lysine. This procedure yielded a preliminary calibration curve. A linear range between 2.5 and 5 μg/ml of gB epitope in solution and a detection limit of 1 μg/ml were measured.     

Site-specific immobilization of a (His)6-tagged acetylcholinesterase on nickel nanoparticles for highly sensitive toxicity biosensors

This paper reports site-specific affinity immobilization of (His)6-tagged acetylcholinesterase (AChE) onto Ni/NiO nanoparticles for the development of an electrochemical screen-printed biosensor for the detection of organophosphate pesticides. The method is based on the specific affinity binding of the His-tagged enzyme to oxidized nickel nanoparticle surfaces in the absence of metal chelators. This approach allows stable and oriented attachment of the enzyme onto the oxidized nickel through the external His residue in one-step procedure, allowing for fast and sensitive detection of paraoxon in the concentration range from 10⁻⁸ to 10⁻¹³ M. A detection limit of 10⁻¹² M for paraoxon was obtained after 20 min incubation. This method can be used as a generic approach for the immobilization of other His-tagged enzymes for the development of biosensors.     

Enhanced sensitivity detection of protein immobilization by fluorescent interference on oxidized silicon

We present a silicon chip-based approach for the enhanced sensitivity detection of surface-immobilized fluorescent molecules. Green fluorescent protein (GFP) is bound to the silicon substrate by a disuccinimidyl terephtalate-aminosilane immobilization procedure. The immobilized organic layers are characterized by surface analysis techniques, like ellipsometry, atomic force microscopy (AFM) and X-ray induced photoelectron spectroscopy. We obtain a 20-fold enhancement of the fluorescent signal, using constructive interference effects in a fused silica dielectric layer, deposited before immobilization onto the silicon. Our method opens perspectives to increase by an order of magnitude the fluorescent response of surface immobilized DNA- or protein-based layers for a variety of biosensor applications.     

N-(Iodoacetyl)-N'-(anthraquinon-2-oyl)-ethylenediamine (IAED): a new heterobifunctional reagent for the preparation of biochips

Design and synthesis of a new heterobifunctional reagent, N-(iodoacetyl)-N'-(anthraquinon-2-oyl)-ethylenediamine (IAED), have been described for the preparation of oligonucleotide-based biochips. The performance of the featured reagent is probed by the immobilization of thiolated and thiophosphorylated oligonucleotides on modified glass microslides via two routes (routes A and B). The immobilization procedure was accelerated by performing a chemical reaction between thiolated oligomers and the iodoacetyl moiety of the reagent under microwaves (MW), where it is completed in just 10 min. The quality of the constructed oligonucleotide microarrays was tested by performing a hybridization assay with a complementary target and subsequently used for the detection of base mismatches. The immobilized probes were found to be thermally stable.     

Development of DNA electrochemical biosensor based on covalent immobilization of probe DNA by direct coupling of sol-gel and self-assembly technologies

A new procedure for fabricating deoxyribonucleic acid (DNA) electrochemical biosensor was developed based on covalent immobilization of target single-stranded DNA (ssDNA) on Au electrode that had been functionalized by direct coupling of sol-gel and self-assembled technologies. Two siloxanes, 3-mercaptopropyltrimethoxysiloxane (MPTMS) and 3-glycidoxypropyltrimethoxysiloxane (GPTMS) were used as precursors to prepare functionally self-assembly sol-gel film on Au electrode. The thiol group of MPTMS allowed assembly of MPTMS sol-gel on gold electrode surface. Through co-condensation between silanols, GPTMS sol-gel with epoxide groups interconnected into MPTMS sol-gel and enabled covalent immobilization of target NH(2)-ssDNA through epoxide/amine coupling reaction. The concentration of MPTMS and GPTMS influenced the performance of the resulting biosensor due to competitive sol-gel process. The linear range of the developed biosensor for determination of complementary ssDNA was from 2.51 x 10(-9) to 5.02 x 10(-7)M with a detection limit of 8.57 x 10(-10)M. The fabricated biosensor possessed good selectivity and could be regenerated. The covalent immobilization of target ssDNA on self-assembled sol-gel matrix could serve as a versatile platform for DNA immobilization and fabrication of biosensors.     

A novel reagentless amperometric immunosensor based on gold nanoparticles/TMB/Nafion-modified electrode

A novel reagentless immunosensor was fabricated by immobilization of redox mediator 3,3',5,5'-tetramethylbenzidine (TMB) on the Nafion (Nf) film modified glassy carbon electrode. Gold nanoparticles were assembled onto the TMB/Nafion film modified electrode to provide active sites for the immobilization of antibody molecules. The antibody (anti-MIgG), in the present study, was fixed on the electrode for the rapid detection of antigen molecules (MIgG as a model analyte). The results showed that the immunosensor based on the immobilized TMB redox mediator exhibited good electrochemical response. A good linear relationship between peak current and the concentration of the MIgG was obtained in the concentration range from 4 to 120ng/mL. The detection limit was estimated to be 1ng/ml. Under the optimized conditions, the immunosensor exhibits good sensitivity, reproducibility and stability.