Microfluidic biosensor for the serotype-specific detection of dengue virus RNA

The development of a microfluidic biosensor with fluorescence detection for the rapid, sensitive, and serotype-specific detection of Dengue virus is presented. The biosensor chip consists of poly(dimethylsiloxane) (PDMS) substrate with fabricated microchannels and a glass substrate used to seal the microchannels. These two substrates are packaged within a pressure-closed Plexiglas housing to provide a watertight reversible sealing at the PDMS-glass interface. The ability to reversibly seal the device permits easy disassembly and quick interchange of the device parts, which is ideal for developmental purposes. The biosensor employs a magnetic bead-based sandwich hybridization system in conjugation with liposome amplification for the specific detection of nucleic acids. The concentrations of the various biosensor components were optimized using a synthesized fragment of Dengue virus RNA. To evaluate the sensitivity of the assay, two detection systems, based on fluorescence measurements of intact and lysed liposomes, were analyzed. The entire analysis was complete within 20 min (including incubation time) with RNA detection limits of 0.125 nM and 50 pM for intact and lysed liposome detection systems, respectively. Subsequently, the biosensor was applied to the analysis of actual RNA obtained from Dengue virus serotypes 1-4. The resulting signals were compared to those obtained using standard electrochemiluminescence detection and shown to correspond perfectly with respect to serotype identification.     

Frequency-selective rotation of two-particle nanoactuators for rapid and sensitive detection of biomolecules

We describe an optomagnetic bionanotechnology for rapid and sensitive solution-based affinity assays. Nanoactuators made from bioactive magnetic nanoparticles undergo rotational motion in the volume of a fluid under frequency-controlled magnetic actuation. The nanoactuators show a time-dependent scattering cross-section to an incoming light beam. We demonstrate that the temporal behavior of the scattered light intensity relates to the number, the magnetic properties and the size distribution of the nanoactuators, independently revealing the average value and variation in the magnetic properties of the nanoparticles as well as the concentration of nanoactuators. The method is applied to detect biomolecules in fluid by interparticle binding. In a total assay time of less than 3 min, we demonstrate a limit of detection lower than 400 fM in buffer and 5 pM in human plasma.     

Raman spectroscopy-based sensitive and specific detection of glycated hemoglobin

In recent years, glycated hemoglobin (HbA1c) has been increasingly accepted as a functional metric of mean blood glucose in the treatment of diabetic patients. Importantly, HbA1c provides an alternate measure of total glycemic exposure due to the representation of blood glucose throughout the day, including post-prandially. In this article, we propose and demonstrate the potential of Raman spectroscopy as a novel analytical method for quantitative detection of HbA1c, without using external dyes or reagents. Using the drop coating deposition Raman (DCDR) technique, we observe that the nonenzymatic glycosylation (glycation) of the hemoglobin molecule results in subtle but discernible and highly reproducible changes in the acquired spectra, which enable the accurate determination of glycated and nonglycated hemoglobin using standard chemometric methods. The acquired Raman spectra display excellent reproducibility of spectral characteristics at different locations in the drop and show a linear dependence of the spectral intensity on the analyte concentration. Furthermore, in hemolysate models, the developed multivariate calibration models for HbA1c show a high degree of prediction accuracy and precision--with a limit of detection that is a factor of ~15 smaller than the lowest physiological concentrations encountered in clinical practice. The excellent accuracy and reproducibility achieved in this proof-of-concept study opens substantive avenues for characterization and quantification of the glycosylation status of (therapeutic) proteins, which are widely used for biopharmaceutical development. We also envision that the proposed approach can provide a powerful tool for high-throughput HbA1c sensing in multicomponent mixtures and potentially in hemolysate and whole blood lysate samples.     

An immuno-wall microdevice exhibits rapid and sensitive detection of IDH1-R132H mutation specific to grade II and III gliomas

