Histamine detection in fish samples based on indirect competitive ELISA method using iron-cobalt co-doped carbon dots labeled histamine antibody.

Due to complex matrixes and specific reagent deficiency, the rapid detection of histamine is still a challenge to date. Based on the high peroxidase-like activity of iron-cobalt co-doped carbon dots, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established for histamine detection using the mimic enzyme labeled with histamine antibody (His-Ab). Through the competitive binding of the labeled His-Ab to solid-phase and sample antigens, histamine content was detected with a linear range of 2.5-150 µg mL-1. The detection limit based on 3σ/K was 0.50 mg kg-1, which was much lower than those of commercial His-kit and HPLC methods. The ic-ELISA method was applied to histamine detection in fish samples with the recovery of (103.4 ± 0.5)%, which was in accord with those of commercial His-kit and HPLC methods. The results indicated that the established ic-ELISA method was suitable for rapid detection of histamine in fish samples with high accuracy, sensitivity and stability.

Aptamer-based fluorometric determination of chloramphenicol by controlling the activity of hemin as a peroxidase mimetic.

A method for the aptamer-based determination of chloramphenicol (CAP) was developed by exploiting the peroxidase mimicking activity of hemin. The method includes two hemin-modified DNA probes termed P1 and P2. P1, which was modified at its 5' end with one hemin monomer, contains the CAP-binding sequence. The hybridization between P1 and P2 brings the two hemin monomers in close proximity, resulting in the formation of a hemin dimer with low peroxidase mimicking activity. The duplex structure was dehybridized in the presence of CAP. The formed hemin monomer featured a strong peroxidase mimicking activity and catalyzed the conversion of non-fluorescent tyramine into fluorescent dityramine by hydrogen peroxide. Fluorescence (with an excitation/emission maxima at 320 and 410 nm, respectively) increased linearly in the 0.1 ng mL-1 to 10 ng mL-1 CAP concentration range. The detection limit based on the 3σ/k criterion reached 0.07 ng mL-1. The proposed assay was successfully employed for CAP detection in (spiked) honey samples with recoveries of 94.3-117.2%. Given its high sensitivity and good stability, this method shows potential in providing a platform for antibiotic detection.

Highly sensitive detection of multiple antibiotics based on DNA tetrahedron nanostructure-functionalized magnetic beads.

Functional DNAs-functionalized magnetic beads (MBs) offer great potential in bioanalysis field because of their target recognition and magnetic separation functions. However, the recognition capability and hybridization affinity of DNA probes often suffer from limited available space, poor probe conformation and non-selective adsorption. To overcome these limitations, we herein used aptamer-pendant DNA tetrahedron nanostructure-functionalized MBs (TETapt-tet MBs) to develop a target-response fluorescence method with tetracycline (TET) as a model. In the absence of TET, 6-carboxy-X-rhodamine-labeled complementary DNAs (ROX-cDNAs) were assembled on the surface of MBs. Upon the addition of target TET, the ROX-cDNAs were separated and released from the MBs to generate fluorescence signal. The limit of detection and limit of quantification for TET were found to be 6 pg mL-1 and 20 pg mL-1, respectively. Compared with ssDNA-functionalized MBs surface, the designed DNA tetrahedron nanostructure-based surface could decrease the hybridization time and reduce false positives, ensuring the accuracy of TET detection in complex samples. The presented method was successfully employed for TET detection in honey samples. Moreover, this functionalization strategy could be extended to detect multiple antibiotics by simply substituting different aptamer sequences. Therefore, the proposed method has great potential in the field of food safety and public health.

A dichromatic label-free aptasensor for sulfadimethoxine detection in fish and water based on AuNPs color and fluorescent dyeing of double-stranded DNA with SYBR Green I.

