Multiplexed sensing of biomolecules with optically detected magnetic resonance of nitrogen-vacancy centers in diamond.

In the past decade, a great effort has been devoted to develop new biosensor platforms for the detection of a wide range of analytes. Among the various approaches, magneto-DNA assay platforms have received extended interest for high sensitive and specific detection of targets with a simultaneous manipulation capacity. Here, using nitrogen-vacancy quantum centers in diamond as transducers for magnetic nanotags (MNTs), a hydrogel-based, multiplexed magneto-DNA assay is presented. Near-background-free sensing with diamond-based imaging combined with noninvasive control of chemically robust nanotags renders it a promising platform for applications in medical diagnostics, life science, and pharmaceutical drug research. To demonstrate its potential for practical applications, we employed the sensor platform in the sandwich DNA hybridization process and achieved a limit of detection in the attomolar range with single-base mismatch differentiation.

New Assay Reveals Vast Excess of Defective over Intact HIV-1 Transcripts in Antiretroviral Therapy-Suppressed Individuals.

Most of the HIV DNA in infected individuals is noninfectious because of deleterious mutations. However, it is unclear how much of the transcribed HIV RNA is potentially infectious or defective. To address this question, we developed and validated a novel intact viral RNA assay (IVRA) that uses droplet digital reverse transcriptase PCR (dd-RT-PCR) for the commonly mutated packaging signal (Psi) and Rev response element (RRE) regions (from the intact proviral DNA assay [IPDA]) to quantify likely intact (Psi+ RRE+), 3' defective (Psi+ RRE-), and 5' defective (Psi- RRE+) HIV RNA. We then applied the IPDA and IVRA to quantify intact and defective HIV DNA and RNA from peripheral CD4+ T cells from 9 antiretroviral therapy (ART)-suppressed individuals. Levels of 3' defective HIV DNA were not significantly different from those of 5' defective HIV DNA, and both were higher than intact HIV DNA. In contrast, 3' defective HIV RNA (median 86 copies/106 cells; 94% of HIV RNA) was much more abundant than 5' defective (2.1 copies/106 cells; 5.6%) or intact (0.6 copies/106 cells; <1%) HIV RNA. Likewise, the frequency of CD4+ T cells with 3' defective HIV RNA was greater than the frequency with 5' defective or intact HIV RNA. Intact HIV RNA was transcribed by a median of 0.018% of all proviruses and 2.2% of intact proviruses. The vast excess of 3' defective RNA over 5' defective or intact HIV RNA, which was not observed for HIV DNA, suggests that HIV transcription is completely blocked prior to the RRE in most cells with intact proviruses and/or that cells transcribing intact HIV RNA are cleared at very high rates. IMPORTANCE We developed a new assay that can distinguish and quantify intact (potentially infectious) as well as defective HIV RNA. In ART-treated individuals, we found that the vast majority of all HIV RNA is defective at the 3' end, possibly due to incomplete transcriptional processivity. Only a very small percentage of all HIV RNA is intact, and very few total or intact proviruses transcribe intact HIV RNA. Though rare, this intact HIV RNA is tremendously important because it is necessary to serve as the genome of infectious virions that allow transmission and spread, including rebound after stopping ART. Moreover, intact viral RNA may contribute disproportionately to the immune activation, inflammation, and organ damage observed with untreated and treated HIV infection. The intact viral RNA assay can be applied to many future studies aimed at better understanding HIV pathogenesis and barriers to HIV cure.

Unexpected finding of a rare pathogenic germline BRCA1 variant in an intrahepatic cholangiocarcinoma using the Oncomine Focus DNA assay: clinical and diagnostic implications.

