Signal DNA Probe Research Articles

Amplified Assembly of G-Quadruplex-Decorated DNA Network Nanostructure toward AIE Signaling-Based Sensitive Biosensing.

Aggregation-induced emission (AIE) has offered a promising approach for developing low-background fluorescent methods; however, its applications often suffer from complex probe synthesis and poor biocompatibility. Herein, a novel AIE biosensing method for kanamycin antibiotic assays was developed by utilizing a DNA network nanostructure assembled from an aptamer recognition reaction to capture a large number of tetraphenylethylene fluorogen-labeled signal DNA (DTPE) probes. Due to the excellent hydrophilicity of the oligonucleotides, DTPE exhibited excellent water solubility without obvious background signal emission. Based on an ingenious nucleotide design, an abundance of G-quadruplex blocks neighboring the captured DTPE were formed on the DNA nanostructure. Because of the greatly restricted free motion of DTPE by this unique nanostructure, a strong AIE fluorescence signal response was produced to construct the signal transduction strategy. Together with target recycling and rolling circle amplification-based cascade nucleic acid amplification, this method exhibited a wide linear range from 75 fg mL-1 to 1 ng mL-1 and a detection limit down to 24 fg mL-1. The excellent analytical performance and effective manipulation improvement of the method over previous approaches determine its promising potential for various applications.

Target-triggered hybridization chain reaction for ultrasensitive dual-signal miRNA detection

A signal amplification sensing system with target-triggered DNA cascade reaction combined with dual-signal readout technology was designed for ultrasensitive analysis of miRNA. The highly conductive metal organic frameworks (MOFs) derivative, N-doped carbon dodecahedron (N-PCD) was deposited with gold nanoparticles as the electrode substrate, which could assist the electron transfer between the molecular probe and the electrode surface, and could remarkably enhance electrochemical response. Tetrahedral DNA nanostructure (T4-DNA) with high structural stability and mechanical stiffness was designed to improve the loading capacity and binding efficiency of the target, thus increasing the sensitivity of the system. The non-enzymatic amplification method based on the DNA cascade reaction allows the electrochemical responses from dual signal DNA probes labeled with ferrocene (Fc) and methylene blue (MB), respectively in turn to improve the reliability of detection. Under optimal conditions, the sensor has a linear range of 5–1.0 × 104 fM, and the limit of detection is as low as 1.92 fM and 3.74 fM for Fc and MB labeled probe, respectively. This strategy raises the promising application for the rapid detection of miRNA targets with low abundance in complex biological systems.

An ultrasensitive carcinoembryonic antigen electrochemical aptasensor based on 3D DNA nanoprobe and Exo III

In this study, a highly ordered three dimensional (3D) DNA nanostructure was self-assembled by label-free DNA nanotweezers, which was used as recognized probe to interact with target. Once the target was recognized by the 3D DNA nanoprobe (3D DNT), DNA nanotweezers opened to release target analog (T1). This recognition process was proceeded in homogeneous solution, which can avoid complex electrode modification and improve reaction efficiency. Then these target analogs were captured by the signal DNA probes (E1) modified on the electrode. In the assistance of Exo III, E1 was digested and the T1 was released to participate in the next cycle to realize signal amplification. Finally, an ultrasensitive carcinoembryonic antigen (CEA) electrochemical biosensing with a detection limit of 4.88 fg mL−1 was developed.

Electrochemical determination of hantavirus using gold nanoparticle-modified graphene as an electrode material and Cu-based metal-organic framework assisted signal generation

An electrochemical biosensor was prepared for nucleic acid-based hantavirus detection using a Cu-based metal-organic framework (CuMOF) as a signal tag. The CuMOF was synthesized by the solvothermal method and then covalently bonded with signal DNA (sDNA) probes. The Au nanoparticles and reduced graphene oxide composite were deposited on the electrode surface by electroreduction as support substrate and was then functionalized with capture DNA (cDNA) probes by self-assembly. Through the complementary base pairing, the target DNA (tDNA) fragment of hantavirus hybridized with the cDNA and the sDNA in a sandwich-type format. The tDNA was detected according to the current signal of the CuMOF catalyzed reaction using o-phenylenediamine as redox substrate. The peak current of the biosensor at - 0.55V increased linearly in proportion to the logarithmic value of the tDNA concentration from 10-15 to 10-9mol/L, with a detection limit of 0.74 × 10-15mol/L. Moreover, the proposed biosensor was successfully applied to detect hantavirus and was able to distinguish hantavirus from other arboviruses.

