Chamras Promptmas

Ultrasensitive detection of Vibrio cholerae O1 using microcantilever-based biosensor with dynamic force microscopy

This work presents the first demonstration of a cantilever based cholerae sensor. Dynamic force microscopy within atomic force microscope (AFM) is applied to measure the cantilevers resonance frequency shift due to mass of cell bound on microcantilever surface. The Vibrio cholerae O1, a food and waterborne pathogen that caused cholera disease in human, is a target bacterium cell of interest. Commercial gold-coated AFM microcantilevers are immobilized with monoclonal antibody (anti-V. cholerae O1) by self-assembled monolayer method. V. cholerae O1 detection experiment is then conducted in concentrations ranging from 1×10(3) to 1×10(7) CFU/ml. The microcantilever-based sensor has a detection limit of ∼1×10(3) CFU/ml and a mass sensitivity, Δm/ΔF, of ∼146.5 pg/Hz, which is at least two orders of magnitude lower than other reported techniques and sufficient for V. cholerae detection in food products without pre-enrichment steps. In addition, V. cholerae O1 antigen-antibody binding on microcanilever is confirmed by scanning electron microscopy. The results demonstrate that the new biosensor is promising for high sensitivity, uncomplicated and rapid detection of V. cholerae O1.

The development of DNA-based quartz crystal microbalance integrated with isothermal DNA amplification system for human papillomavirus type 58 detection.

To address the effect of dramatic change in temperature and viscosity during PCR process on quartz crystal microbalance (QCM) sensor and to increase the sensitivity, isothermal amplification was employed in the system. We combined loop-mediated isothermal amplification (LAMP) technique with QCM, called as LAMP-QCM, for detection of high-risk human papillomavirus viral DNA type 58 (HPV-58) which is commonly found in Asian women. The liquid-phase LAMP-QCM prototype comprised the frequency counter, a temperature control device and housing of the quartz crystal with polished gold electrodes on both sides. QCM detection signal was monitored in real-time based on an avidin-biotin binding between avidin coated QCM surface and specific biotinylated LAMP products. Analytical performance was evaluated for precision, sensitivity and specificity. A plasmid clone containing the HPV-58 sequence was diluted from 10(6) to 1 copy and used for detection limit. Cut-off value was estimated at 28.8 Hz from negative viral template. The system could detect 100 copies with Δf at 34.0±3.6 Hz compared to 1000 copies detected by conventional LAMP. No cross-reaction was observed with other HPV types. The HPV-58 detection was compared among LAMP-QCM, conventional LAMP and nested PCR in 50 cervical cancer tissues. The positive rate of LAMP-QCM was higher than that of conventional LAMP with 100% sensitivity and 90.5% specificity. The integrated LAMP-QCM system has improved the detection limit up to ten times compared to conventional LAMP with less-time consuming.

Surface modification of silicon dioxide, silicon nitride and titanium oxynitride for lactate dehydrogenase immobilization

Three different types of surface, silicon dioxide (SiO2), silicon nitride (Si3N4), and titanium oxynitride (TiON) were modified for lactate dehydrogenase (LDH) immobilization using (3-aminopropyl)triethoxysilane (APTES) to obtain an amino layer on each surface. The APTES modified surfaces can directly react with LDH via physical attachment. LDH can be chemically immobilized on those surfaces after incorporation with glutaraldehyde (GA) to obtain aldehyde layers of APTES-GA modified surfaces. The wetting properties, chemical bonding composition, and morphology of the modified surface were determined by contact angle (CA) measurement, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM), respectively. In this experiment, the immobilized protein content and LDH activity on each modified surface was used as an indicator of surface modification achievement. The results revealed that both the APTES and APTES-GA treatments successfully link the LDH molecule to those surfaces while retaining its activity. All types of tested surfaces modified with APTES-GA gave better LDH immobilizing efficiency than APTES, especially the SiO2 surface. In addition, the SiO2 surface offered the highest LDH immobilization among tested surfaces, with both APTES and APTES-GA modification. However, TiON and Si3N4 surfaces could be used as alternative candidate materials in the preparation of ion-sensitive field-effect transistor (ISFET) based biosensors, including lactate sensors using immobilized LDH on the ISFET surface.