BACKGROUND: Cholangiocarcinoma (CCA) is a malignant tumor arising from the epithelial cells of the bile ducts and is the second most common liver cancer after hepatocellular carcinoma. Recently, our Institution launched a Comprehensive Genomic Profiling (CGP) program (named FPG500 program), set up to provide a complete molecular characterization through the TruSight Oncology 500 High Throughput (TSO500HT) solution and samples that do not reach pre-set sample quantity and/or quality thresholds required for TSO500HT, are addressed to Oncomine Focus DNA Assay (OFA) and the Archer's FusionPlex Lung Panel (AFL). METHODS AND RESULTS: Here we report the case of a patient with iCCA enrolled in the FPG500 program and screened by the orthogonal workflow (OFA/AFL). Although BRCA1 is not among the genes declared in the OFA panel, we unexpectedly detected a pathogenic variant in this gene (c.5278-2del, rs878853285). CONCLUSIONS: This case highlights the diagnostic capabilities of CGP, now widely used in both clinical practice and academic setting. The incidental involvement of BRCA1 focuses attention on the role of BRCA genes in biliary tract cancers. Finally, as an orthogonal test confirmed the germline origin of BRCA1 c.5278-2del variant, the germline implications of CGP need to be considered.

Neutral Red-carbon nanodots for selective fluorescent DNA sensing.

Carbon nanodots modified with Neutral Red covalently inserted in the nanostructure (NR-CDs) have been prepared by a simple synthesis method based on microwave irradiation under controlled temperature and pressure. The synthetized NR-CDs have been characterized by different techniques, demonstrating the covalent bonding of Neutral Red molecules to the carbon dots nanostructure. Fluorescence activity of the prepare NR-CDs has been explored showing different interaction pathways with singled and doubled stranded DNA. These studies have been successfully applied to develop a new fluorescence DNA hybridization assay to the detection of a specific DNA sequence of Escherichia coli bacteria.

Restriction Endonuclease-Based Assays for DNA Detection and Isothermal Exponential Signal Amplification.

Application of restriction endonuclease (REase) enzymes for specific detection of nucleic acids provides for high assay specificity, convenience and low cost. A direct restriction assay format is based on the specific enzymatic cleavage of a target-probe hybrid that is accompanied with the release of a molecular marker into the solution, enabling target quantification. This format has the detection limit in nanomolar range. The assay sensitivity is improved drastically to the attomolar level by implementation of exponential signal amplification that is based on a cascade of self-perpetuating restriction endonuclease reactions. The cascade is started by action of an amplification "trigger". The trigger is immobilized through a target-specific probe. Upon the target probe hybridization followed with specific cleavage, the trigger is released into the reaction solution. The solution is then added to the assay amplification stage, and the free trigger induces cleavage of amplification probes, thus starting the self-perpetuating cascade of REase-catalyzed events. Continuous cleavage of new amplification probes leads to the exponential release of new triggers and rapid exponential signal amplification. The proposed formats exemplify a valid isothermal alternative to qPCR with similar sensitivity achieved at a fraction of the associated costs, time and labor. Advantages and challenges of the approach are discussed.

A dual signal amplification strategy combining thermally initiated SI-RAFT polymerization and DNA-templated silver nanoparticles for electrochemical determination of DNA.

A highly sensitive method is described for determination of DNA. It is based on dual signal amplification, viz. (a)DNA-templated metal deposition, and (b) thermally initiated surface-initiated reversible addition-fragmentation chain transfer (SI-RAFT) polymerization. A peptide nucleic acid (PNA) with a terminal thiol group was grasped onto a gold electrode by self-assembly. The modified electrode serves as a probe to selectively capture target DNA (tDNA). In the next step, Zr(IV) ions are bound to the phosphate groups of the tDNA. A chain-transfer agent (CTA) for thermally initiated SI-RAFT polymerization, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPAD), was immobilized on tDNA by conjugation of the carboxy group to Zr(IV) ions. Subsequently, numerous monomers of glycosyloxyethyl methacrylate (GEMA) were connected to the CPAD by thermally initiated SI-RAFT polymerization with azobisisobutyronitrile (AIBN) serving as the free-radical thermal initiator. Afterwards, hydroxyl groups of the GEMA were oxidized to aldehyde groups reacting with sodium periodate, and silver nanoparticles were further introduced on the surface of electrode via "silver mirror reaction". This results in a large electrochemical signal amplification. Under optimized conditions, the electrochemical signal (best measured at a working potential of 0 V vs. SCE (KCl; 3 M)) increases linearly with the logarithm of tDNA concentration in the 10 to 106 aM concentration range. The detection limit is as low as 5.6 aM (~34 molecules in a 10 µL sample). This is lower by factors between 2 and 1800 times than detection limits of most other ultra-sensitive electrochemical DNA assays. Graphical abstractSchematic representation of a dual signal amplification strategy combining thermally initiated surface-initiated reversible addition-fragmentation chain transfer polymerization (SI-RAFT) and DNA-templated silver nanoparticles for electrochemical determination of DNA.

