Avidin-biotin System Research Articles

Cyclic arginine-glycine-aspartic acid-modified red blood cells for drug delivery: Synthesis and in vitro evaluation

Red blood cells (RBCs) are an excellent choice for cell preparation research because of their biocompatibility, high drug loading, and long half-life. In this study, doxorubicin (DOX) was encapsulated with RBCs as the carrier. The biotin-avidin system binding principle was used to modify biotinylated cyclic arginine-glycine-aspartic acid (cRGD) onto RBC surfaces for accurate targeting, high drug loading, and sustained drug release. The RBC drug delivery system (DDS) was characterized, and the concentration of surface sulfur in the energy spectrum was 6.330%. The physical and chemical properties of RBC DDS were as follows: drug content, 0.857 mg/mL; particle size, 3339 nm; potential value, −12.5 mV; and cumulative release rate, 81.35%. There was no significant change in RBC morphology for up to seven days. The results of the targeting and cytotoxicity studies of RBC DDS showed that many RBCs covered the surfaces of U251 cells, and the fluorescence intensity was higher than that of MCF-7 cells. The IC50 value of unmodified drug-loaded RBCs was 2.5 times higher than that of targeted modified drug-loaded RBCs, indicating that the targeting of cancer cells produced satisfactory inhibition. This study confirms that the RBC DDS has the characteristics of accurate targeting, high drug loading, and slow drug release, which increases its likelihood of becoming a clinical cancer treatment in the future.

Electrofluorochromic Imaging Analysis of Glycan Expression on Living Single Cell with Bipolar Electrode Arrays.

The in situ glycan profiling of a single tumor cell plays an important role in personalized cancer treatment. Herein, an integrated microfluidic system was designed for living single-cell trapping and real-time monitoring of galactosyl expression on the surface, combining closed bipolar electrode (BPE) arrays and electrofluorochromic (EFC) imaging. Galactosyl groups on human liver cancer HepG2 cells were used as the model analysts, galactose oxidase (GAO) could selectively oxidize hydroxyl sites of galactosyl groups on the cell surface to aldehydes, and then biotin hydrazide (BH) was used to label the aldehydes by aniline-catalyzed hydrazone ligation. With the biotin-avidin system, nanoprobes were finally introduced to the galactosyl groups on the cell surface with avidin as a bridge, which was prepared by simultaneously assembling ferrocene-DNA (Fc-DNA) and biotin-DNA (Bio-DNA) on gold nanoparticles (AuNPs) due to their large surface area and excellent electrical conductivity. After a labeled single cell was captured in the anodic microchannel, the Fc groups attached on the cell surface were oxidized under suitable potential, and the nonfluorescent resazurin on the cathode was correspondingly reduced to produce highly fluorescent resorufin, collected by fluorescence confocal microscope. The combination of EFC imaging and BPE realized monitoring galactosyl group expression of 5.0 × 108 molecules per cell. Furthermore, the proposed platform had the ability to distinguish a single cancer cell from a normal cell according to the expression level of galactosyl groups and to dynamically monitor the galactosyl group variation on the cell surface, providing a simple and accessible method for the single-cell analysis.

DNA building blocks for AFM tip functionalization: An easy, fast and stable strategy.

Biosensing atomic force microscopy (AFM) offers the unique feature to determine the energy landscape of a bimolecular interaction at the real single molecule level. Furthermore, simultaneous and label-free mapping of molecular recognition and the determination of sample topography at the nanoscale gets possible. A prerequisite and one of the major parts in biosensing AFM are the bio-functionalized AFM tips. In the past decades, different approaches for tip functionalization have been developed. Using these functionalization strategies, several biological highly relevant interactions at the single molecule level have been explored. For the most common approach, the use of a heterobifunctional poly(ethylenglycol) crosslinker, a broad range of linkers for different chemical coupling strategies is available. Nonetheless, the time consuming functionalization protocol as well as the broad distribution of rupture length reduces the possibility of automation and may reduce the accuracy of the results. Here we present a stable and fast forward approach based on tetra-functional DNA tetrahedra. A fast functionalization and a sharp defined distribution of rupture length gets possible with low effort and high success rate. We tested the performance on the classical avidin biotin system by using tetrahedra with three disulfide legs for stable and site directed coupling to gold coated tips and a biotinylated end at the fourth vertex. A special advantage appears when working with a DNA aptamer as sensing molecule. In this case, the fourth strand can be extended by a certain DNA sequence complementary to the linkage part of an aptamer. This AFM tip functionalization protocol was applied on thrombin using DNA aptamers directed against the fibrinogen binding side of human thrombin.

