Contractile Hairpin DNA-Mediated Dual-Mode Strategy for Simultaneous Quantification of Lactoferrin and Iron Ion by Surface-Enhanced Raman Scattering and Fluorescence Analysis.

DNA-mediated self-assembly technology with good sensitivity and affinity ability has been rapidly developed in the field of probe sensing. The efficient and accurate quantification of lactoferrin (Lac) and iron ions (Fe3+) in human serum and milk samples by the probe sensing method can provide useful clues for human health and early diagnosis of anemia. In this paper, contractile hairpin DNA-mediated dual-mode probes of Fe3O4/Ag-ZIF8/graphitic quantum dot (Fe3O4/Ag-ZIF8/GQD) NPs were prepared to realize the simultaneous quantification of Lac by surface-enhanced Raman scattering (SERS) and Fe3+ by fluorescence (FL). In the presence of targets, these dual-mode probes would be triggered by the recognition of aptamer and release GQDs to produce FL response. Meanwhile, the complementary DNA began to shrink and form a new hairpin structure on the surface of Fe3O4/Ag, which produced hot spots and generated a good SERS response. Thus, the proposed dual-mode analytical strategy possessed excellent selectivity, sensitivity, and accuracy due to the dual-mode switchable signals from "off" to "on" in SERS mode and from "on" to "off" in FL mode. Under the optimized conditions, a good linear range was obtained in the range of 0.5-100.0 µg/L for Lac and 0.01-5.0 µmol/L for Fe3+ and with detection limits of 0.14 µg/L and 3.8 nmol/L, respectively. Finally, the contractile hairpin DNA-mediated SERS-FL dual-mode probes were successfully applied in the simultaneous quantification of iron ion and Lac in human serum and milk samples.

Sensitive Imaging of Cellular RNA via Cascaded Proximity-Induced Fluorogenic Reactions.

Owing to its important biological functions, RNA has become a promising molecular biomarker of various diseases. With a dynamic change in its expression level and a relatively low amount within the complicated biological matrix, signal amplification detection based on DNA probes has been put forward, which is helpful for early diagnosis and prognostic prediction. However, conventional methods are confined to cell lysates or dead cells and are not only time-consuming in sample preparation but also inaccessible to the spatial-temporal information of target RNAs. To achieve live-cell imaging of specific RNAs, both the detection sensitivity and intracellular delivery issues should be addressed. Herein, a new cascaded fluorogenic system based on the combination of hybridization chain reactions (HCRs) and proximity-induced bioorthogonal chemistry is developed, in which a bioorthogonal reaction pair (a tetrazine-quenched dye and its complementary dienophile) is brought into spatial proximity upon target RNA triggering the HCR to turn on and amplify the fluorescence in one step, sensitively indicating the cellular distribution of RNA with minimal false positive results caused by unspecific degradation. Facilitated by a biodegradable carrier based on black phosphorus with high loading capacity and excellent biocompatibility, the resulting imaging platform allows wash-free tracking of target RNAs inside living cells.

Insights into the effects of acetate on the community structure of Candidatus Accumulibacter in biological phosphorus removal system using DNA stable-isotope probing (DNA-SIP).

Sodium acetate has been most commonly used as the external carbon source to achieve successful performance of full-scale enhanced biological phosphorus removal (EBPR) processes, but its microbial mechanism for the improvement of phosphorus removal performance was still unclear. DNA based stable-isotope probing (DNA-SIP) is able to discriminate the metabolic activity of different microbes for specific substrates, thus it was applied to explore the different effects of sodium acetate on the community structure of Candidatus Accumulibacter (hereafter called Accumulibacter) and Candidatus Competibacter (hereafter called Competibacter) in a modified University of Cape Town (MUCT) process treating the real domestic sewage. Results showed that acetate addition significantly improved the abundance of Accumulibacter and Competibacter in MUCT. Accumulibacter clade IID exhibited the highest proportion in all clades before and after acetate supplementation but the proportion decreased from 95.4 % on day 23-66.3% on day 95. Contrarily, the proportion of clade IIF increased from 0.9% to 24%. DNA-SIP incubation found that the ratio of Accumulibacter in the heavy fractions to the total quantities increased faster than that of Competibacter, which successfully revealed the acetate assimilating precedence of Accumulibacter over Competibacter. Besides, the ratios of Accumulibacter clade IIF in heavy fraction increased by 22.3 %, exhibited a higher metabolic activity than other clades. Adequate acetate accomplied with high temperature possibly promoted the preferential proliferation of clade ⅡF, which provided a way to enrich clade IIF. This is the first study that successfully applied DNA-SIP to discriminate the acetate metabolic activity of Accumulibacter and Competibacter, and Accumulibacter clades.