A dichromatic label-free aptasensor was described for sulfadimethoxine (SDM) detection. Compared with the binding of SDM-aptamer to SDM, the higher affinity of aptamer to cDNA may result in the hybridization of dsDNA. In the presence of SDM, the aptamer specifically binds to SDM, leading to a blue color of AuNPs in deposit and fluorescence at 530 nm in supernatant after adding cDNA and SGI. With no target of SDM, AuNPs protected with the aptamer re-disperse in PBS with a red color, and no fluorescence occurs in supernatant. Based on the principle, SDM can be quantitatively detected through both fluorescent emission and AuNPs color changes with recoveries ranging from 99.2% to 102.0% for fish and from 99.5% to 100.5% for water samples. An analytical linear range of 2-300 ng mL-1 was achieved with the detection limits of 3.41 ng mL-1 for water and 4.41 ng g-1 for fish samples (3σ, n = 9).

Detection of Malachite Green using a colorimetric aptasensor based on the inhibition of the peroxidase-like activity of gold nanoparticles by cetyltrimethylammonium ions.

A specific and sensitive colorimetric aptasensor is described for the determination of Malachite Green (MG). It is exploiting the inhibition of the peroxidase-like activity of gold nanoparticles (AuNPs). The AuNPs act as enzyme mimics that catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 to yield a dark blue solution. The catalytic activity is inhibited by hexadecyl trimethyl ammonium ion, specifically by cetyltrimethylammonium bromide (CTAB), which causes the aggregation of AuNPs. If a (negatively charged) RNA-aptamer against MG is added, it binds to the positively charged CTAB and prevents aggregation. This enhances the enzyme mimicking activity of the AuNPs and leads to the formation of a dark blue solution. However, in the presence of MG, the aptamer binds to MG, and leads to the aggregation of AuNPs again. The aggregated AuNPs possess a light blue color. A colorimetric method (best performed at 650 nm) was work out that can detect MG in a concentration range from 10 to 500 nmol L-1. The detection limit based on 3σ/k criterion is 1.8 nmol L-1. The assay is highly specific and accurate. Recoveries from spiked real samples (aquaculture water) ranged from 80% to 120%. Graphical abstract Based on the inhibition of cetyltrimethyal ammonium ion and the enhancement of RNA-aptamer, the differences of the peroxidase-like activities of AuNPs can be greatly enlarged with and without MG, by which a colorimetric aptasensor can be constructed for the detection of Malachite Green (MG).

Label-Free Fluorescence-Based Aptasensor for the Detection of Sulfadimethoxine in Water and Fish.

Fluorescence-based aptasensors possess high sensitivity but are complicated and usually require multistep labeling and modification in method design, which severely limit the practical applications. Here, a label-free fluorescence-based aptasensor, consisting of aptamer, gold nanoparticles (AuNPs), and cadmium telluride (CdTe) quantum dots (QDs), was developed for the detection of sulfadimethoxine (SDM) in water and fish based on the specific recognition of SDM-aptamer and the inner filter effect of QDs and AuNPs. In the absence of a target, AuNPs dispersed in salt solution because of the aptamer protection, which could effectively quench the fluorescence emission of QDs, while in the presence of SDM, AuNPs aggregated due to the specific recognition of SDM-aptamer to SDM, which resulted in fluorescence recovery. A linear response of SDM concentrations in the range of 10-250 ng mL-1 ( R2 = 0.99) was obtained, and the detection limit was 1.54 ng mL-1 (3σ, n = 9), far below the maximum residue limit (100 ng mL-1) of SDM in edible animal tissues regulated by China and the European Commission. The fluorescence-based aptasensor was applied to the detection of SDM in aquaculture water and fish samples with high accuracy, excellent precision, and ideal selectivity. The results indicated that the developed aptasensor was simple in design, easy to operate, and could be used to detect rapidly and accurately SDM in water and fish samples.

Ratiometric fluorescence probe of MIPs@CdTe QDs for trace malachite green detection in fish.