Target-induced molecular-switch on triple-helix DNA-functionalized carbon nanotubes for simultaneous visual detection of nucleic acids and proteins.

We report an easy and efficient approach based on a target-induced molecular-switch on triple-helix DNA (THD)-functionalized carbon nanotubes (CNTs) for the simultaneous visual detection of nucleic acids and proteins with a lateral flow nucleic acid biosensor. The assay had the capability to detect a minimum of 25 pM target DNA and 0.25 nM thrombin simultaneously within 20 min.

A novel sandwich aptasensor for detecting T-2 toxin based on rGO-TEPA-Au@Pt nanorods with a dual signal amplification strategy

T-2 toxin is a mycotoxin that can cause chronic illnesses, and the detection of T-2 toxin in food is critical for human health. Herein, a novel sandwich aptasensor with a dual signal amplification strategy was developed for the detection of T-2 toxin. Molybdenum disulfide-polyaniline-chitosan-gold nanoparticles (MoS2-PANI-Chi-Au) were processed to the modified glassy carbon electrode (GCE) and used as the aptasensor platform to expedite the electronics transport and immobilize the amino-terminated capture DNA probe by Au–N bonds. The reduced graphene oxide-tetraethylene pentamine-gold@platinum nanorods (rGO-TEPA-Au@Pt NRs) were first synthesized and immobilized with a signal DNA probe. Once T-2 toxin was added into the biosensing system, the aptamer would trap T-2 toxin to turn the signal off. Next, dissociative aptamer hybridized with the capture DNA probe in GCE and linked simultaneously to the signal DNA probe on rGO-TEPA-Au@Pt NRs with another end sequence of aptamer to turn the signal on. Owing to the efficient catalytic ability of bimetallic Au@Pt nanorods, the signal was perfectly amplified through the catalysis of hydrogen peroxide (H2O2) and recorded by chronoamperometry. With the outstanding augment response, the limit of detection reached 1.79 fg mL−1 (3SB/m) and a wide linear range from 10 fg mL−1 to 100 ng mL−1 was presented. The sensitivity of the aptasensor was 19.88 μA⋅μM−1⋅cm−2. Meanwhile, the DNA aptamer-bimetallic nanorod based sensing system presented excellent specificity. The developed aptasensor provides a new platform for T-2 toxin detection with low cost for real sample assays.

Ultrasensitive electrochemical genosensor for detection of CaMV35S gene with Fe3O4-Au@Ag nanoprobe

We report an attomolar sensitive electrochemical genosensor for the detection of cauliflower mosaic virus 35S (CaMV35S) gene. The sandwich-type genosensor uses gold-silver core-shell (Au@Ag)-loaded iron oxide (Fe3O4) nanocomposite (Fe3O4-Au@Ag) as label of signal DNA probe (sDNA). Electrochemical sensing is accomplished at interface of electrodeposited AuNPs and carboxylated multiwalled carbon nanotubes-modified glassy carbon electrode through the specific interaction between the capture probe and target CaMV35S (tDNA), and tDNA and the labeled sDNA. The detection sensitivity was improved by the amplified reduction signal of hydrogen peroxide (H2O2), which takes advantage of the enhanced electrocatalytic activity of Fe3O4-Au@Ag. Under the optimal experimental conditions, an ultralow limit of detection was calculated to be 1.26 × 10−17 M (S/N = 3), and the blank value subtracted reduction signal of H2O2 of the sensor increased linearly with the logarithm of CaMV35S concentration over a wide range (1 × 10−16 M to 1 × 10−10 M). This genosensor displayed excellent stability, selectivity and reproducibility, and was successful in detecting the target CaMV35S in genetically modified tomato samples.