Detection of Non-Amplified Mycobacterium tuberculosis Genomic DNA Using Piezoelectric DNA-Based Biosensors

Piezoelectric DNA-based biosensor technology was developed as a new method for detection of M. tuberculosis. This method consists of immobilizing a thiol-modified oligonucleotide probe on the gold electrode surface of a quartz crystal, using a self-assembled monolayer method. The advantage of this study is that a non-amplified genomic bacterial DNA target was used. Instead, the genomic DNA was digested by restriction enzyme to obtain DNA fragments containing the target sequence. The fabricated biosensor was evaluated through an examination of 200 samples. No cross hybridization were observed against M. avium complex and other microorganisms. This target DNA preparation, without PCR amplification, will reduce time, costs, and the tedious step of amplification.

Correlative effect on the toxicity of three surface-exposed loops in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin.

Surface-exposed loop residues, Pro(389) (beta(6)-beta(7) loop), Glu(417) (beta(8)-beta(9) loop), Tyr(455) and Asn(456) (beta(10)-beta(11) loop), in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin have been previously demonstrated to be crucial for toxicity. Herein, five combinations of two-loop mutants, P389A/E417A (beta(6)-beta(7)/beta(8)-beta(9) loops), P389A/Y455A, P389A/N456A (beta(6)-beta(7)/beta(10)-beta(11) loops), E417A/Y455A and E417A/N456A (beta(8)-beta(9)/beta(10)-beta(11) loops), were constructed as a means of examining a correlative effect of these three critical loops on Cry4Ba toxicity. All 130-kDa mutant protoxins were overexpressed as inclusion bodies in Escherichia coli with yields comparable to the wild-type toxin. In addition, all mutant toxins were structurally stable upon solubilization and trypsin activation in carbonate buffer, pH 9.0. Interestingly, E. coli cells expressing all the double-loop mutants showed an almost complete loss in toxicity against Aedes aegypti mosquito larvae while their corresponding single-loop mutants exhibited reduced activity of approximately 50%. Moreover, in situ binding analysis revealed that the 65-kDa purified toxins representing each pairwise mutant exhibited reduced binding to apical microvilli of A. aegypti larval midgut when compared with the single mutants. Altogether, the data demonstrate for the first time that all these three surface-exposed loops of the Cry4Ba toxin are equally involved in receptor binding and hence toxicity.

Silver quartz crystal microbalance for differential diagnosis of Plasmodium falciparum and Plasmodium vivax in single and mixed infection.

The most severe form of malaria is cerebral malaria caused by Plasmodium falciparum. Standard malaria diagnosis is Giemsa stained peripheral blood smear but false negative findings are always reported. Moreover, mixed infections are underestimated by routine microscopy. Many methods have been developed to overcome these disadvantages and the most specific and sensitive is molecular diagnosis. Specific malaria genes are amplified by polymerase chain reaction (PCR) and many post-PCR methods have been created. We developed a gold fabricated quartz crystal microbalance (QCM) as a post-PCR method of malaria diagnosis. In this work a cheaper silver fabricated QCM was developed to identify both single and mixed infection of P. falciparum and Plasmodium vivax. The biotinylated malaria probe was immobilized on silver surface via specific avidin-biotin interaction. The target DNA fragment of 18s rRNA gene was amplified and hybridized with a QCM immobilized probe. Mass changes due to DNA hybridization were indicated by changes of quartz resonance frequencies. Validation showed that malaria silver QCM had high diagnostic potency. Evaluation of suspected 67 febrile blood samples from malaria endemic area demonstrated that the malaria silver QCM could identify both false negative and misdiagnosis cases of routine microscopy. The analysis cost of malaria silver QCM was

Diagnosis and genotyping of Plasmodium falciparum by a DNA biosensor based on quartz crystal microbalance (QCM)

1/sample and analysis time was 30 min after blood collection. The malaria silver QCM is stable at tropical temperature for up to 6 months. Thus, it can be transported to be used in a remote endemic area. Thus, the malaria silver QCM is accurate, precise, rapid, cheap, and field applicable.