A Flexible PI/Si/SiO2 Piezoresistive Microcantilever for Trace-Level Detection of Aflatoxin B1.

In this paper, a polyimide (PI)/Si/SiO2-based piezoresistive microcantilever biosensor was developed to achieve a trace level detection for aflatoxin B1. To take advantage of both the high piezoresistance coefficient of single-crystal silicon and the small spring constant of PI, the flexible piezoresistive microcantilever was designed using the buried oxide (BOX) layer of a silicon-on-insulator (SOI) wafer as a bottom passivation layer, the topmost single-crystal silicon layer as a piezoresistor layer, and a thin PI film as a top passivation layer. To obtain higher sensitivity and output voltage stability, four identical piezoresistors, two of which were located in the substrate and two integrated in the microcantilevers, were composed of a quarter-bridge configuration wheatstone bridge. The fabricated PI/Si/SiO2 microcantilever showed good mechanical properties with a spring constant of 21.31 nN/μm and a deflection sensitivity of 3.54 × 10−7 nm−1. The microcantilever biosensor also showed a stable voltage output in the Phosphate Buffered Saline (PBS) buffer with a fluctuation less than 1 μV @ 3 V. By functionalizing anti-aflatoxin B1 on the sensing piezoresistive microcantilever with a biotin avidin system (BAS), a linear aflatoxin B1 detection concentration resulting from 1 ng/mL to 100 ng/mL was obtained, and the toxic molecule detection also showed good specificity. The experimental results indicate that the PI/Si/SiO2 flexible piezoresistive microcantilever biosensor has excellent abilities in trace-level and specific detections of aflatoxin B1 and other biomolecules.

Controlling water transport between micelles and aqueous microdroplets during sample enrichment

Droplet microfluidics technologies have advanced rapidly, but enrichment in droplets has still been difficult. To deterministically control the droplet enrichment, the water transport from an aqueous microdroplet in organic continuous phase containing span 80 micelles was investigated. Organic phase containing Span-80-micelles contacted a NaCl aqueous solution to control hydration degree of the micelles, prior to being used in the microfluidic device. Then, the organic phase was continuously applied to the microdroplets trappled in microwells. Here, water was transported from the microdroplet to the organic phase micelles. This spontaneous emulsification process induced the droplet shrinkage and stopped when the microdroplet reached a certain diameter. The micelle hydration degree correlated well with the final water activity of droplets. The enrichment factor can be determined by the initial microdroplet salt concentration and by the micelle hydration degree. As a proof-of-concept experiment, enrichment of fluorescent nanoparticles and dye was demonstrated, and fluorescent resonance energy transfer was observed as expected. Another demonstration of bound-free separation was performed utilizing the avidin-biotin system. This technique has the potential to be a powerful pretreatment method for bioassays in droplet microfluidics.

Avidin-Biotin ELISA-Based Detection of 5hmC.

The enzyme-linked immunosorbent assay (ELISA) technique has been developed half a century ago, and yet its role in molecular biology remains significant. Among the most sensitive of immunoassays, it offers high throughput, combined with affordability and ease of use. This chapter provides the procedure of a highly reproducible indirect sandwich ELISA protocol, which can be applied to a variety of semi-quantitative assays for the investigation of the molecular biology of 5-hydroxymethylcytosine (5hmC) or TET enzymes. Three variations of this protocol are described: assessment and validation of 5hmC-binding proteins, screening and validation of anti-5hmC antibodies, or a readout of TET catalytic activity in in vitro experiments. The assay principle is based on the use of a high affinity avidin-biotin system for efficient immobilization of DNA fragments for further detection by high specificity antibodies. A colorimetric enzymatic reaction is ultimately developed with intensity correlating with the amount of attached antigen.