Chameleon silver nanoclusters for ratiometric sensing of miRNA

Chameleon silver nanoclusters (AgNCs) have attracted substantial attention over traditional AgNCs, which provide new possibilities for the construction of ratiometric fluorescent biosensors. Herein, a novel method for sensitive and selective detection of miRNA is developed coupling chameleon AgNCs with hybridization chain reaction. Generally, the signal DNA probe is designed to contain two pieces of DNA templates for the synthesis of AgNCs, which are in close proximity due to the hairpin structure. A series of hybridization reactions can be triggered by target miRNA, which open the hairpin of signal probe and separate the two pieces of segmental AgNCs. Controllable color switching from red to yellow is thus achieved. Note that signal generation and amplification are integrated into one step, which is a great advance compared with previous amplified systems. The proposed strategy provides a sensitive fluorescent detection of miRNA down to 2.8 pM with high selectivity. Therefore, this work not only brings novel insights into fluorescence regulation of DNA-templated AgNCs, but also holds great potential for the application of miRNA diagnostics.

A palladium-platinum bimetal nanodendritic melamine network for signal amplification in voltammetric sensing of DNA.

A sandwich-type electrochemical DNA sensor is described for the detection of oligonucleotides typical for MECP2 gene mutations. Palladium nanoparticles (PdNPs) and platinum nanoparticles (PtNPs) were used to synthesize flower-like PdPt nanodendrites (NDs) by a one-pot method. The PdPt NDs possess a high specific surface area and excellent catalytic capabilities. They served as the carrier for the signal DNA probe (SP) and simultaneously catalyze the reduction of hydrogen peroxide (H2O2). The PdPt NDs were modified with melamine, and this results in the formation of a PdPt-melamine network through stable interactions between the PdPt NDs and the three amino groups of each melamine molecule. The network exhibits excellent catalytic ability in enhancing the current signal response in the voltammetric detection of MECP2 gene mutation, best measured at -0.4V vs. SCE and using H2O2 as the electrochemical probe. In addition, gold nanoflowers were electrodeposited on the electrode interface in order to accelerate electron transfer and to capture the capture probe. The sensor is stable and can detect MECP2 gene mutations in the 1 fmol·L-1 to 1nmol·L-1 concentration range, with a 0.33 fmol·L-1 lower detection limit at an S/N ratio of 3. Graphical abstract Schematic presentation of electrodes for the determination of the X-linked gene methyl-CpG-binding protein 2 (MECP2). The sensor is based on the electrooxidation of added H2O2 by using the melamine modified palladium platinum bimetal nanodendrites as network signal amplification strategy. This versatile platform expands studies on the detection of monogenic disease.

Ultrasensitive chemiluminescence biosensors using nucleic acid-functionalized silver-cysteine nanowires as signal amplifying labels.

Ultrasensitive chemiluminescence (CL) sensors for biomolecules (DNA and proteins) have been developed by adopting DNA-functionalized silver-cysteine hybrid nanowires (p-SCNWs) as signal amplifying labels. The sensing is established from a sandwich-type DNA hybridization, where the target DNA strands are initially hybridized with the capture DNA located at paramagnetic microspheres (PMs) and subsequently hybridized with p-SCNWs functionalized with the signal DNA probe. After magnetic separation, p-SCNWs on the hybrids were completely decomposed with HNO3 to release numerous silver ions. The powerful catalysis of silver ions toward the redox reaction of K2S2O8-Mn2+-H3PO4 causes the generation of KMnO4 that is capable of oxidizing luminol at high pH, triggering an amplified chemiluminescent signal emission. The sensing combines the extraordinary sensitivity of the catalytic chemiluminescence technology and the amplifying strategy via releasing large quantities of silver ions as the catalyst from each hybrid, enabling the assay of target DNA strands at a concentration as low as 0.34 fM. The CL signals associated with single-base pair mismatched DNA strands and non-complementary DNA strands are able to be discriminated well from the CL signal related to the complementary DNA hybridization. Likewise, the combination of p-SCNWs functionalized with an aptamer and PMs/aptamer/thrombin complex allowed the chemiluminescence sensing of thrombin with a low limit of detection corresponding to 0.17 pM.