Colorimetric DNA assay by exploiting the DNA-controlled peroxidase mimicking activity of mesoporous silica loaded with platinum nanoparticles.

A nanozyme composed of mesoporous silica and platinum nanoparticles (MS-PtNPs) was synthesized and is shown to display peroxidase-like activity. Its activity can be controlled by loading with single-stranded DNA. The PtNPs on the MS are homogeneously distributed and act as enzyme mimics. The adsorption of DNA probe on the MS blocks the nucleation sites of PtNPs. This leads to a decrease in the peroxidase-mimicking activity. After introduction of target DNA that is complementary to the DNA probe, the activity of the nanozyme is recovered. By using the 3,3,5,5-tetramethylbenzidine/H2O2 chromogenic system, a rapid method was developed for colorimetric determination of DNA. The assay, best performed at 450 nm, has a linear response in the 5 nM to 100 nM DNA concentration range and a 2.6 nM detection limit. It possesses high selectivity and can distinguish even a single-base mismatch. Graphical abstract The peroxidase-like activity of mesoporous silica and platinum nanoparticles (MS-PtNPs) was depressed when noncovalent ssDNA-MS was in-situ deposited on the PtNPs. After introduction of target DNA, the complementary dsDNA releases from the MS, and then its activity is recovered.

Digital triplex DNA assay based on plasmonic nanocrystals.

A new analytical method has been developed to detect three kinds of DNA simultaneously based on magnetic beads and color-encoded plasmonic nanocrystals. Magnetic beads modified with capture DNA are employed to collect the specific target DNA, and color-encoded plasmonic nanocrystals are applied to signal the target through DNA hybridization. As a proof of concept, three types of representative metal nanocrystals of gold nanoparticle (AuNP), gold nanorod (AuNR), and gold/silver nanoparticle (Au/AgNP) were employed to signal three dissimilar virus-related protective antigen genes, Ebola virus (EV), Variola virus (VV), and Bacillus anthracis (BA), respectively. Detection limits of 0.5-3 fM were obtained showing the high sensitivity for DNA detection. The microscopic discrimination of the encoded nanoparticles allows simple, rapid, accurate, and cost-effective detection of multiple DNA molecules, indicative of the potential in practical applications. Graphical abstract Development of a novel digital triplex DNA assay based on single-countable color-encoded plasmonic nanocrystals.

A Comparison of Fresh Frozen vs. Formalin-Fixed, Paraffin-Embedded Specimens of Canine Mammary Tumors via Branched-DNA Assay.

Mammary neoplasms are the tumors most affecting female dogs and women. Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable source of archived biological material. Fresh frozen (FF) tissue is considered ideal for gene expression analysis. However, strategies based on FFPE material offer several advantages. Branched-DNA assays permit a reliable and fast workflow when analyzing gene expression. The aim of this study was to assess the comparability of the branched-DNA assay when analyzing certain gene expression patterns between FF and FFPE samples in canine mammary tumors. RNA was isolated from 109 FFPE samples and from 93 FF samples of different canine mammary tissues. Sixteen (16) target genes (Tp53; Myc; HMGA1; Pik3ca; Mcl1; MAPK3; FOXO3; PTEN; GATA4; PFDN5; HMGB1; MAPK1; BRCA2; BRCA1; HMGA2; and Her2) were analyzed via branched-DNA assay (b-DNA). ACTB, GAPDH, and HPRT1 were used as data normalizers. Overall, the relative gene expression of the two different origins of samples showed an agreement of 63%. Still, care should be taken, as FFPE specimens showed lower expression of the analyzed targets when compared to FF samples. The fact that the gene expression in FFPE proved to be lower than in FF specimens is likely to have been caused by the effect of storage time. ACTB had the best performance as a data normalizer.