Electrochemical Immunosensor for the Early Detection of Rheumatoid Arthritis Biomarker: Anti-Cyclic Citrullinated Peptide Antibody in Human Serum Based on Avidin-Biotin System.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that produces a progressive inflammatory response that leads to severe pain, swelling, and stiffness in the joints of hands and feet, followed by irreversible damage of the joints. The authors developed a miniaturized, label-free electrochemical impedimetric immunosensor for the sensitive and direct detection of arthritis Anti-CCP-ab biomarker. An interdigitated-chain-shaped microelectrode array (ICE) was fabricated by taking the advantage of microelectromechanical systems. The fabricated ICE was modified with a self-assembled monolayer (SAM) of Mercaptohexanoic acid (MHA) for immobilization of the synthetic peptide bio-receptor (B-CCP). The B-CCP was attached onto the surface of SAM modified ICE through a strong avidin-biotin bio-recognition system. The modified ICE surface with the SAM and bio-molecules (Avidin, B-CCP, Anti-CCP-ab and BSA) was morphologically and electrochemically characterized. The change in the sensor signal upon analyte binding on the electrode surface was probed through the electrochemical impedance spectroscopy (EIS) property of charge-transfer resistance (Rct) of the modified electrodes. EIS measurements were target specific and the sensor response was linearly increased with step wise increase in target analyte (Anti-CCP-ab) concentrations. The developed sensor showed a linear range for the addition of Anti-CCP-ab between 1 IU mL−1 → 800 IU mL−1 in phosphate buffered saline (PBS) and Human serum (HS), respectively. The sensor showed a limit of detection of 0.60 IU mL−1 and 0.82 IU mL−1 in the PBS and HS, respectively. The develop bio-electrode showed a good reproducibility (relative standard deviation (RSD), 1.52%), selectivity and stability (1.5% lost at the end of 20th day) with an acceptable recovery rate (98.0% → 101.18%) and % RSD’s for the detection of Anti-CCP-ab in spiked HS samples.

Research progress in signal amplification for immunoassays

With the requirements of early diagnosis, biomarker development and functional definition, the challenge of sensitivity of immunoassay has become increasingly prominent. How to improve it to break the bottleneck has become a major challenge in the field of bioassays. Amplifying the immunosignal is the most direct method to improve detection sensitivity. Biotin-avidin system (BAS), tyramide signal amplification (TSA) and immuno-polymerase chain reaction (Im-PCR) are the most classic signal amplification techniques which significantly improved the sensitivity of immunoassays. In recent years, studies have confirmed that the sensitivity of immunoassays can be further increased by approximately three orders of magnitude with the invention of techniques including catalyzed reporter deposition-based signal amplification, nanotechnologies-based signal amplification and hybridization chain reaction-based signal amplification. Herein, we will summarize the techniques that have been developed in recent years for amplifying the signals of immunodetection and comparatively analyze their advantages and disadvantages in order to provide reference for the developed techniques transformed to clinical application and further research on ultrasensitive immunoassays. Key words: Immunoassay; Sensitivity; Biomarkers; Signal amplification

Size-adaptable and ligand (biotin)-sheddable nanocarriers equipped with avidin scavenging technology for deep tumor penetration and reduced toxicity

The conventional active-targeting nano-chemotherapy suffers from poor tumor tissue penetration and non-negligible toxicity due to the size/ligand dilemmas and insufficient target selectivity. In this report, a stimuli-responsive size-adaptable and ligand (biotin)-sheddable drug delivery system (DDS) combined with two-step strategy of biotin-avidin system was designed to seek a balance between tumor targeting and penetration as well as to self-scavenge the nonresponsive nanocarriers in normal tissues. This DDS was composed of ‘multi-seed’ polymeric liposomes (ASL-BIO-MPL) with asulacrine-loaded micelles as seeds in their aqueous cavities. The shell of such liposomes was modified with MMP-9 cleavable polymer-polypeptide functionalized with the tumor targeting ligand biotin. ASL-BIO-MPL could disintegrate into mixture of irregularly-shaped liposomes (~200 nm) and scattered tiny micelles (~40 nm) after incubation with MMP-9. The fluorescence-labeled BIO-MPL could travel to the center of the 4T1 breast tumor spheroids under the action of MMP-9, possibly benefited from the relay of released tiny micelles. Conversely, neither the biotin-modified micelles nor non-MMP-9-responsive multi-seed liposomes could penetrate into the spheroids possibly due to the potent binding-site barrier of biotin and large size, respectively. In tumor-bearing mice, ASL-BIO-MPL exhibited the strongest drug penetrability and thus the optimal inhibition of tumor growth compared to other formulations. Following administration of avidin with a rational dosage regimen, the number of apoptotic cells in normal tissues induced by ASL-BIO-MPL reduced without affecting their targeting effect, suggesting the followed administration of adivin could scavenge the DDS in non-target site. Overall, the size/ligand adapting MPL system combined with two-step strategy of biotin-avidin may provide potential avenues for nanocarriers to enhance deep tumor tissue targeting and protect normal tissues.