Invertase-labeling gold-dendrimer for in situ amplified detection mercury(II) with glucometer readout and thymine-Hg(2+)-thymine coordination chemistry.

A simple, low-cost transducer with glucometer readout was designed for sensitive detection of mercury(II) (Hg(2+)), coupling with thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination chemistry and invertase-functionalized gold-dendrimer nanospheres for the signal amplification. Initially, nanogold-encapsulated poly(amidoamine) dendrimers (Au DENs) were synthesized by in-situ reduction of gold(III). Thereafter, the as-prepared Au DENs were utilized for the labeling of invertase and T-rich signal DNA probe. In the presence of target Hg(2+), the functionalized Au DENs were conjugated to capture DNA probe-modified electrode via T-Hg(2+)-T coordination chemistry. Accompanying the Au DENs, the labeled invertase could hydrolyze sucrose into glucose, which could be quantitatively monitored by an external personal glucometer (PGM). The PGM signal increased with the increasing target Hg(2+) in the sample. Under the optimal conditions, our designed sensing platform exhibited good PGM responses toward target Hg(2+), and allowed the detection of Hg(2+) at a concentration as low as 4.2 pM. This sensing system also displayed remarkable specificity relative to target Hg(2+) against other competing ions, and could be applied for reliable monitoring of spiked Hg(2+) into the environmental water samples with satisfactory results. With the advantages of cost-effectiveness, simplicity, portability, and convenience, our strategy provides a tremendous potential to be a promising candidate for point-of-use monitoring of non-glucose targets by the public.

Sensitive, simultaneous quantitation of two unlabeled DNA targets using a magnetic nanoparticle-enzyme sandwich assay.

We report herein the development of a simple, sensitive colorimetric magnetic nanoparticle (MNP)–enzyme-based DNA sandwich assay that is suitable for simultaneous, label-free quantitation of two DNA targets down to 50 fM level. It can also effectively discriminate single-nucleotide polymorphisms (SNPs) in genes associated with human cancers (KRAS codon 12/13 SNPs). This assay uses a pair of specific DNA probes, one being covalently conjugated to an MNP for target capture and the other being linked to an enzyme for signal amplification, to sandwich a DNA target, allowing for convenient magnetic separation and subsequent efficient enzymatic signal amplification for high sensitivity. Careful optimization of the MNP surfaces and assay conditions greatly reduced the background, allowing for sensitive, specific detection of as little as 5 amol (50 fM in 100 μL) of target DNA. Moreover, this sensor is robust, it can effectively discriminate cancer-specific SNPs against the wild-type noncancer target, and it works efficiently in 10% human serum. Furthermore, this sensor can simultaneously quantitate two different DNA targets by using two pairs of unique capture- and signal-DNA probes specific for each target. This general, simple, and sensitive DNA sensor appears to be well-suited for a wide range of genetics-based biosensing and diagnostic applications.

DNAzyme-guided polymerization of aniline for ultrasensitive electrochemical detection of nucleic acid with bio-bar codes-initiated rolling circle amplification

A novel and versatile amplified electrochemical strategy for ultrasensitive detection of nucleic acid has been developed on the basis of polyaniline (PANI) as the redox label which was guided and deposited by multiple HRP-mimicking DNAzyme as a powerful catalyst and template generated by bio-bar codes-initiated rolling circle amplification (RCA). The “sandwich-type” DNA biosensor was fabricated with the thiol-functionalized capture DNA first immobilized on an Au electrode and hybridized with one end of target DNA, the other end of which was recognized with a signal DNA probe that on the bio-bar codes, respectively. After the RCA reaction, multiple HRP-mimicking DNAzyme were successfully formed, which subsequently catalyzed the oxidation of aniline to PANI and lead to a readily measurable “turn-on” electrochemical signal. As a result of multiple amplification effects, the constructed platform exhibited a good linear response toward nucleic acid over a wide range of concentration from 6.0fM to 1.0nM.