An electrochemical DNA biosensor fabricated from graphene decorated with graphitic nanospheres.

Graphene decorated with graphitic nanospheres functionalized with pyrene butyric acid (PBA) is used for the first time to fabricate a DNA biosensor. The electrode was formed by attaching a DNA probe onto PBA, which had been stacked onto a graphene material decorated with graphene nanospheres (GNSs). The nanomaterial was drop-coated onto a carbon screen-printed electrode (SPE) to create the GNS-PBA modified electrode (GNS-PBA/SPE). A simple method was used to produce GNS by annealing graphene oxide (GO) solution at high temperature. Field emission scanning electron micrographs confirmed the presence of a spherical shape of GNS with a diameter range of 40-80 nm. A stable and uniform PBA-modified GNS (GNS-PBA) was obtained with a facile ultrasonication step. Thus allowing aminated DNA probes of genetically modified (GM) soybean to be attached to the nanomaterials to form the DNA biosensor. The GNS-PBA/SPE exhibited excellent electrical conductivity via cyclic voltammetry (CV) and differential pulse voltammetry (DPV) tests using potassium ferricyanide (K3[Fe(CN)6]) as the electroactive probe. By employing an anthraquinone monosulfonic acid (AQMS) redox intercalator as the DNA hybridization indicator, the biosensor response was evaluated using the DPV electrochemical method. A good linear relationship between AQMS oxidation peak current and target DNA concentrations from 1.0 × 10-16 to 1.0 × 10-8 M with a limit of detection (LOD) of less than 1.0 × 10-16 M was obtained. Selectivity experiments revealed that the voltammetric GM DNA biosensor could discriminate complementary sequences of GM soybean from non-complementary sequences and hence good recoveries were obtained for real GM soybean sample analysis. The main advantage of using GNS is an improvement of the DNA biosensor analytical performance.

Calcium-cation-doped polydopamine-modified 2D black phosphorus nanosheets as a robust platform for sensitive and specific biomolecule sensing.

Many polymer decorated/modified 2D nanomaterials have been developed as enhanced drug delivery systems and photothermal theranostic nanoagents. However, few reports describe the use of these novel nanomaterials as nanoplatforms for biomolecule sensing. Herein, we used calcium-cation-doped polydopamine-modified (PDA-modified) 2D black phosphorus (BP) nanosheets (BP@PDA) as a sensing nanoplatform for the detection of nucleic acids and proteins in complex biological samples. Fluorescent-dye-labeled single-strand DNA aptamer/probes are adsorbed by the Ca2+-doped BP@PDA mediated by calcium-cation coordination. The PDA coating enhances the stability of the inner BP, provides binding sites to DNA nucleobases, and quenches fluorescence. Without any chemical conjugation, this sensing nanoplatform selectively and specifically detects protein (human thrombin, linear range: 10-25 nM, detection limit: 0.02 nM), single-strand DNA (linear range: 1-10 nM, detection limit: 0.52 nM) in 1% serum diluted samples, and senses intracellular mRNAs (C-myc, and actin) in living cells. The nanoplatform exhibits the advantages of both the 2D nanomaterial (BP) and the coating polymer (PDA), naturally enters living cells unaided by transfection agents, resists enzymatic lysis and shows high biocompatibility. This nanoplatform design contributes towards future biomolecule analytical method development based on polymer decorated/modified 2D nanomaterials.

DNA-templated copper nanoparticles as signalling probe for electrochemical determination of microRNA-222.