World Health Organization grade II and III gliomas most frequently occur in the central nervous system (CNS) in adults. Gliomas are not circumscribed; tumor edges are irregular and consist of tumor cells, normal brain tissue, and hyperplastic reactive glial cells. Therefore, the tumors are not fully resectable, resulting in recurrence, malignant progression, and eventual death. Approximately 69–80% of grade II and III gliomas harbor mutations in the isocitrate dehydrogenase 1 gene (IDH1), of which 83–90% are found to be the IDH1-R132H mutation. Detection of the IDH1-R132H mutation should help in the differential diagnosis of grade II and III gliomas from other types of CNS tumors and help determine the boundary between the tumor and normal brain tissue. In this study, we established a highly sensitive antibody-based device, referred to as the immuno-wall, to detect the IDH1-R132H mutation in gliomas. The immuno-wall causes an immunoreaction in microchannels fabricated using a photo-polymerizing polymer. This microdevice enables the analysis of the IDH1 status with a small sample within 15 min with substantially high sensitivity. Our results suggested that 10% content of the IDH1-R132H mutation in a sample of 0.33 μl volume, with 500 ng protein, or from 500 cells is theoretically sufficient for the analysis. The immuno-wall device will enable the rapid and highly sensitive detection of the IDH1-R132H mutation in routine clinical practice.     

面制品中痕量铝的快速高灵敏光电化学检测方法的建立

本实验采用新型的光电化学检测手段实现了对于面制品中痕量铝的快速高灵敏检测。实验中通过优化相应检测条件,探索出一种针对面制品中痕量铝检测的高灵敏、高稳定性的新的定量检测方法,并将该方法运用于实际样品检测,结果令人满意。     

Magnetic self-assembled zeolite clusters for sensitive detection and rapid removal of mercury(II)

We reported here the fabrication of a hierarchical mesoporous zeolite nanocomposite using 20 nm crystalline domins of zeolite L as building "bricks" by a simple and general one-step synthetic approach. By taking advantages of the large pore volumes, superparamagnetic iron oxide nanocrystals could be encapsulated into the nanocomposite conveniently for further facilitate separation and detection. In addition, by covalent coupling of fluorescent receptor (rhodamine-hydrazine), the combination of well-defined inorganic nanomaterials and organic receptors could be applied to selective detection of Hg(2+). Importantly, the unique adsorption capacity enabled by the hierarchical mesoporous zeolite and the efficient removal ability form complex multiphase systems by the magnetic characteristic made this multifunctional nanomaterial an excellent probe for detection, adsorption, and removal of Hg(2+) from waste aqueous solution.     

The optimization of magnetosandwich assays for the sensitive and specific detection of proteins in serum

Over the past decade, the use of magnetic particles (MPs) as labels in magnetic biosensors has attracted increasing interest because it provides a highly sensitive platform that can meet the diagnostic needs that are currently not met by existing technologies. However, preparing magnetic biosensors for a specific diagnostic application is a challenging task, and the (bio)chemical aspects are often neglected. Hence, one of the major remaining bottlenecks in the development of magnetic biosensors is the lack of an optimized magnetosandwich assay for the highly sensitive and specific detection of proteins in complex sample matrices. Therefore, in this article, we report on the impact of several different aspects of magnetosandwich assay development, that is, surface chemistry, MP size, rinsing procedure, sample matrix, and blocking procedure on the total-assay performance using quartz crystal microbalance and optical microscopy analysis. The optimization focused on the diagnostically relevant protein S100betabeta, a marker for stroke and minor head injury. It was observed that small MPs in combination with a strong rinsing and a BSA/Tween-20 blocking allows for the most specific and sensitive detection of S100betabeta in serum over a wide concentration range.     

Facile preparation of a DNA sensor for rapid herpes virus detection

In this paper, a simple DNA sensor platform was developed for rapid herpes virus detection in real samples. The deoxyribonucleic acid (DNA) sequences of the herpes simplex virus (DNA probe) were directly immobilized on the surface of interdigitated electrodes by electrochemical polymerization along with pyrrole monomers. The potential was scanned from ? 0.7 to + 0.6 V, and the scanning rate was 100 mV/s. Fourier transform infrared spectroscopy was employed to verify specific DNA sequence binding and the conducting polymer. The morphology of the conducting polymer doped with DNA strands was characterized using a field emission scanning electron microscope. As-obtained DNA sensor was used to detect the herpes virus DNA in the real samples. The results show that the current DNA sensors detected the lowest DNA concentration of 2 nM. This sensitivity appears to be better than that of the DNA sensors prepared by immobilization of the DNA probe on the 3-aminopropyl-triethoxy-silance (APTS) membrane.     