Multiplex Gene Expression Profiling of 16 Target Genes in Neoplastic and Non-Neoplastic Canine Mammary Tissues Using Branched-DNA Assay.

Mammary gland tumors are one of the most common neoplasms in female dogs, and certain breeds are prone to develop the disease. The use of biomarkers in canines is still restricted to research purposes. Therefore, the necessity to analyze gene profiles in different mammary entities in large sample sets is evident in order to evaluate the strength of potential markers serving as future prognostic factors. The aim of the present study was to analyze the gene expression of 16 target genes (BRCA1, BRCA2, FOXO3, GATA4, HER2, HMGA1, HMGA2, HMGB1, MAPK1, MAPK3, MCL1, MYC, PFDN5, PIK3CA, PTEN, and TP53) known to be involved in human and canine mammary neoplasm development. Expression was analyzed in 111 fresh frozen (FF) and in 170 formalin-fixed, paraffin-embedded (FFPE) specimens of neoplastic and non-neoplastic canine mammary tissues using a multiplexed branched-DNA (b-DNA) assay. TP53, FOXO3, PTEN, and PFDN5 expression revealed consistent results with significant low expression in malignant tumors. The possibility of utilizing them as predictive factors as well as for assisting in the choice of an adequate gene therapy may help in the development of new and improved approaches in canine mammary tumors.

Microarray on digital versatile disc for identification and genotyping of Salmonella and Campylobacter in meat products.

Highly portable, cost-effective, and rapid-response devices are required for the subtyping of the most frequent food-borne bacteria; thereby the sample rejection strategies and hygienization techniques along the food chain can be tailor-designed. Here, a novel biosensor is presented for the generic detection of Salmonella and Campylobacter and the discrimination between their most prevalent serovars (Salmonella Enteritidis, Salmonella Typhimurium) and species (Campylobacter jejuni, Campylobacter coli), respectively. The method is based on DNA microarray developed on a standard digital versatile disc (DVD) as support for a hybridization assay and a DVD driver as scanner. This approach was found to be highly sensitive (detection limit down to 0.2 pg of genomic DNA), reproducible (relative standard deviation 4-19 %), and high working capacity (20 samples per disc). The inclusivity and exclusivity assays indicated that designed oligonucleotides (primers and probes) were able to discriminate targeted pathogens from other Salmonella serovars, Campylobacter species, or common food-borne pathogens potentially present in the indigenous microflora. One hundred isolates from meat samples, collected in a poultry factory, were analyzed by the DVD microarraying and fluorescent real-time PCR. An excellent correlation was observed for both generic and specific detection (relative sensitivity 93-99 % and relative specificity 93-100 %). Therefore, the developed assay has been shown to be a reliable tool to be used in routine food safety analysis, especially in settings with limited infrastructure due to the excellent efficiency-cost ratio of compact disc technology. Graphical Abstract DNA microarray performed by DVD technology for pathogen genotyping.

HIV-1 proviral DNA in purified peripheral blood CD34+ stem and progenitor cells in individuals with long-term HAART; paving the way to HIV gene therapy.