Expression of MYC, IgM, As Well As Non-Germinal Centre B-Cell Like Immunophenotype and Positive Immunofish Index Predict a Worse Progression Free Survival and Overall Survival in a Series of 670 De Novo Diffuse Large B-Cell Lymphomas Included in Clinical Trials: A GELA Study of the 2003 Program

AbstractAbstract 1539 IntroductionDiffuse large B-cell lymphomas (DLBCL) represent a heterogeneous disease with variable clinical outcome. Identifying phenotypic biomarkers of tumor cells on paraffin sections that predict different clinical outcome remain an important goal that may also help to better understand the biology of this lymphoma. Differentiating non-germinal centre B-cell-like (non-GCB) from Germinal Centre B-cell-like (GCB) DLBCL according to Hans algorithm has been considered as an important immunohistochemical biomarker with prognostic value among patients treated with R-CHOP although not reproducibly found by all groups. Gene expression studies have also shown that IgM expression might be used as a surrogate for the GCB and ABC subtypes with a strong preferential expression of IgM in ABC DLBCL subtype. ImmunoFISH index based on the differential expression of MUM-1, FOXP1 by immunohistochemistry and on the BCL6 rearrangement by FISH has been previously reported (C Copie-Bergman, J Clin Oncol. 2009;27:5573-9) as prognostic in an homogeneous series of DLBCL treated with R-CHOP. In addition, oncogenic MYC protein overexpression by immunohistochemistry may represent an easy tool to identify the consequences of MYC deregulation in DLBCL.Our aim was to analyse by immunohistochemistry the prognostic relevance of MYC, IgM, GCB/nonGCB subtype and ImmunoFISH index in a large series of de novo DLBCL treated with Rituximab (R)-chemotherapy (anthracyclin based) included in the 2003 program of the Groupe d'Etude des Lymphomes de l'Adulte (GELA) trials. MethodsThe 2003 program included patients with de novo CD20+ DLBCL enrolled in 6 different LNH-03 GELA trials (LNH-03-1B, −B, −3B, 39B, −6B, 7B) stratifying patients according to age and age-adjusted IPI. Tumor samples were analyzed by immunohistochemistry using CD10, BCL6, MUM1, FOXP1 (according to Barrans threshold), MYC, IgM antibodies on tissue microarrays and by FISH using BCL6 split signal DNA probes. Considering evaluable Hans score, 670 patients were included in the study with 237 (35.4%) receiving intensive R-ACVBP regimen and 433 (64.6%) R-CHOP/R-mini-CHOP. Results304 (45.4%) DLBCL were classified as GCB and 366 (54.6%) as non-GCB according to Hans algorithm. 337/567 cases (59.4%) were positive for the ImmunoFISH index (i.e. two out of the three markers positive: MUM1 protein positive, FOXP1 protein Variable or Strong, BCL6 rearrangement). Immunofish index was preferentially positive in the non-GCB subtype (81.3%) compared to the GCB subtype (31.2%), (p<0.001). IgM was recorded as positive in tumor cells in 351/637 (52.4%) DLBCL cases with a preferential expression in non-GCB 195 (53.3%) vs GCB subtype 100(32.9%), p<0.001). MYC was positive in 170/577 (29.5%) cases with a 40% cut-off and in 44/577 (14.2%) cases with a cut-off of 70%. There was no preferential expression of MYC among GCB or non-GCB subtype (p>0.4) for both cut-offs.Progression-free Survival (PFS) was significantly worse among patients with high IPI score (p<0.0001), IgM positive tumor (p<0.0001), MYC positive tumor with a 40% threshold (p<0.001), ImmunoFISH positive index (p<0.002), non-GCB DLBCL subtype (p<0.0001).Overall Survival (OS) was also significantly worse among patients with high IPI score (p<0.0001), IgM positive tumor (p=0.02), MYC positive tumor with a 40% threshold (p<0.01), ImmunoFISH positive index (p=0.02), non-GCB DLBCL subtype (p<0.0001).All significant parameters were included in a multivariate analysis using Cox Model and in addition to IPI, only the GCB/non-GCB subtype according to Hans algorithm predicted significantly a worse PFS among non-GCB subgroup (HR 1.9 [1.3—2.8] p=0.002) as well as a worse OS (HR 2.0 [1.3–3.2], p=0.003). This strong prognostic value of non-GCB subtyping was confirmed considering only patients treated with R- CHOP for PFS (HR 2.1 [1.4–3.3], p=0.001) and for OS (HR 2.3 [1.3–3.8], p=0.002). ConclusionOur study on a large series of patients included in trials confirmed the relevance of immunohistochemistry as a useful tool to identify significant prognostic biomarkers for clinical use. We show here that IgM and MYC might be useful prognostic biomarkers. In addition, we confirmed in this series the prognostic value of the ImmunoFISH index. Above all, we fully validated the strong and independent prognostic value of the Hans algorithm, daily used by the pathologists to subtype DLBCL. Disclosures:No relevant conflicts of interest to declare.