Generating a High Valency Biotin Binder by Selecting Uniform Protein Assemblies via Crystallization

Crystallization is a common practice in the purification process in small molecule synthesis while selecting the wanted product. For proteins it is rarely applied due to the methodological predicaments in obtaining crystals. Our observation of the stabilized octamers in the crystal structure of hoefavidin, a novel dimeric member of the avidin family, led to the notion of developing a novel biotechnological tool via covalent crosslinking. The avidin–biotin system has been exploited for decades utilizing the ultra-high affinity between avidin and biotin as a basis for numerous applications. Optimizing the system led to the discovery of a novel group of dimeric avidins including hoefavidin. Hoefavidin has a dynamic quaternary structure, where a dimer is the basis for generating the octamer via crystallographic symmetry operation. Upon biotin binding in solution hoefavidin dissociates solely into dimers. In order to stabilize the octamer, we designed the P61C mutant to form a disulfide bridge stabilizing the octamer and preventing dissociation upon biotin binding. The process of selecting P61C hoefavidin uniform octamers includes crystallization followed by dissolving the crystals. The P61C modified hoefavidin octamer can have a substantial added value to the various biotechnological applications and advances of the biotin based high affinity systems.

Ultrasensitive electrochemical detection of Mycobacterium tuberculosis IS6110 fragment using gold nanoparticles decorated fullerene nanoparticles/nitrogen-doped graphene nanosheet as signal tags

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains the top fatal infection continuing to threat public health, and the present detection method for MTB is facing great challenges with the global TB burden. In response to this issue, a novel electrochemical DNA biosensor was developed for detecting the IS6110 fragment within MTB. For the first time, the nanohybrid of gold nanoparticles decorated fullerene nanoparticles/nitrogen-doped graphene nanosheet (Au-nano-C60/NGS) directly served as a new signal tag to generate signal response without additional redox molecules and subsequently labeled with signal probes (SPs) to form tracer label to achieve signal amplification. Additionally, a biotin-avidin system was introduced to immobilize abundant capture probes (CPs), further improving the sensitivity of the proposed biosensor. After a typical sandwich hybridization, the proposed electrochemical DNA biosensor was incubated with tetraoctylammonium bromide (TOAB), which was used as a booster to induce the intrinsic redox activity of the tracer label, resulting in a discriminating current response. The proposed electrochemical DNA biosensor shows a broad linear range for MTB determination from 10 fM to 10 nM with a low limit of detection (LOD) of 3 fM. In addition, this proposed biosensor not only distinguishes mismatched DNA sequence, but also differentiates MTB from other pathogenic agents. More importantly, it has been preliminarily applied in clinical detection and displayed excellent ability to identify the PCR products of clinical samples. There is great potential for this developed method to be used in early diagnosis and monitor of TB.

Aberrant expression of N-glycolyl GM3 ganglioside is associated with the aggressive biological behavior of human sarcomas