TiO2 nanowire bundle microelectrode based impedance immunosensor for rapid and sensitive detection of Listeria monocytogenes

A novel TiO 2 nanowire bundle microelectrode based immunosensor was demonstrated as a more sensitive, specific, and rapid technology for detection of Listeria monocytogenes. TiO 2 nanowire bundle was prepared through a hydrothermal reaction of alkali with TiO 2 powder and connected to gold microelectrodes with mask welding. Monoclonal antibodies were immobilized on the surface of a TiO 2 nanowire bundle to specifically capture L. monocytogenes. Impedance change caused by the nanowire-antibody-bacteria complex was measured and correlated to bacterial number. This nanowire bundle based immunosensor could detect as low as 10 (2) cfu/ml of L. monocytogenes in 1 h without significant interference from other foodborne pathogens.     

Robust and highly sensitive fluorescence approach for point-of-care virus detection based on immunomagnetic separation

In this work, robust approach for a highly sensitive point-of-care virus detection was established based on immunomagnetic nanobeads and fluorescent quantum dots (QDs). Taking advantage of immunomagnetic nanobeads functionalized with the monoclonal antibody (mAb) to the surface protein hemagglutinin (HA) of avian influenza virus (AIV) H9N2 subtype, H9N2 viruses were efficiently captured through antibody affinity binding, without pretreatment of samples. The capture kinetics could be fitted well with a first-order bimolecular reaction with a high capturing rate constant k(f) of 4.25 × 10(9) (mol/L)(-1) s(-1), which suggested that the viruses could be quickly captured by the well-dispersed and comparable-size immunomagnetic nanobeads. In order to improve the sensitivity, high-luminance QDs conjugated with streptavidin (QDs-SA) were introduced to this assay through the high affinity biotin-streptavidin system by using the biotinylated mAb in an immuno sandwich mode. We ensured the selective binding of QDs-SA to the available biotin-sites on biotinylated mAb and optimized the conditions to reduce the nonspecific adsorption of QDs-SA to get a limit of detection low up to 60 copies of viruses in 200 μL. This approach is robust for application at the point-of-care due to its very good specificity, precision, and reproducibility with an intra-assay variability of 1.35% and an interassay variability of 3.0%, as well as its high selectivity also demonstrated by analysis of synthetic biological samples with mashed tissues and feces. Moreover, this method has been validated through a double-blind trial with 30 throat swab samples with a coincidence of 96.7% with the expected results.     

Amplification strategy using aggregates of ferrocene-containing cationic polythiophene for sensitive and specific electrochemical detection of DNA

We report a new electrochemical amplification strategy for an ultrasensitive electrochemical detection of DNA sequences using aggregates composed of a water-soluble, ferrocene-functionalized polythiophene. A two-step hybridization is performed at one addressing surface with PNA capture probes whereas the electrochemical detection is done on an electrode nearby. Specific and quantitative detection of DNA targets with a detection limit of 4 × 10(-16) M (about 4 zeptomoles or about 2500 copies of oligonucleotides) was achieved.     

Thermospray methods for rapid, sensitive, and nonchromatographic speciation of chromium oxidation States

A sensitive technique for speciation and quantification of Cr(III) and Cr(VI) has been developed using thermospray (TSP) sample introduction with inductively coupled plasma atomic emission spectrometry (ICPAES). For unacidified solutions, the sensitivity for Cr(III) was found to be lower than that for Cr(VI). The sensitivity for Cr(III) was further depressed to a negligible level by adjusting sample and thermospray operating parameters. The low sensitivity for Cr(III) was thought to result from the precipitation of that species to form Cr(OH)(3), which deposited within the vaporizer. For acidic solutions (1% v/v HNO(3)), the sensitivities for both species were essentially identical. On the basis of these results, methods for speciation of Cr(III) and Cr(VI) were developed. With samples buffered to pH 4.4, Cr(VI) could be selectively determined. With acidic sample aliquots (1% v/v HNO(3)), the total chromium concentration could also be determined, and the Cr(III) concentration could be calculated by difference. Parameters affecting Cr(III) sensitivity, such as control temperature, pH, and pump flow rate, were studied in addition to optimal TSP-ICPAES parameters. The limits of detection (LODs) for Cr(VI) and for total Cr were 0.47 and 0.61 μg/L with standard deviations of 1.5% and 2.0%, respectively. Good accuracy and precision of the method were demonstrated for analysis of spiked tap water and lake water samples. Mobile phase ion-pairing chromatography with ICPAES detection provided comparable results for moderately high concentration samples. Accuracy of measurements for Cr(VI) was within 1% of the certified value for NIST standard reference material 2109.     