An ultrasensitive electrochemical biosensor is described for the determination of microRNAs. It is based on the use of DNA-templated copper nanoparticles (Cu NPs) as signalling probe. MicroRNA-222 was selected as the model analyte. The probe was obtained from two different oligonucleotides (containing complementary bases) via hybridization chain reaction to form long DNA concatemers as template. The Cu NPs were formed by reaction of ascorbate with copper sulfate. The biosensor was fabricated as follows: (a) Capture probe (cDNA) with a thiolated group was immobilized on reduced graphene oxide modified with gold nanoparticles (rGO/Au NPs), (b) materials was placed on a glassy carbon electrode (GCE); (c) the modified electrode (cDNA/rGO/Au NPs/GCE) was sequentially hybridized with microRNA-222 and signal probe; this results in the formation of a sandwich structure of cDNA-microRNA-signal probe on surface of the modified electrode. Differential pulse voltammetry was employed to record the electrochemical response of biosensor in pH 6.0 solution. As a result, a sensitive oxidation current with a peak potential at 0.10 V (vs. SCE) was obtained corresponding to Cu NPs. The experimental conditions were optimized. Under optimal conditions, the biosensor exhibited wide linear response range (0.5 fM to 70 nM) and low limit of detection (0.03 fM; at S/N = 3). The assay possesses high selectivity and can discriminate analyte microRNA from single-base mismatched microRNA. Graphical abstractA sensitive electrochemical biosensor is described for the determination of microRNA-222 by using a dsDNA-templated Cu NPs as signalling probe. (A) represents the preparation of signal probe, and (B) represents the fabrication of electrochemical microRNA sensor.

Exonuclease III-assisted signal amplification strategy for sensitive fluorescence detection of polynucleotide kinase based on poly(thymine)-templated copper nanoparticles.

A sensitive and label-free fluorometric method has been developed for the determination of polynucleotide kinase (PNK) activity, by employing exonuclease III (Exo III)-assisted cyclic signal amplification and poly(thymine)-templated copper nanoparticles (polyT-CuNPs). In the presence of PNK, cDNA with 5'-hydroxyl termini was phosphorylated and then hybridized with tDNA to form the cDNA/tDNA duplex, which subsequently triggered the λ exonuclease cleavage reaction, eventually resulting in the release of tDNA. The released tDNA could unfold the hairpin structure of HP DNA to generate partially complementary duplex (tDNA/HP DNA), wherein the HP DNA possessed T-rich sequences (T30) and tDNA recognition sequence. With the help of Exo III digestion, the tDNA was able to initiate the cycle for the generation of T-rich sequences, the template for the formation of fluorescent CuNPs. Conversely, the cDNA could not be cleaved by λ exonuclease without PNK and individual HP DNA could not be hydrolyzed by Exo III. The T-rich sequence was caged in HP DNA, resulting in a weak fluorescence signal. Under optimized conditions, the fluorescence intensity was linearly correlated to a concentration range of 0.001 to 1 U mL-1 with a low detection limit of 2 × 10-4 U mL-1. Considering the intriguing analytical performance, this approach could be explored to screen T4 PNK inhibitors and hold promising applications in drug discovery and disease therapy.

A Multicolor Fluorescence Nanoprobe Platform Using Two-Dimensional Metal Organic Framework Nanosheets and Double Stirring Bar Assisted Target Replacement for Multiple Bioanalytical Applications.

Multicolor fluorescence probes can show fluorescence of different colors when detecting different targets, and the excellent feature can create a highly differentiated multicolor sensing platform. However, most of the previously reported multicolor luminescent materials usually suffer from high toxicity and photobleaching, complex preparation procedures, and poor water solubility, which may not be conducive to bioanalytical applications. Two-dimensional metal organic frameworks (2D MOFs), which have large specific surface areas with long-range fluorescence quenching coupled with biomolecular recognition events, have encouraged innovation in biomolecular probing. Here, we propose a 2D-MOF-based multicolor fluorescent aptamer nanoprobe using a double stirring bar assisted target replacement system for enzyme-free signal amplification. It utilizes the interaction between 2D MOFs and DNA molecules to detect multiple antibiotics quickly, sensitively, and selectively. Since 2D MOFs have excellent quenching efficiency for luminescence of fluorescent-dye-labeled single-strand DNA (ssDNA), the background fluorescence can be largely reduced and the signal-to-noise ratio can be improved. When the adsorbed ssDNA formed double helix double-stranded DNA with its complementary ssDNA, its fluorescence can be almost fully recovered. The assay was tested by detecting chloramphenicol (CAP), oxytocin (OTC), and kanamycin (KANA) in biological samples. The developed aptasensor was sufficiently sensitive to detect the antibiotic residues as low as 1.5 pM CAP, 2.4 pM OTC, and 1 pM KANA (S/N = 3). It has been preliminarily used for multicolor imaging of three different antibiotics in fish tissue slices with satisfactory results.