BackgroundThe aberrant expression of N-glycolyl GM3 ganglioside (NeuGcGM3) in patients with sarcomas was reevaluated by assessing the relation of this molecule with some clinicopathological features and overall survival (OS) of patients.MethodsFifty formalin-fixed and paraffin-embedded specimens from patients diagnosed with sarcomas were included. For the evaluation of NeuGcGM3, the 14F7 monoclonal antibody followed by a peroxidase avidin-biotin system was used. Clinicopathological features were obtained from patient records. Survival rates were estimated by the Kaplan-Meier method and compared with the log-rank test. For multivariate analyses, the Cox regression model was used to identify independent prognostic factors for OS.ResultsThe majority of samples had high levels of NeuGcGM3 expression (66.0%) that showed statistical correlation with age (p = 0.014), TNM stage (p = 0.022), histological grade (p = 0.013) and proliferation rates (p = 0.012). In addition, a tendency for association with tumor depth (p = 0.070) was evidenced. In univariate survival analysis, TNM stage (p = 0.000), occurrence of metastasis (p = 0.000) and expression of NeuGcGM3 (p = 0.034) were significant prognostic factors for OS, while a tendency for association was evidenced for histological grade (p = 0.091). Among these variables, only the presence of metastasis (p = 0.001) was an independent prognostic factor on multivariate analysis.ConclusionsThe present research suggests the evaluation of NeuGcGM3 expression as a complementary prognostic factor in sarcoma, although our results need to be validated in a larger series and prospective studies. Moreover, our results could support the use of this molecule as a target for immunotherapy.

A Cautionary Tale: Endogenous Biotinylated Proteins and Exogenously-Introduced Protein A Cause Antibody-Independent Artefacts in Western Blot Studies of Brain-Derived Proteins

Antibodies are commonly used to detect or isolate proteins from biological samples. Much attention has been paid to the potential for poorly-characterized antibodies to lead to misleading results, but antibody-independent artefacts may also occur. Here, we recount two examples of antibody-independent artefacts that have confounded the interpretation of results in our search for molecular entities associated with memory loss in Alzheimer’s disease (AD). First, when using biotin-avidin systems for antibody detection, endogenous biotinylated proteins created spurious bands in Western blots of brain lysates from AD patients and transgenic mouse models of AD. These artefactual bands occurred in a transgene- and strain-dependent manner. A second, unexpected artefact occurred when Protein A-conjugated Sepharose beads were used to deplete lysates of endogenous immunoglobulins prior to immunopurification of target proteins. In these assays, Protein A shed from the beads, then bound to (and was eluted from) an immunoaffinity matrix designed to capture AD-related proteins. The Protein A then bound detection antibodies when the immunoaffinity eluates were analyzed by Western blot. Both of these artefacts–the endogenous biotinylated proteins and the Protein A artefact–can be monitored by including an “irrelevant” antibody as an experimental control (e.g., running a parallel protocol in which the antibody directed against the target of interest is replaced by a non-specific antibody).

Egg autofluorescence and options for detecting peanut agglutinin binding for the identification of Haemonchus contortus eggs in fecal samples

Quantifying eggs from Haemonchus and other trichostrongyle genera in sheep and goat fecal samples is important for evaluating control and treatment strategies for this family of nematodes with divergent pathologies, capabilities for anthelmintic resistance and environmental susceptibilities. Unfortunately, egg morphology among most of the genera do not differ enough to support the accurate identification of these genera with standard microscopic techniques. Several studies have identified specific lectins which bind selectively to sugars located on the egg surfaces for individual genera among the trichostrongyles. To detect lectins binding to these eggs, they must be directly or indirectly bound to fluorophores, and observed with an epi-fluorescence microscope. The binding of multiple lectins to isolated eggs from a fecal sample can be simultaneously detected if fluorophores are used whose excitation and emission spectra do not overlap, and this would enable the development of a fluorescence-based diagnostic test that identifies multiple trichostrongyle genera within each sample. The present study compared the usefulness of different, commercially available detection systems for use in detecting lectin binding to trichostrongyle eggs. Comparisons were made using the detection of PNA binding to H. contortus eggs with the goal of finding three systems with color spectra that do not overlap. These evaluations included both fluorophores directly conjugated to PNA in a one-step incubation protocol and a two-step incubation protocol involving biotinylated PNA and streptavidin conjugated to different fluorophores. Autofluorescence can affect the efficiency of any fluorescence-based detection system, and significant autofluorescence was observed among the unstained H. contortus eggs with the DAPI-type fluorescence filter, but it was significantly lower with the FITC-type filter and was virtually absent with the rhodamine-type filter. This study demonstrated that all the PNA detection methods tested with H. contortus eggs generated fluorescence intensities (FIs) that were significantly above the autofluorescence generated by the eggs among the three different fluorescence filters. Fluorescence intensities from PNA directly conjugated to either the FITC or rhodamine fluorophores were not different, but the lower autofluoresence in the rhodamine-type filter will enable this fluorophore to be detected more efficiently. Use of biotinylated PNA combined with streptavidin-conjugated to synthetic fluorophores (Alexa Fluor 405, 488 and 546) significantly increased FIs over that of the directly conjugated PNA, but there were no significant differences in FIs among these three biotin-avidin conjugation fluorophores. This biotin-avidin system required two incubation steps. Doubling the concentration of PNA also provided increased FI, at least for the biotin-avidin system. Adding an additional amplification step to the biotin-avidin system involving biotinylated anti-streptavidin followed by the streptavidin-Alexa Fluor complex also provided additional fluorescence.