Human immunodeficiency virus (HIV)-1 infection is an important public health problem worldwide. After primary HIV-1 infection, transcribed HIV-1 DNA is integrated into the host genome, serving as a reservoir of the virus and hindering a definite cure. Although highly active antiretroviral therapy suppresses active viral replication, resulting in undetectable levels of HIV RNA in the blood, a viral rebound can be detected after a few weeks of treatment interruption. This supports the concept that there is a stable HIV-1 reservoir in people living with HIV-1. Recently, a few individuals with HIV infection were reported to be probably cured by hematopoietic stem transplantation (HSCT). The underlying mechanism for this success involved transfusion of uninfected hematopoietic stem and progenitor cells (HSPCs) from CCR5-mutated donors who were naturally resistant to HIV infection. Thus, gene editing technology to provide HIV-resistant HSPC has promise in the treatment of HIV infections by HSCT. In this study, we aimed to find HIV-infected individuals likely to achieve a definite cure via gene editing HSCT. We screened for total HIV proviral DNA by Alu PCR in peripheral blood mononuclear cells (PBMCs) of 20 HIV-infected individuals with prolonged viral suppression. We assessed the amount of intact proviral DNA via a modified intact proviral DNA assay (IPDA) in purified peripheral CD34+ HSPCs. PBMCs from all 20 individuals were positive for the gag gene in Alu PCR, and peripheral CD34+ HSPCs were IPDA-negative for six individuals. Our results suggested that these six HIV-infected individuals could be candidates for further studies into the ability of gene editing HSCT to lead to a definite HIV cure.

Target-triggered cascade signal amplification in nanochannels: An ingenious strategy for ultrasensitive photoelectrochemical DNA bioanalysis.

Artificial solid-state nanochannels have aroused intense interests in biosensors and bioelectronics because of their special architectures. Herein, we pioneered an ingenious approach of target-triggered cascade signal amplification in porous anodic aluminum oxide (AAO) nanochannels for ultrasensitive photoelectrochemical (PEC) DNA bioanalysis. In the design, AAO nanochannels were modified initially with capture DNA (cDNA) and then incorporated with a photoelectrode, yielding the desired architecture of highly ordered nanoarrays on top of the signal transducer. For target DNA (tDNA) probing, exonuclease III (Exo-III) mediated target recycling (ETR) was first activated to generate plenty of output DNA (oDNA) fragments. After oDNA and the conjugate of Au-labeled probe DNA (Au-pDNA) were anchored within the nanochannels via DNA hybridization, in-situ synthesis of Ag shells on tethered Au nanoparticles was conducted. The resulting large-sized Au@Ag core-shell nanostructure within the nanochannels would cause conspicuous blocking effect to hinder the transportation of electrons accessing the photoelectrode. Since the signal inhibition was directly related to tDNA concentration, an innovative nanochannels PEC DNA assay was exploited and qualified for ultrasensitive detection. The anti-interference ability of this platform was also emphasized by the split AAO membrane for biological incubation without participation of the photoelectrode. This featured nanochannels PEC strategy with cascade amplification launched a novel detecting platform for trace levels of DNA, and it could spark more inspiration for a follow-up exploration of other smart nanochannels PEC bioassays.

Intact Proviral DNA Analysis of the Brain Viral Reservoir and Relationship to Neuroinflammation in People with HIV on Suppressive Antiretroviral Therapy.

HIV establishes a persistent viral reservoir in the brain despite viral suppression in blood to undetectable levels on antiretroviral therapy (ART). The brain viral reservoir in virally suppressed HIV+ individuals is not well-characterized. In this study, intact, defective, and total HIV proviral genomes were measured in frontal lobe white matter from 28 virally suppressed individuals on ART using the intact proviral DNA assay (IPDA). HIV gag DNA/RNA levels were measured using single-copy assays and expression of 78 genes related to inflammation and white matter integrity was measured using the NanoString platform. Intact proviral DNA was detected in brain tissues of 18 of 28 (64%) individuals on suppressive ART. The median proviral genome copy numbers in brain tissue as measured by the IPDA were: intact, 10 (IQR 1-92); 3' defective, 509 (225-858); 5' defective, 519 (273-906); and total proviruses, 1063 (501-2074) copies/106 cells. Intact proviral genomes accounted for less than 10% (median 8.3%) of total proviral genomes in the brain, while 3' and 5' defective genomes accounted for 44% and 49%, respectively. There was no significant difference in median copy number of intact, defective, or total proviruses between groups stratified by neurocognitive impairment (NCI) vs. no NCI. In contrast, there was an increasing trend in intact proviruses in brains with vs. without neuroinflammatory pathology (56 vs. 5 copies/106 cells, p = 0.1), but no significant differences in defective or total proviruses. Genes related to inflammation, stress responses, and white matter integrity were differentially expressed in brain tissues with >5 vs. +5 intact proviruses/106 cells. These findings suggest that intact HIV proviral genomes persist in the brain at levels comparable to those reported in blood and lymphoid tissues and increase CNS inflammation/immune activation despite suppressive ART, indicating the importance of targeting the CNS reservoir to achieve HIV cure.