Novel phage amplified multichannel series piezoelectric quartz crystal sensor for rapid and sensitive detection of Mycobacterium tuberculosis

The key factors that control the spread and mortality rate of tuberculosis (TB) are rapid detection and diagnosis. However, the current detection of Mycobacterium tuberculosis (M. tuberculosis) cannot meet the recommended requirements for clinical diagnosis in turnaround time. In this paper, the feature of phage D29 that infects M. tuberculosis and Mycobacterium smegmatis (M. smegmatis) was combined with the sensitivity of multichannel series piezoelectric quartz crystal sensor (MSPQC) to detect M. tuberculosis. The phage D29 played a role of inhibiting the growth of M. tuberculosis and M. smegmatis. M. tuberculosis is used to protect phage D29 from being killed by ferrous ammonium sulfate (FAS) and carries phage D29 into the detection medium containing M. smegmatis. The action of M. smegmatis indicated the existence state of phage D29 in the detection medium. The growth curve of M. smegmatis obtained by MSPQC indicated the state of the growth of M. tuberculosis. Therefore, M. tuberculosis in the sample could be rapidly detected by evaluating the extent of inhibiting the growth of M. smegmatis compared with the normal growth of M. smegmatis. The detection of M. tuberculosis was transformed into the detection of M. smegmatis, which is more rapid and sensitive than that of M. tuberculosis. For 10(2) cfu/mL of M. tuberculosis in clinical sample, the turnaround time was less than 30 h. Although statistical analysis showed that no significant difference existed between the results of the proposed method here and the BACTEC960 MGIT method in clinical M. tuberculosis detection, the phage amplified MSPQC (PA MSPQC) method presented here was faster and more economical.     

Biosensor based on cellobiose dehydrogenase for detection of catecholamines

A cellobiose dehydrogenase (CDH)-modified graphite electrode was designed for amperometric detection of catecholamines in the flow injection mode, by their recycling between the graphite electrode (+300 mV vs Ag|AgCl) and the reduced FAD cofactor of adsorbed CDH, resulting in an amplified response signal. The high efficiency of the enzyme-catecholamine reaction leads to a detection limit below 1 nM and a sensitivity of 15.8 A.M(-1) x cm(-2) (approximately 1150 nA/microM) for noradrenaline, with a coverage of less than 2.5 microg of CDH adsorbed on the electrode surface (0.073 cm(2)). Working parameters such as pH, cellobiose concentration, carrier buffer, and applied potential were optimized, using hydroquinone as a model analyte. The sensitivity, linear range, and amplification factor can be modulated by the steady-state concentration of cellobiose in the flow buffer. The response of the sensor decreases only 2% when run continuously for 4 h in the flow injection mode. The response peak maximum is obtained within 6 s at a flow rate of 0.5 mL/min, representing the time of the entire sample segment to pass the electrode. CDH enzymes from Phanerochaete chrysosporium and Sclerotium rolfsii were investigated, providing different characteristics of the sensor, with sensors made with CDH from P. chrysosporium being the better ones.     

Rapid and highly sensitive method for influenza A (H1N1) virus detection

In this study, we applied the developed paired surface plasma waves biosensor (PSPWB) in a dual-channel biosensor for rapid and sensitive detection of swine-origin influenza A (H1N1) virus (S-OIV). In conjunction with the amplitude ratio of the signal and the reference channel, the stability of the PSPWB system is significantly improved experimentally. The theoretical limit of detection (LOD) of the dual-channel PSPWB for S-OIV is 30 PFU/mL (PFU, plaque-forming unit), which was calculated from the fitting curve of the surface plasmon resonance signal with a S-OIV clinical isolate concentration in phosphate-buffered saline (PBS) over a range of 18-1.8 × 10(6) PFU/mL. The LOD is 2 orders of magnitude more sensitive than the commercial rapid influenza diagnostic test at worst and an order of magnitude less sensitive than real-time quantitative polymerase chain reaction (PCR) whose LOD for S-OIV in PBS was determined to be 3.5 PFU/mL in this experiment. Furthermore, under in vivo conditions, this experiment demonstrates that the assay successfully measured S-OIV at a concentration of 1.8 × 10(2) PFU/mL in mimic solution, which contained PBS-diluted normal human nasal mucosa. Most importantly, the assay time took less than 20 min. From the results, the dual-channel PSPWB potentially offers great opportunity in developing an alternative PCR-free diagnostic method for rapid, sensitive, and accurate detection of viral pathogens with epidemiological relevance in clinical samples by using an appropriate pathogen-specific antibody.     