A facile deoxyuridine/biotin-modified molecular beacon for simultaneous detection of proteins and nucleic acids via a label-free and background-eliminated fluorescence assay.

Simultaneous detection of different types of cancer biomarkers (nucleic acids and proteins) could facilitate early diagnosis of cancer and clinical treatment. Herein, a simultaneous detection platform of proteins and nucleic acids has been developed using a single substrate probe combining a label-free and background-eliminated fluorescence assay. Telomerase and telomerase RNA (TR) were chosen as the models. The molecular beacon (dU-BIO-HP) that contains deoxyuridine/biotin in its side arm, a TR recognition sequence in the loop and a telomerase substrate primer at the stem end was ingeniously designed. In the presence of telomerase, the stem of dU-BIO-HP is elongated by the addition of telomere repeats complementary to the assistant DNA. Furthermore, the formed dsDNA performed as engaging primers to initiate a SDA reaction, generating abundant G-quadruplex monomers. Similarly, on TR, the hybridization between TR and dU-BIO-HP can open its stem, triggering another SDA reaction, producing abundant short ssDNAs. With the G-quadruplex binding with ZnPPIX and ssDNA binding with SG for specific fluorescence responses, the label-free multiple detection can be achieved. In our strategy, the deoxyuridine of dU-BIO-HP acts as a barrier to block the DNA extension due to its strong inhibitory effects on DNA polymerase activity and to make sure that the two SDA reactions occurred independently. The biotin of dU-BIO-HP enables the reduction of the background from the binding between SG, ZnPPIX and dU-BIO-HP through streptavidin-biotin interaction. This method showed an excellent sensitivity with telomerase and TR detection limit of 2.18 HeLa cells per mL and 0.16 × 10-12 M, respectively. Furthermore, the telomerase and TR in different cell lines have been evaluated as powerful tools for biomedical research and clinical diagnosis.

Electrochemical nucleic acid hybridization biosensor based on poly(L-Aspartic acid)-modified electrode for the detection of short oligonucleotide sequences related to hepatitis C virus 1a.

This work describes, for the first time, the fabrication of poly(L-aspartic acid) (PAA) film modified pencil graphite electrode (PGE) for the detection of hepatitis C Virus 1a (HCV1a). The presence of PAA on the electrode surface can provide free carboxyl groups for covalent binding of biomolecules. The PGE surface was first coated with PAA via electropolymerization of the L-aspartic acid, and avidin was subsequently attached to the PAA modified electrode by covalent attachment. Biotinylated HCV1a probes were immobilized on avidin/PAA/PGE via avidin-biotin interaction. The morphology of PAA/PGE was examined using a scanning electron microscope. The hybridization events were monitored with square wave voltammetry using Meldola's blue (MDB). Compared to non-complementary oligonucleotide sequences, when hybridization was carried out between the probe and its synthetic targets or the synthetic polymerase chain reaction analog of HCV1a, the highest MDB signal was observed. The linear range of the biosensor was 12.5 to 100 nM and limit of detection was calculated as 8.7 nM. The biosensor exhibited favorable stability over relatively long-term storage. All these results suggest that PAA-modified electrode can be used to nucleic acid biosensor application and electropolymerization of L-aspartic acid can be considered as a good candidate for the immobilization of biomolecules.

A colorimetric ATP assay based on the use of a magnesium(II)-dependent DNAzyme.