An Electrochemical Sense Array Based on Aptamer and Biotin-Avidin System for the Selective Detection of Glucagon-Like Peptide-1.

GLP-1 as an incretin, has the ability to decrease blood sugar levels in a glucose-dependent manner by enhancing the secretion of insulin. Besides the insulinotropic effects, GLP-1 has been associated with numerous regulatory and protective effects. Thus, the action of GLP-1 is preserved in patients with type 2 diabetes and substantial pharmaceutical research has therefore been directed towards the development of GLP-1-based treatment. In this work, we reported an electrochemical sense array based on the aptamer and biotin-avidin system for the detection of glucagon-like peptide-1 (GLP-1). The sense array employed a "stem-loop" conformation ap-tamer which was immobilized on the electrode of the 16-unit gold array via pre-labeled thiol group (-SH). Pre-labeled biotin serves as an affinity tag for the binding of avidin-horseradish peroxidase (avidin-HRP). The stem-loop structure of the aptamer kept the biotin from being approached by a bulky avidin-HRP by means of the steric hindrance. After the interaction of the target (GLP-1) and the aptamer, the aptamer would undergo a significant conformational change to force biotin away from the electrode, giving the avidin-HRP easy access to the labeled biotin. The HRP in the substrate could sensitively transduce the concentration of GLP-1 into the electrical signals, which were then measured by electrochemical technology of cyclic voltammetry and amperometric i-t curve. Under the optimal experimental conditions, the proposed sense array for GLP-1 had a good linear relationship from 0.1 pmol/L to 20 pmol/L with a detection limit of 0.05 pmol/L and can be used to accurately detect the GLP-1 in serum. The experimental results show that GLP-1 could be selectively detected by the electrochemical sense array, indicating that the proposed sense array based on the biotin-avidin system and the stem-loop aptamer has great potential in the detection of GLP-1.

Bioconjugation in Drug Delivery: Practical Perspectives and Future Perceptions.

For the past three decades, pharmaceutical research has been mainly converging to novel carrier systems and nanoparticulate colloidal technologies for drug delivery, such as nanoparticles, nanospheres, vesicular systems, liposomes, or nanocapsules to impart novel functions and targeting abilities. Such technologies opened the gate towards more sophisticated and effective multi-acting platform(s) which can offer site-targeting, imaging, and treatment using a single multifunctional system. Unfortunately, such technologies faced major intrinsic hurdles including high cost, low stability profile, short shelf-life, and poor reproducibility across and within production batches leading to harsh bench-to-bedside transformation.Currently, pharmaceutical industry along with academic research is investing heavily in bioconjugate structures as an appealing and advantageous alternative to nanoparticulate delivery systems with all its flexible benefits when it comes to custom design and tailor grafting along with avoiding most of its shortcomings. Bioconjugation is a ubiquitous technique that finds a multitude of applications in different branches of life sciences, including drug and gene delivery applications, biological assays, imaging, and biosensing.Bioconjugation is simple, easy, and generally a one-step drug (active pharmaceutical ingredient) conjugation, using various smart biocompatible, bioreducible, or biodegradable linkers, to targeting agents, PEG layer, or another drug. In this chapter, the different types of bioconjugates, the techniques used throughout the course of their synthesis and characterization, as well as the well-established synthetic approaches used for their formulation are presented. In addition, some exemplary representatives are outlined with greater emphasis on the practical tips and tricks of the most prominent techniques such as click chemistry, carbodiimide coupling, and avidin-biotin system.