Detection and haplotype differentiation of Southeast Asian α-thalassemia using polymerase chain reaction and a piezoelectric biosensor immobilized with a single oligonucleotide probe

Abstract Background: Malaria infection with Plasmodium falciparum is an important basic health problem in the tropical and sub-tropical countries. The standard diagnostic method is blood film examination to visualize parasite morphology. However, in cases of low parasitemia or mixed infection with very low cryptic species, microscopy is not sensitive enough. Therefore, molecular techniques have been widely employed. Methods: A label-free DNA biosensor based on quartz crystal microbalance (QCM) to diagnose and genotype P. falciparum was developed. Avidin-biotin interaction was used to coat the specific biotinylated probe on the gold surface of QCM. The gene encoding merozoite surface protein 2 (msp2) was amplified and the PCR products were then cut with restriction enzyme (MwoI). Enzymatic cutting made the PCR products suitable for QCM development. Hybridization between probe and enzymatic cutting DNA fragments resulted in frequency changes of the QCM. Results: The newly developed QCM was tested for its diagnosis ability using both malaria laboratory strains and clinical isolates. The biosensor was sensitive at the sub-nanogram level, specific for only P. falciparum detection, no cross-reaction with P. vivax, and stable at room temperature for up to 6 months. Selection of msp2 as a target gene and a geno-typing marker made the QCM potentially useful for falciparum diagnosis simultaneously with genotyping. Potency was tested by genotyping two allelic families of P. falciparum, FC27 and IC1, using malaria laboratory strains, K1 and 3D7, respectively. Conclusions: The dual function QCM was successfully developed with high sensitivity and specificity, and was cost-effective, stable and field adaptable.

Development of an Amperometric Immunosensor for the Determination of Methamphetamine in Urine

DNA-based diagnosis of alpha-thalassemias routinely relies on polymerase chain reaction (PCR) and gel electrophoresis. Here, we developed a new procedure for the detection and haplotype differentiation of Southeast Asian (SEA) alpha-thalassemia using a 3-primer system for PCR coupling with a DNA-based piezoelectric biosensor. PCR products amplified from genomic DNA were differentiated directly by using a quartz crystal microbalance immobilized with a single oligonucleotide probe. The frequency changes after hybridization of the PCR products amplified from a representative sample of normal alpha-globin, SEA alpha-thalassemia heterozygote, and homozygote were 206+/-11, 256+/-5, and 307+/-3 Hz, respectively. The fabricated biosensor was evaluated through an examination of 18 blind specimens. It could accurately discriminate between normal and SEA alpha-thalassemic samples, which suggests that this biosensor system is a promising alternative technique to detect SEA alpha-thalassemia because of its specificity and less hazardous exposure as compared with conventional methods.

Bacillus thuringiensis Cry4Ba toxin employs two receptor-binding loops for synergistic interactions with Cyt2Aa2

An amperometric immunosensor in the competitive format was developed for the detection of methamphetamine in urine. The electrodes consisted of carbon paste and Ag/AgCl screen printed on heat sealing film, respectively, and of monoclonal anti-methamphetamine antibody as the biorecognition element. Optimum amounts of methamphetamine- N -bovine serum albumin conjugate, monoclonal antibody and alkaline phosphatase-goat anti-mouse immunoglobulin G were 20, 10 ng and 1:10,000 dilution in 10 w l each, respectively. Methamphetamine was detected by the conversion of p -aminophenyl phosphate to electroactive p -aminophenol in the range of 200 ng/ml (lower detection limit) to 1,500 ng/ml methamphetamine in a nearly linear dose response curve. Within amphetamine concentrations of 0-1,500 ng/ml cross-reaction with methamphetamine was not observed. Working with urine samples spiked with methamphetamine, the accuracy and precision of the assay were 91.5-104.4% and 15.8-24.4%, respectively. This is a proof of concept in the clinical perspective for an amperometric immunosensor whose electrodes are amenable to future mass production.