A facile and practical ratiometric fluorescence probe based on two CdTe quantum dots (QDs) coated with molecularly imprinted polymers (MIPs) was prepared for the detection of trace malachite green (MG) in fish. Two CdTe QDs coated with MIPs were fabricated by a one-pot method using MG, (3-aminopropyl) triethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) as template, functional monomer, and cross-linker, respectively. CdTe QDs with λem 530 nm (gQDs) and 630 nm (rQDs) were used as the referential fluorophore and target sensitive fluorophore, respectively. The fluorescence intensity of gQDs remained unchanged in the presence of MG, while the fluorescence of rQDs could be quantitatively quenched by MG based on the strategy of fluorescence resonance energy transfer. The ratiometric fluorescence probe (MIPs@gQDs&rQDs) was characterized by transmission electron microscopy and Fourier transform infrared spectroscopy. The linear range of MG detection was 0.1-32 µmol L-1 with a detection limit of 8.8 µg kg-1. The constructed probe has been successfully applied to the detection of MG in fish with the recoveries of 92.3-109.1%, which were validated by the method of HPLC. The result indicated that the probe possessed rapid response, wide linear range, high sensitivity, and relatively high selectivity, and was low-cost and easy in operation in the detection of MG in fish samples.

Semipermeable Functional DNA-Encapsulated Nanocapsules as Protective Bioreactors for Biosensing in Living Cells.

The development of functional DNA-based nanosensors in living cells has experienced some design challenges, including, for example, poor cellular uptake, rapid nuclease degradation, and high false positives. Herein, we designed selectively permeable poly(methacrylic acid) (PMA) nanocapsules to encapsulate functional DNAs for metal ions and small-molecules sensing in living cells. Since functional DNAs are concentrated in the nanocapsules, an increasing reaction rate is obtained in vitro. During endocytosis, polymeric capsules simultaneously improve cellular uptake of functional DNAs and preserve their structural integrity inside the confined capsule space. More importantly, selective shell permeability allows for the free diffusion of small molecular targets through capsule shells but limits the diffusion of large biomolecules, such as nuclease and nonspecific protein. Compared to the free DNAzyme, PMA nanocapsules could reduce false positives and enhance detection accuracy. Furthermore, PMA nanocapsules are biocompatible and biodegradable. Through the controllability of wall thickness, permeability, and size distribution, these nanocapsules could be expanded easily to other targets, such as microRNAs, small peptides, and metabolites. These nanocapsules will pave the way for in situ monitoring of various biological processes in living cells and in vivo.

Facile Phase Transfer and Surface Biofunctionalization of Hydrophobic Nanoparticles Using Janus DNA Tetrahedron Nanostructures.

Hydrophobic nanoparticles have shown substantial potential for bioanalysis and biomedical applications. However, their use is hindered by complex phase transfer and inefficient surface modification. This paper reports a facile and universal strategy for phase transfer and surface biofunctionalization of hydrophobic nanomaterials using aptamer-pendant DNA tetrahedron nanostructures (Apt-tet). The Janus DNA tetrahedron nanostructures are constructed by three carboxyl group modified DNA strands and one aptamer sequence. The pendant linear sequence is an aptamer, in this case AS1411, known to specifically bind nucleolin, typically overexpressed on the plasma membranes of tumor cells. The incorporation of the aptamers adds targeting ability and also enhances intracellular uptake. Phase-transfer efficiency using Apt-tet is much higher than that achieved using single-stranded DNA. In addition, the DNA tetrahedron nanostructures can be programmed to permit the incorporation of other functional nucleic acids, such as DNAzymes, siRNA, or antisense DNA, allowing, in turn, the construction of promising theranostic nanoagents for bioanalysis and biomedical applications. Given these unique features, we believe that our strategy of surface modification and functionalization may become a new paradigm in phase-transfer-agent design and further expand biomedical applications of hydrophobic nanomaterials.

Direct detection of circulating microRNAs in serum of cancer patients by coupling protein-facilitated specific enrichment and rolling circle amplification.