A colorimetric assay for ATP is described that uses a strategy that combines the concept of split Mg(II)-dependent DNAzyme, split aptamer, and hybridization-induced aggregation of gold nanoparticles (AuNPs). Both ATP aptamer and Mg(II)-dependent DNAzyme are split into two fragments which are allocated to two well-designed DNA probes. The probes also possess mutually complementary stem sequences and spacer sequences. In the presence of ATP, the separated DNAzyme sequences in the two probes assemble via the synchronous recognition of ATP with two fragments of the aptamer. Then, the activated DNAzyme catalyzes multiple cycles of the cleavage of its substrate DNA sequence. The latter acts as a linker and induces the aggregation of two types of ssDNA-modified AuNP through the hybridization between the complementary sequences. Thus, the color of the AuNP solution remains red. However, in the absence of ATP, the detached aptamer cannot induce the assembly of DNAzyme to cleave the linker DNA. This results in the aggregation of AuNP and a concomitant color transition from red to purple. This ATP assay, performed at a wavelength of 530 nm, has a linear detection range that extends from 10 pM to 100 nM, with a detection limit of 5.3 pM. It was applied to the detection of ATP in human serum. Conceivably, the strategy has a wide scope in that it may be applied to the colorimetric detection of various other analytes through the split aptamer configuration. Graphical abstract Schematic presentation of colorimetric assay for adenosine triphosphate (ATP) based on the use of a split Mg(II)-dependent DNAzyme, a split aptamer, and by exploiting the hybridization-induced aggregation of gold nanoparticles that leads to a color change from red to purple.

Highly efficient Polyaniline-MoS2 hybrid nanostructures based biosensor for cancer biomarker detection.

In this work, polyaniline nanospindles have been synthesized using iron oxide as sacrificial template. These nanospindles were utilized for the fabrication of PANI-MoS2 nanoflower architectures via hydrothermal route. The electrostatic interaction between PANI and MoS2 improves the conductivity and provides more direct paths for charge transportation. SEM, TEM, XRD, Raman Spectroscopy techniques were employed to explore the crystal structure, and morphological properties of the PANI-MoS2 nanocomposite. Furthermore, an electrochemical biosensing platform based on PANI-MoS2 nanocomposite was fabricated for the specific detection of chronic myelogenous leukemia (CML) by using electrochemical impedance spectroscopy technique. The binding interactions between the pDNA/PANI-MoS2/ITO bioelectrode and target DNA sequence were also studied. The biosensor exhibits high sensitivity and wide detection range (10-6  M to 10-17  M) of target DNA with low detection limit (3 × 10-18  M). Additionally, the specificity studies of the genosensor with various target DNA sequences (complementary, noncomplementary and one base mismatch) and real samples analysis of CML shows its potential for clinical diagnostics.

Novel homo Yin-Yang probes improve sensitivity in RT-qPCR detection of low copy HIV RNA.

Nucleic acids labeled with a fluorophore/quencher pair are widely used as probes in biomedical research and molecular diagnostics. Here we synthesized novel DNA molecular beacons double labeled with the identical dyes (R6G, ROX and Cy5) at 5'- and 3'-end and studied their photo physical properties. We demonstrated that fluorescence quenching by formation of the homo dimer exciton in such molecular beacons allows using them in homogeneous assays. Further, we developed and evaluated homo Yin-Yang DNA probes labeled with identical dyes and used them for detection of low copy HIV RNA by RT-qPCR. They demonstrated improved sensitivity (LLQ: 10 vs 30 copies mL-1) in comparison to commercially available Abbott RealTime HIV-1 kit based on VIC-BHQ dyes both for model mixtures (naive human plasma with added deactivated HIV-1 virus) and for preliminarily confirmed 36 clinical samples (4 vs 1 positive ones for low-copy samples).

A novel fluorescence method for activity assay and drug screening of T4 PNK by coupling rGO with ligase reaction.

T4 polynucleotide kinase (PNK) is the primary member of the 5'-kinase family that can transfer the γ-phosphate residue of ATP to the 5'-hydroxyl group of oligonucleotides. In this article, using the differential quenching ability of reduced graphene oxide (rGO) towards the fluorophore-labeled DNA probe, we propose a novel method for detecting T4 PNK activity assisted by ligase reaction. Under the optimized conditions, the detection limit of T4 PNK was estimated to be 0.0002 U µL-1 in the linear region of 0.001 U µL-1-0.1 U µL-1. Additionally, the developed method was used to screen regulators of T4 PNK from natural compounds. The compound f isolated from the root of Kadsura coccinea (Lem.) A.C. Smith was found to stimulate T4 PNK activity in a concentration-dependent manner in vitro. Finally, the method was used to monitor the relation of T4 PNK activity with pelvic inflammatory disease (PID). The results demonstrated that the development of this disease could inhibit T4 PNK activity to some extent. In summary, the above data indicate that the method not only provides a universal platform for monitoring T4 PNK activity, but also shows great potential to be used in drug screening and clinic diagnosis.