Short DNA-catalyzed formation of quantum dot-DNA hydrogel for enzyme-free femtomolar specific DNA assay.

Fast, sensitive, specific, and user-friendly DNA assay is a key technique for the next generation point-of-care molecular diagnosis. However, high-cost, time-consuming, and complicated enzyme-based DNA amplification step is essential to achieve high sensitivity. Herein, a short target DNA-catalyzed formation of quantum dot (QD)-DNA hydrogel is proposed as a new DNA assay platform satisfying the above requirements. A single-stranded target DNA catalyzes the opening cycle of DNA hairpin loops, which are quickly self-assembled with DNA-functionalized QDs to generate QD-DNA hydrogel. The three-dimensional hydrogel network allows efficient resonance energy transfer, dramatically lowering the limit of detection down to ~6 fM without enzymatic DNA amplification. The QD-DNA hydrogel also enables a rapid detection (1 h) with high specificity even for a single-base mismatch. The clinical applicability of the QD-DNA hydrogel is demonstrated for the Klebsiella pneumoniae carbapenemase gene, one of the key targets of drug-resistant pathogenic bacteria.

A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir.

Quantifying the replication-competent HIV reservoir is essential for evaluating curative strategies. Viral outgrowth assays (VOAs) underestimate the reservoir because they fail to induce all replication-competent proviruses. Single- or double-region HIV DNA assays overestimate it because they fail to exclude many defective proviruses. We designed two triplex droplet digital PCR assays, each with 2 unique targets and 1 in common, and normalize the results to PCR-based T cell counts. Both HIV assays are specific, sensitive, and reproducible. Together, they estimate the number of proviruses containing all five primer-probe regions. Our 5-target results are on average 12.1-fold higher than and correlate with paired quantitative VOA (Spearman's ρ = 0.48) but estimate a markedly smaller reservoir than previous DNA assays. In patients on antiretroviral therapy, decay rates in blood CD4+ T cells are faster for intact than for defective proviruses, and intact provirus frequencies are similar in mucosal and circulating T cells.

Babylonia spirata (Linnaeus, 1758) on biochemical and nutritional composition levels are altered by Aeromonas hydrophila infection.

The present study comprises the biochemical and nutritional composition level of control and infected host of B. spirata with A. hydrophila. The healthy species were collected from the Therespuram coast, Southeast coast of India. After the acclimatization period, 15 snails were selected and infected with seven different bacterial pathogens by intramuscular injection. The snails which shows the maximum mortality rate after the bacterial infection was selected for the biochemical composition nutritional level. It was then analyzed and compared to the control group. Based on this result, the FTIR spectrum, DNA fragmentation, SDS PAGE Profile, amino acids (phenylalanine), fatty acids (linolenic acids), minerals (aluminum and copper) was recorded maximum in control and minimum in infected tissue of B. spirata. The result of the present study showed, presences of rich nutrition composition good protein profile in this species add more value of economic importance.

Nanoparticle Beacons: Supersensitive Smart Materials with On/Off-Switchable Affinity to Biomedical Targets.