We have developed a simple method for direct detection of circulating microRNAs in serum by using the p19 protein-functionalized magnetic beads (PFMBs) for specific enrichment and rolling circle amplification (RCA). The detection limit for microRNAs is 1 fM. Therefore, the proposed method has the potential of being used in the analysis of circulating microRNAs and clinical diagnosis.

Ultrasensitive electrochemical detection of cancer-associated circulating microRNA in serum samples based on DNA concatamers.

MicroRNAs (miRNAs), a kind of endogenous, noncoding RNAs (19-24 nucleotides), play vital roles in regulating gene expression and cellular processes. In recent years, it has been found that circulating miRNAs are differentially expressed in patients and healthy controls. This leads to the suggestion that circulating miRNAs are promising biomarkers for cancer classification and prognosis. However, it is still difficult to detect circulating miRNAs directly from real samples such as human serum without prior extraction and purification. In this work, we developed an ultrasensitive electrochemical biosensor for detection of cancer-associated circulating miRNAs based on DNA concatamers amplification. The proposed biosensor showed a high sensitivity for target miRNA-21 in a concentration range from 100 aM to 100 pM with a detection limit of 100 aM. Furthermore, the biosensor was successfully employed for direct detection of circulating miRNAs in human serum. Due to the high sensitivity, good selectivity and stability, the proposed electrochemical biosensor might have potential clinical application for circulating miRNAs in relation to diagnosis and prognosis.

Enzyme-free and label-free ultrasensitive electrochemical detection of human immunodeficiency virus DNA in biological samples based on long-range self-assembled DNA nanostructures.

Biosensors based on nanomaterials have been used for detection of various biological molecules with high sensitivity and selectivity. Herein, we developed a simple and ultrasensitive electrochemical DNA biosensor using long-range self-assembled DNA nanostructures as carriers for signal amplification, which can achieve an impressive detection limit of 5 aM human immunodeficiency virus (HIV) DNA even in complex biological samples. In this study, we designed two auxiliary probes. A cascade of hybridization events between the two auxiliary probes can lead to long-range self-assembly and form micrometer-long one-dimensional DNA nanostructures. In the presence of target DNA, each copy of the target can act as a trigger to connect a DNA nanostructure to a capture probe on the electrode surface. Then, a great amount of redox indicator [Ru(NH(3))(6)](3+) can be electrostatically bound to the DNA nanostructures and eventually result in significantly amplified electrochemical signals.

Experimental study on bone defect treated by combined autologous bone marrow transplantation, cuttlebone, and sodium hyaluronate / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the feasibility of repairing bone defect by combined autologous bone marrow transplantation, cuttebone, and sodium hyaluronate.</p><p><b>METHODS</b>Forty-eight New Zealand rabbits were randomly divided into four groups. The 10-mm bone defect of the radial shaft animal model was established, with the periosteum remained. Rabbits of Group A were treated by autologous bone marrow transplantation, cuttlebone, and sodium hyaluronate. Those of Group B were treated by autologous bone marrow transplantation and cuttlebone. Rabbits of Group C were implanted with cuttlebone and sodium hyaluronate. And rabbits of Group D were taken as the blank control. There were twelve rabbits in each group. All rabbits were sacrificed, and the general histological examination, X-ray test, the pathohistological observation and scoring, the new born formation area measurement were performed at 2-week, 4-week, 8-week, and 12-week after transplantation respectively. The capacities for bone transplantation and defect repairing were compared and analyzed as well.</p><p><b>RESULTS</b>The bone defect of Group A was completely repaired at week 12. The comprehensive indices at each time point were superior to those of the rest groups, showing statistical significance (P<0.05). The bone repair in Group B and Group C were somewhat poor, with the repairing effect inferior to that of Group A. The bone repairing was better in Group B than in Group C. Most portion of the bone defect in Group D was filled with fibrous tissue and muscular tissue, with little bone repairing.</p><p><b>CONCLUSIONS</b>The combined autologous bone marrow transplantation, cuttlebone, and sodium hyaluronate showed obviously synergistically bone forming capacities. It could be taken as a substitute material for transplantation.</p>