A reversible fluorescent probe for directly monitoring protein-small molecules interaction utilizing vibration-induced emission

The interactions between proteins and small molecules play an important role in the regulation of various cellular processes and modern drug discovery. Herein, we have developed a novel strategy for real-time monitoring the protein-ligand interaction based on vibration-induced emission (VIE) mechanism. These results demonstrated that the probe DPAC-DB could directly visualize the binding process in biotin-avidin system, which was undisturbed to other environmental stimuli, such as pH, interference species and temperature. Furthermore, the unique VIE effect caused by dramatic and reversible intramolecular vibrations enabled our strategy possible to build up the bio-logic gate. This method to efficiently control the emission of VIE-based probe through the reversible noncovalent protein-small molecules interactions opens new avenues for the development of potent protein pharmaceuticals and small molecule drugs.

Preparation of monoclonal antibodies against KHV and establishment of an antigen sandwich ELISA for KHV detection

Infections with koi herpesvirus (KHV) in carp are still a severe problem worldwide. Detection and elimination of infected fish are necessary for control of the Koi herpesvirus disease (KHVD). Serum is an excellent specimen for KHV testing because of high survivability of KHV in serum and ease of collection, storage, and handling. The direct detection of fish viruses based on the sandwich ELISA has emerged as a practical and reliable means of diagnosis. Thus, it is important to create monoclonal antibodies (MAbs) against purified KHV. By using hybridoma-monoclonal antibody technology, two hybridoma cell lines secreting MAbs against the KHV were established. By Western blot and IFAT analysis, the secreted MAbs from cell line IB7IB4 and cell line 7C72F7 recognized proteins of KHV. The result demonstrated that the MAbs were highly specific and sensitive to the KHV, and can be used for monitoring the virus quantification of carp, for example, the direct KHV diagnosis by sandwich enzyme-linked immunosorbent assay(ELISA). An antigen sandwich ELISA applying the biotin-avidin system was established using the biotinylated MAb IB7IB4 and 7C72F7 to detect virus in koi sera. These MAbs did not react with any of the tested other viruses by ELISA except KHV. The detection limit of the test was 3.923ng/ml KHV. Thus, this antigen sandwich ELISA is suitable for recognition of KHV.

Design of a microfluidic chip consisting of micropillars and its use for the enrichment of nasopharyngeal cancer cells.

The aim of the present study was to discuss the design of a microfluidic chip consisting of columns, and its use for the enrichment of nasopharyngeal cancer (NPC) cells. A microfluidic chip experiment was simulated using FLUENT software. Within the microfluidic chip, aptamers were bound to the reaction chamber (consisting of columns) using a biotin-avidin system. Cell suspension was introduced into the reaction chamber to capture NPC cells. NPC cells were subsequently eluted, and the capture rate of the cells was calculated. The modified aptamer-bound microfluidic chip was able to capture NPC cells with a capture rate of ~90%. The modified aptamer-bound microfluidic chip has a wide range of potential applications for the diagnosis of NPC.

Kinetic Mechanism of Metal Enhanced Fluorescence by Gold Nanoparticle with Avidin–Biotin as Spacer and by Gold–Silver Core–Shell Nanoparticle Using Fluorescence Lifetime Image Microscopy

We study the effects of metal-enhanced fluorescence (MEF) on rhodamine B fluorophore by nanoparticles of varied shapes. The avidin–biotin system was used as spacer to connect the fluorophore to the surface of nanoparticles. Fluorescence lifetime image microscopy (FLIM) was used to detect emission lifetime for dye molecules on single nanoparticles. Spherical gold particles diameter of 60 and 170 nm, respectively, cube length of 70 nm, and rhombic dodecahedron (RD) diameter of 63 nm were used. In the measured emission curves of rhodamine B, we obtained a short component with lifetime 16–26 ps attributed to the fluorophore under influence of the local electric field of gold nanoparticle and energy dissipation to nanoparticle. The second lifetime is 200, 270, 280, and 330 ps for 170 nm sphere, 70 nm cube, 63 nm RD, and 60 nm sphere, respectively. This component is referred to as the bright mode of nanoparticle which is coupled to the excited dye molecule and transfers energy back to the fluorophore. On the ba...