Smart materials that can switch between different states under the influence of chemical triggers are highly demanded in biomedicine, where specific responsiveness to biomarkers is imperative for precise diagnostics and therapy. Superior selectivity of drug delivery to malignant cells may be achieved with the nanoagents that stay "inert" until "activation" by the characteristic profile of microenvironment cues (e.g., tumor metabolites, angiogenesis factors, microRNA/DNA, etc.). However, despite a wide variety and functional complexity of smart material designs, their real-life applications are hindered by very limited sensitivity to inputs. Here, we present ultrasensitive smart nanoagents with input-dependent On/Off switchable affinity to a biomedical target based on a combination of gold nanoparticles with low-energy polymer structures. In the proposed method, a nanoparticle-based agent is surface coated with a custom designed flexible polymer chain, which has an input-switchable structure that regulates accessibility of the terminal receptor for target binding. Implementation of the concept with a DNA-model of such polymer has yielded nanoagents that have input-dependent cell-targeting capabilities and responsiveness to as little as 30 fM of DNA input in 15 min lateral flow assay. Thus, we show that surface phenomena can augment nanoagents with capability for switchable affinity without compromising the sensitivity to inputs. The proposed approach is promising for development of next-generation theranostic agents and ultrasensitive nanosensors for point-of-care diagnostics.

Novel Competitive Chemiluminescence DNA Assay Based on Fe3O4@SiO2@Au-Functionalized Magnetic Nanoparticles for Sensitive Detection of p53 Tumor Suppressor Gene.

A simple, rapid response time and ultrahigh sensitive chemiluminescence (CL) DNA assay based on Fe3O4@SiO2@Au-functionalized magnetic nanoparticles (Au-MNPs) was developed for detection of p53 tumor suppressor gene. In this study, 2',6'-dimethylcarbonylphenyl-10-sulfopropyl acridinium-9-carboxylate 4'-NHS ester (NSP-DMAE-NHS), as a new kind of highly efficient luminescence reagent, was immobilized on the complementary sequence of the wild-type p53 (ssDNA) to improve the detection sensitivity. The optimal concentration of ssDNA-(NSP-DMAE-NHS) conjugates mixed with the wild-type p53 (wtp53) samples respectively. Then, the wtp53-Au-MNPs conjugates were added to continue the competitive reaction in the above solution. Subsequently, the Au-MNPs separated under magnetic field, measured by a homemade luminescent measurement system. Under optimal conditions, the method exhibited ultrasensitive sensitivity with a detection limit of 0.001 ng mL-1 (0.16 pM), a wide range of liner response from 0.001 ng mL-1~6.6 µg mL-1. Therefore, the immunomagnetic nanocomposites-based detection strategy was rapid, low-cost, and highly sensitive that can be easily extended to the early diagnosis of cancer development and monitoring of patient therapy.

High-performance nanogap electrode-based impedimetric sensor for direct DNA assays.

The rapid and sensitive detection of pathogen DNA (Deoxyribonucleic acid) would be essential for diagnosis and appropriate antibiotic treatment time. Herein, we report a novel direct DNA detectable impedimetric sensor. Direct assay of the amplified target DNA (mecA gene from methicillin-resistant Staphylococcus aureus (MRSA)) was performed using the PCR (polymerase chain reaction) product without any purification. Even though there are lots of PCR reagents and excess salts in sample PCR product, the nanogap electrode-based impedimetric sensor was able to detect DNA amplification fast in 5th PCR cycle which had 260 fM mecA gene in sample originally. The 70 nm gap electrode sensor yielded over 20% signal increase at the 5th PCR cycle and the impedance change grew up to about 60% at 25th in case of sample with 260 fM mecA gene template originally. The increased concentration of target DNA template led to the rise in impedance change such as 60% up at 5th and 120% up at 25th cycle with 260 pM, respectively. It is very outstanding result as compared with the traditional PCR agarose gel. Besides, it is 7-fold superior sensitivity to the microgap electrode. Furthermore, genomic DNA sample extracted from MRSA was detected rapidly. The nanogap electrode-based impedimetric sensor could be a good candidate for a rapid, sensitive, and low-cost electrical biosensor for DNA characterization in diagnostics and disease monitoring.