Salmonella Typhimurium Sensing Strategy Based on the Loop-Mediated Isothermal Amplification Using Retroreflective Janus Particle as a Nonspectroscopic Signaling Probe.

Loop-mediated isothermal amplification (LAMP) is a powerful gene amplification method, which has many advantages, including high specificity, sensitivity, and simple operation. However, quantitative analysis of the amplified target gene with the LAMP assay is very difficult. To overcome this limitation, we developed a novel biosensing platform for molecular diagnosis by integrating the LAMP method and retroreflective Janus particle (RJP) together. The final amplified products of the LAMP assay are dumbbell-shaped DNA structures, containing a single-stranded loop with two different sequences. Therefore, the concentration of the amplified products can be measured in a manner similar to the sandwich-type immunoassay. To carry out the sandwich-type molecular diagnostics using the LAMP product, two DNA probes, with complementary sequences to the loop-regions, were prepared and immobilized on both the sensing surface and the surface of the RJPs. When the amplified LAMP product was applied to the sensing surface, the surface-immobilized DNA probe hybridized to the loop-region of the LAMP product to form a double-stranded structure. When the DNA probe-conjugated RJPs were injected, the RJPs bound to the unreacted loop-region of the LAMP product. The number of RJPs bound to the loop-region of the LAMP product was proportional to the concentration of the amplified LAMP product, indicating that the concentration of the target gene can be quantitatively analyzed by counting the number of observed RJPs. Using the developed system, a highly sensitive and selective quantification of Salmonella was successfully performed with a detection limit of 102 CFU.

DNA-Hybridization Detection on Si(100) Surfaces Using Light-Activated Electrochemistry: A Comparative Study between Bovine Serum Albumin and Hexaethylene Glycol as Antifouling Layers.

Light can be used to spatially resolve electrochemical measurements on a semiconductor electrode. This phenomenon has been explored to detect DNA hybridization with light-addressable potentiometric sensors and, more recently, with light-addressable amperometric sensors based on organic-monolayer-protected Si(100). Here, a contribution to the field is presented by comparing sensing performances when bovine serum albumin (BSA) and hexaethylene glycol (OEG6) are employed as antifouling layers that resist nonspecific adsorption to the DNA-modified interface on Si(100) devices. What is observed is that both sensors based on BSA or OEG6 initially allow electrochemical distinction among complementary, noncomplementary, and mismatched DNA targets. However, only surfaces based on OEG6 can sustain electroactivity over time. Our results suggest that this relates to accelerated SiO x formation occasioned by BSA proteins adsorbing on monolayer-protected Si(100) surfaces. Therefore, DNA biosensors were analytically explored on low-doped Si(100) electrodes modified on the molecular level with OEG6 as an antifouling layer. First, light-activated electrochemical responses were recorded over a range of complementary DNA target concentrations. A linear semilog relation was obtained from 1.0 × 10-11 to 1.0 × 10-6 mol L-1 with a correlation coefficient of 0.942. Then, measurements with three independent surfaces indicated a relative standard deviation of 4.5%. Finally, selectivity tests were successfully performed in complex samples consisting of a cocktail mixture of four different DNA sequences. Together, these results indicate that reliable and stable light-activated amperometric DNA sensors can be achieved on Si(100) by employing OEG6 as an antifouling layer.

Diagnosis of EGFR exon21 L858R point mutation as lung cancer biomarker by electrochemical DNA biosensor based on reduced graphene oxide /functionalized ordered mesoporous carbon/Ni-oxytetracycline metallopolymer nanoparticles modified pencil graphite electrode.