Chemiluminescent Detection of DNA Hybridization Based on Signal DNA Probe Modified with Gold and Cobalt Nanoparticles

Chemiluminescent Detection of DNA Hybridization Based on Signal DNA Probe Modified with Gold and Cobalt Nanoparticles

The electrogenerated chemiluminescence detection of IS6110 of Mycobacterium tuberculosis based on a luminol functionalized gold nanoprobe

A novel electrogenerated chemiluminescence (ECL) DNA sensor was developed for a fast test for Mycobacterium tuberculosis (MTB) without using the polymerase chain reaction amplification, and was based on luminol functionalized gold nanoprobes with excellent signal amplification functionality. This nanoprobe was formed by the conjugation of luminol functionalized gold nanoparticles (lum–AuNPs) with signal DNA probes. An 81bp segment derived from the Mycobacterium tuberculosis specific insertion sequence IS6110 was chosen as the target strand and corresponding probes were designed to specifically hybridize with it. Biotinylated capture probes can be effectively immobilized directly on a streptavidin coated AuNP modified indium tin oxide electrode. After catching the TB target strand, signal probes tagged with lum–AuNPs were attached to the assembled electrode surface to form a sandwich-type TB sensor. Treating the resulting electrode surface with a carbonate buffer solution containing 1.0 mmol L−1 of H2O2 and applying a double-step potential to the electrode, the ECL response was generated which realized the detection of the TB target strand. The detection limit was estimated to be 6.7 × 10−15 mol L−1 (equal to approximately 1.7 × 10−10 g L−1) of the synthetic TB target strand, which is superior to other genetic methodologies for TB tests based on gold nanoprobes. Genomic DNA from other pathogenic bacterias (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) had a negligible effect on its detection, which guaranteed the good selectivity of the TB sensor. The efficacy of the TB sensor was also evaluated for genomic DNA extracted from cultured M. tuberculosis. The TB sensor is sensitive, highly selective, convenient and cost-effective. The simplicity of the assay and the lack of a requirement for sophisticated equipment render the TB sensor a promising candidate as a rapid molecular test for the detection ofM. tuberculosis.

Sensitive fluorescent detection of Staphylococcus aureus using nanogold linked CdTe nanocrystals as signal amplification labels

A sensitive fluorescent assay was developed for the detection of DNA specifically for Staphylococcus aureus. A sandwich-type detection system was fabricated by first immobilizing biotinylated capture DNA on avidin-modified wells of microplates, then hybridizing the capture DNA with one end of the target DNA, and then recognizing the other end of the target DNA with a signal probe labeled with CdTe nanocrystals and gold nanoparticles (Au-NPs) at the 3′- and 5′-terminus, respectively. Hybridization was monitored by measuring the fluorescent intensity of the assembly. The experimental results demonstrated that the incorporation of Au-NPs in this detection system can significantly enhance the sensitivity and the selectivity because a single Au-NP can be loaded with hundreds of signal DNA probe strands modified with CdTe nanocrystals. Under the optimized conditions, a detection limit of 10 fmol of DNA per L can be achieved and at least 50 colony forming units of Staph. aureus per mL of sample can be detected. The method was assessed by analyzing real samples, and it was validated by comparing it to an official standard method.