Minimally stable nanoparticle-based colorimetric assay for simple, rapid, and sensitive antibody structure and activity evaluation

A gold nanoparticle-based colorimetric antibody structure and activity evaluation method is developed without using complicated and expensive instrumentation. In this assay, a minimum number of antibodies to stabilize nanoparticles are conjugated to gold nanoparticles to prepare minimally stable nanoparticle probes, and the addition of salt rapidly induced particle aggregation and a color change of the solution from red to blue (25-min assay time). It is found that the solution color change is affected by the degree of structural denaturation of antibodies, and the conformational change of antibodies affects the modification of antibodies to nanoparticles and particle stability. Importantly, the colorimetric method can be applied to different types of antibodies (IgG, IgA, and IgM) and it shows comparable or better structural sensitivity than conventional circular dichroism spectroscopy. Moreover, immunoassay results show that these structural changes of antibodies are highly correlated with their antigen-binding activities. Rapid particle aggregation and high structural sensitivity are achieved in this assay because particles are modified with a minimum number of antibodies to stabilize particles in solution. This nanoparticle-based colorimetric method could be useful in evaluating the structural and activity changes of an array of antibodies in an easy, rapid, and sensitive manner.     

Biosensor based on imaging ellipsometry for serotype-specific detection of Riemerella anatipestifer

In this study, a practicable method for the detection of Riemerella anatipestifer (R. anatipestifer) using biosensor based on imaging ellipsometry (BIE) is described. The method is performed by immobilizing anti-R. anatipestifer egg yolk immunoglubilin (IgY) onto modified chemistry surface to form sensing layer. Antigen captured by sensing layer can then be quantitatively measured through imaging ellipsometry in grayscale format. The results demonstrate that it can detect R. anatipestifer as low as 5.2 × 10³ CFU/mL. Furthermore, the proposed method can realize specific discrimination for multiple serotypes of 1, 2, 4, and 14 of R. anatipestifer. It has the advantages of rapid, simple, high sensitivity and low cost.     

Asynchronous magnetic bead rotation microviscometer for rapid, sensitive, and label-free studies of bacterial growth and drug sensitivity

The long turnaround time in antimicrobial susceptibility testing (AST) endangers patients and encourages the administration of wide spectrum antibiotics, thus resulting in alarming increases of multidrug resistant pathogens. A method for faster detection of bacterial proliferation presents one avenue toward addressing this global concern. We report on a label-free asynchronous magnetic bead rotation (AMBR) based viscometry method that rapidly detects bacterial growth and determines drug sensitivity by measuring changes in the suspension's viscosity. With this platform, we observed the growth of a uropathogenic Escherichia coli isolate, with an initial concentration of 50 cells per drop, within 20 min; in addition, we determined the gentamicin minimum inhibitory concentration (MIC) of the E. coli isolate within 100 min. We thus demonstrated a label-free, microviscometer platform that can measure bacterial growth and drug susceptibility more rapidly, with lower initial bacterial counts than existing commercial systems, and potentially with any microbial strains.     

Catalog-library approach for the rapid and sensitive structural elucidation of oligosaccharides.

We obtained the nearly complete structural elucidation of oligosaccharide components, including sequence, linkage, and even stereochemistry in the picomolar levels. The "catalog-library" approach is used for elucidating the structures of minor components in a mixture of oligosaccharides. Oligosaccharides released from a family of glycoproteins are often composed of a small finite set of monosaccharides. In this regard, the numerous oligosaccharide species are analogous to the products found in syntheses involving combinatorial libraries. The great structural diversity in the library is the result of the nearly infinite combinations in which even a small number of monosaccharides can be arranged. Fortunately, structural similarities exist between different oligosaccharides, as specific substructural motifs are preserved among different compounds. We propose that a catalog of substructural motifs can be identified and characterized by collision-induced dissociation mass spectrometry. The catalog is constructed from a set of known compounds that have been fully structurally elucidated by, for example, nuclear magnetic resonance. The catalog consists of the characteristic fragmentation patterns belonging to a set of specific substructural motifs. Collision-induced dissociation is used to determine the presence of these motifs and reconstruct the structures of less abundant components.