In this present work we made a novel, fast, selective and sensitive electrochemical genobiosensor to detection of EGFR exon 21 point mutation based on two step electropolymerization of Ni(II)-oxytetracycline conducting metallopolymer nanoparticles (Ni-OTC NPs) on the surface of pencil graphite electrode (PGE) which was modified by reduced graphene oxide/carboxyl functionalized ordered mesoporous carbon (rGO/f-OMC) nanocomposite. ssDNA capture probe with amine groups at the5' end which applied as recognition element was immobilized on the rGO/f-OMC/PGE surface via the strong amide bond. Ni-OTC metallopolymer NPs were electropolymerized to rGO/ssDNA-OMC/PGE surface and then hybridization fallows through the peak current change in differential pulse voltammetry (DPV) using Ni-OTC NPs as a redox label. The biosensor was characterized by field emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), FT-IR spectroscopy, energy dispersive X-ray spectroscopy (EDX), cyclic voltammetry and Nitrogen adsorption-desorption analysis. The Ni-OTC current response verified only the complementary sequence indicating a significant reduction current signal in comparison to single point mismatched and non-complementary and sequences. Under optimal conditions, the prepared biosensor showed long-term stability (21 days) with a wide linear range from 0.1 µM to 3 µM with high sensitivity (0.0188 mA/µM) and low detection limit (120 nM).

Rapid and Efficient FISH using Pre-Labeled Oligomer Probes.

Fluorescence in situ hybridization (FISH) is used to visualize the distribution of DNA elements within a genome. Conventional methods for FISH take 1-2 days. Here, we developed a simplified, rapid FISH technique using pre-labeled oligonucleotide probes (PLOPs) and tested the procedure using 18 PLOPs from 45S and 5S rDNA, Arabidopsis-type telomere, and newly-identified Panax ginseng-specific tandem repeats. The 16 developed rDNA PLOPs can be universally applied to plants and animals. The telomere PLOPs can be utilized in most plants with Arabidopsis-type telomeres. The ginseng-specific PLOP can be used to distinguish P. ginseng from related Panax species. Differential labeling of PLOPs allowed us to simultaneously visualize different target loci while reducing the FISH hybridization time from ~16 h to 5 min. PLOP-FISH is efficient, reliable, and rapid, making it ideal for routine analysis, especially of newly sequenced genomes using either universal or specific targets, such as novel tandem repeats identified from whole-genome sequencing data.

Development of an upconversion fluorescence DNA probe for the detection of acetamiprid by magnetic nanoparticles separation.

An upconversion fluorescence DNA probe which consists of aptamer-conjugated magnet nanoparticles (apt-MNPs) and complementary DNA-conjugated upconversion nanoparticles (cDNA-UCNPs) was developed to detect acetamiprid. Acetamiprid can specifically conjugate with the apt-MNPs to dissociate the cDNA-UCNPs from the apt-MNPs and resulted in reduced fluorescence intensity through an external magnet. The change of fluorescence intensity (△I) is positively related to the concentration of acetamiprid, which can be applied for the quantification of acetamiprid. Under optimal conditions, a linear detection range and detection limit are 0.89-114.18 µg/L and 0.65 µg/L, respectively. The probe was successfully used to detect acetamiprid in spiked paddy water, soil, pear, apple, wheat and cucumber. Average recoveries are 78.2%-103.5% with intra-day relative standard deviations (RSDs) of 2.6%-10.9% and inter-day RSDs of 4.3%-10.2%. The amounts of acetamiprid in the authentic paddy water and pear samples detected by the DNA probe are significantly correlated with that detected by high-performance liquid chromatography (HPLC).

Spiky gold shells on magnetic particles for DNA biosensors.

Combined separation and detection of biomolecules has the potential to speed up and improve the sensitivity of disease detection, environmental testing, and biomolecular analysis. In this work, we synthesized magnetic particles coated with spiky nanostructured gold shells and used them to magnetically separate out and detect oligonucleotides using SERS. The distance dependence of the SERS signal was then harnessed to detect DNA hybridization using a Raman label bound to a hairpin probe. The distance of the Raman label from the surface increased upon complementary DNA hybridization, leading to a decrease in signal intensity. This work demonstrates the use of the particles for combined separation and detection of oligonucleotides without the use of an extrinsic tag or secondary hybridization step.