Probe Amplification Research Articles

Aptamer-Functionalized and Gold Nanoparticle Array-Decorated Magnetic Graphene Nanosheets Enable Multiplexed and Sensitive Electrochemical Detection of Rare Circulating Tumor Cells in Whole Blood.

The identification and monitoring of circulating tumor cells (CTCs) in human blood plays a pivotal role in the convenient diagnosis of different cancers. However, it remains a major challenge to monitor these CTCs because of their extremely low abundance in human blood. Here, we describe the synthesis of a new aptamer-functionalized and gold nanoparticle (AuNP) array-decorated magnetic graphene nanosheet recognition probe to capture and isolate rare CTCs from human whole blood. In addition, by employing the aptamer/electroactive species-loaded AuNP signal amplification probes, multiplexed electrochemical detection of these low levels of CTCs can be realized. The incubation of the probes with the sample solutions containing the target CTCs can lead to the efficient separation of the CTCs and result in the generation of two distinct voltammetric peaks on a screen-printed carbon electrode, whose potentials and current intensities, respectively, reflect the identity and number of CTCs for the multiplexed detection of the Ramos and CCRF-CEM cells with detection limits down to 4 and 3 cells mL-1. With the successful demonstration of the concept, further extension of the developed sensing strategy for the determination of various CTCs in human whole blood for the screening of different cancers can be envisioned in the near future.

DNA synergistic enzyme-mediated cascade reaction for homogeneous electrochemical bioassay

Enzyme-mediated cascade reaction is applied to amplify signal and decrease the background because enzyme can catalyze inactive substrates into active substrates to generate the signal. In this work, Au nanoparticles, as signal probe, are used to load DNA probe and ALP for dual signal amplification. Based on enzyme-mediated cascade reaction, a homogeneous biosensor is constructed for bioassay by employing thrombin as target molecule. When the target is present in the solution, ALP catalyzes the PPi into Pi and then reacts with molybdate in conjunction with Pi in the DNA backbone to produce redox precipitates on the surface of the reduced graphene oxide modified electrode with the help of magnetic separation. Compared with the conventional heterogeneous biosensor, the immobilization-free strategy, proposed in this homogeneous biosensor, improves the sensitivity because of its lower steric hindrance. As a result, this biosensor displayed a great sensitivity with a wide linear range from 1 fM to 10 nM and a detection limit of 0.26 fM, providing a promise and easy operating method for various proteins detection.

A sensitive and specific real-time PCR targeting DNA from wheat, barley and rye to track gluten contamination in marketed foods

This work reports a real-time PCR assay to specifically detect the presence of DNA from the main gluten-containing cereals wheat, barley and rye (WBR) in foodstuffs. The WBR real-time PCR uses two specific primers and a Taqman probe for amplification of a 118 bp DNA fragment common to the three target cereals on the ribosomal internal transcribed spacer (ITS) region. In-house validation of the method was performed employing three binary arrays containing different concentrations (100 000–10 mg/kg) of a wheat/barley/rye mixture in maize flour: untreated, soft heat-treated at 160 °C/13 min and intense heat-treated at 200 °C/20 min. Results showed optimal PCR amplification efficiency, reproducibility and sensitivity with an overall limit of detection of 10–50 mg/kg of the target cereals, theoretically equating to approximately 1–5 mg/kg of gluten. Applicability of the assay was further assessed through a market analysis of 220 food products by the real-time PCR assay and the official R5-based ELISA. Comparative results highlighted the ability of the proposed methodology as a highly sensitive and robust procedure to detect the presence of potentially harmful concentrations of undeclared gluten-containing cereals in some of the tested samples, supporting results of the immunological assays currently used for gluten determination in foods.

Electrochemical aptasensor based on conductive supramolecular polymer hydrogels for thrombin detection with high selectivity

Herein, we synthesized a kind of conductive supramolecular polymer hydrogel (CSPH) based on polyaniline (PANI) which can not only improve the conductivity but also promote antifouling performance of the aptasensor for the specific recruitment of thrombin (TB) from complex samples. With the electrochemical copolymerization of aniline (AN) and 3-aminophenylboronic acid (ABA) on glassy carbon electrode (GCE), the electrode was then inserted into the polyvinyl alcohol (PVA) solution to obtain robust CSPH through boric acid groups incorporated onto PANI to cause gelation of PVA solution, owing to the hydrophilicity of CSPH and nearly electrical neutrality, the modified electrode is antifouling without integration of other antifouling materials. A sandwich-type electrochemical aptasensor was constructed on the CSPH based electrode interfaces. Thrombin aptamer 1 (TBA1) were modified on the CSPH through amide bond, and thrombin aptamer 2 modified magnetic nanoparticles (MNP-TBA2) are used as signal amplification probes, the aptasensor has good sensitivity with a linear range from 1 pmol/L to 10 nmol/L and has a detection limit down to 0.64 pmol/L. The strategy of utilizing eletropolymerization of CSPH films to undergo highly selective thrombin recognition is, of course, readily extended to a broad range of targets in the real samples, and the recovery was ranging from 95.2% to 106.3% and RSDs varying from 2.3% to 4.5%.