Electrochemical detection of DNA hybridization based on signal DNA probe modified with Au and apoferritin nanoparticles

A novel and ultrasensitive electrochemical approach for sequence-specific DNA detection based on signal dual-amplification with Au NPs and marker-loaded apoferritin NPs was reported. Target DNA was sandwiched between capture DNA coupled to magnetic beads and signal DNA self-assembled on Au NPs which were incorporated with marker-loaded apoferritin NPs. Subsequent electrochemical stripping analysis of the electroactive markers released from apoferritin NPs in acidic buffers provided a means to quantify the concentration of target DNA. In this means, one target signal could be transformed into multiple redox signals of the markers since a single Au NP could be loaded with dozens of apoferritin NPs, and an apoferritin NP could be loaded with thousands of markers. Under the optimum conditions, the linear range was from 2.0 × 10 −16 to 1.0 × 10 −14 M and the detection limit was 5.1 × 10 −17 M by using the cadmium as a model marker. The proposed DNA biosensor not only exhibited excellent sensitivity but also had good reproducibility and selectivity against two-base mismatched DNA.

Construction of DNA sandwich electrochemical biosensor with nanoPbS and nanoAu tags on magnetic microbeads

A novel and sensitive sandwich electrochemical biosensor based on the amplification of magnetic microbeads and Au nanoparticles (NPs) modified with bio bar code and PbS nanoparticles was constructed in the present work. In this method, the magnetic microspheres were coated with 4 layers polyelectrolytes in order to increase carboxyl groups on the surface of the magnetic microbeads, which enhanced the amount of the capture DNA. The amino-functionalized capture DNA on the surface of magnetic microbeads hybridized with one end of target DNA, the other end of which was hybridized with signal DNA probe labelled with Au NPs on the terminus. The Au NPs were modified with bio bar code and the PbS NPs were used as a marker for identifying the target oligoncleotide. The modification of magnetic microbeads could immobilize more amino-group terminal capture DNA, and the bio bar code could increase the amount of Au NPs that combined with the target DNA. The detection of lead ions performed by anodic stripping voltammetry (ASV) technology further improved the sensitivity of the biosensor. As a result, the present DNA biosensor showed good selectivity and sensitivity by the combined amplification. Under the optimum conditions, the linear relationship with the concentration of the target DNA was ranging from 2.0 × 10 −14 M to 1.0 × 10 −12 M and a detection limit as low as 5.0 × 10 −15 M was obtained.

Ultrasensitive Flow Injection Chemiluminescence Detection of DNA Hybridization Using Signal DNA Probe Modified with Au and CuS Nanoparticles

A novel and sensitive flow injection chemiluminescence assay for sequence-specific DNA detection based on signal amplification with nanoparticles (NPs) is reported in the present work. The "sandwich-type" DNA biosensor was fabricated with the thiol-functionalized capture DNA first immobilized on an Au electrode and hybridized with one end of target DNA, the other end of which was recognized with a signal DNA probe labeled with CuS NPs and Au NPs on the 3'- and 5'-terminus, respectively. The hybridization events were monitored by the CL intensity of luminol-H2O2-Cu(2+) after the cupric ions were dissolved from the hybrids. We demonstrated that the incorporation of Au NPs in this sensor design significantly enhanced the sensitivity and the selectivity because a single Au NP can be loaded with hundreds of signal DNA probe strands, which were modified with CuS NPs. The ratios of Au NPs, signal DNA probes, and CuS NPs modified on the gold electrode were approximately 1/101/103. A preconcentration process of cupric ions performed by anodic stripping voltammetry technology further increased the sensor performance. As a result of these two combined effects, this DNA sensor could detect as low as femtomolar target DNA and exhibited excellent selectivity against two-base mismatched DNA. Under the optimum conditions, the CL intensity was increased with the increase of the concentration of target DNA in the range of 2.0 x 10(-14)-2.0 x 10(-12) M. A detection limit of 4.8 x 10(-15) M target DNA was achieved.