Magnetic-assisted self-assembled aptamer/protein hybrid probes for efficient capture and rapid detection of cancer cells in whole blood

Self-assembly of building blocks for constructing multifunctional materials has opened prospects for sensing applications in the biomedical fields. In particular, the combination of aptamer with DNA assembly-based nanotechnology has greatly improved the performance of cancer cell detection. Nevertheless, the cancer cell detection strategies of integrating aptamer with protein are relatively sparse. So we have developed a self-assembled aptamer method to realize the efficient capture and rapid detection of cancer cells by ingeniously combining aptamer modified magnetic nanoparticles as capture nanoprobes with self-assembled aptamer/protein hybrid probes (SAPPs) as signal amplification probes. By merely mixing the component materials together simultaneously, the SAPPs, integrating aptamer for cancer cell recognition with protein for amplifying signal, were fabricated by DNA-governed one-step assembly. In addition, the SAPPs-based method exhibits efficient capture, rapid (about 45 min) and specific CCRF-CEM detection performance, with limits of detection down to 75 cells/mL in buffer and 200 cells/mL in whole blood.

SAT-072 DYSREGULATION OF CIRCULATORY MICRO-RNAS IN PATIENTS WITH CHRONIC ANTIBODY MEDIATED REJECTION (CABMR) OF RENAL TRANSPLANT

MicroRNAs (miRNAs) are non-coding RNA that play pivotal role in modulating expression of multiple target genes at post-transcriptional level and have potential to modulate physiological & pathological processes thus can be used as potential therapeutic targets. In kidney, role of miR have been involved in renal fibrosis, organogenesis and in pathogenesis of many diseases including diabetic, IgA nephropathies, glomerulopathies etc thus opening the possibility of their use as biomarkers in CABMR. The impact of miRNA regulation involved in allograft function and immunity has not been investigated thoroughly thus we studied the miRNAs expression involved in CABMR. Patients with CABMR (Banff’s classification- 2017) were included. 11 blood donors (M=10) with a mean age of 48.7Y were served as controls. Samples were processed for RNA isolation. RNA integrity was checked by running over 1% agarose gel and quantity and qualities were checked using nanodrop with 260/230 and 260/280 ≥ 1.8. The miRNAs expressions, miR-21, miR-155, miR-210, miR-146a, miR-126, miR-34a, miR-150 and RNU6B (control) were detected by quantitative miRNA stem loop RT-PCR technology (TaqMan MicroRNA Assays, Applied Biosystems, CA) as per manufactures protocol. Briefly, we used highly target-specific stem loop structure and reverse transcription primer, and after reverse transcription, used specific TaqMan hybridization probes for miRNA amplification. This allowed for a high specificity only for the mature miRNA, and formation of a reverse transcription primer/mature miRNA chimera extending to the 5’ end of the miRNA. Expression was measured by calculating fold change. Total 24 patients (Male=22 with mean age of 42.63 Y) were enrolled for the study with post transplant duration of 32.25 months. The demographic details with mean values were: S Creatinine = 1.75 mg/dl, BUN = 27.97 mg/dl, Tac level = 6.56 ng/ml, S Uric Acid = 6.778 mg/dl, S Albumin = 3.68 g/dl, Na+/K+ = 135.9/4.21 mmol/L, S Phosphorus = 3.250 mg/dl, S Alkaline phosphate = 81.65 u/L. Induction regimen (Basiliximab=20, ATG= 4) Baseline Immunosuppression MMF+Pred (Tacrolimus/Cyclosporin) = 22/2. C4d were positive in 22 and DSA+ in 2 patients. The expression of different microRNAs, miR-21 (p≤0.0001), miR-155 (p=0.0212), miR-210 (p=0.0001), miR-146a (p=0.0055), miR-126 (p=0.0001) were increased in CABMR patients compared to healthy controls whereas expression of miR-150 (p=0.0006) was shown to be reduced. Expression of miR-34a did not show any significant change. Altered miRNA expression profiling was found in CABMR compared to healthy controls. MicroRNAs are crucial regulators of cell function. They are easy to detect and represent potentially good targets for novel therapies.

Electrochemical Biosensor for Acetamiprid Based on Aptamer As Captured Probe and Horseradish Peroxidase for Signal Amplification

Nowadays, people pay more attention to the environmental protection and food safety problems caused by pesticide residues. In this paper, a biosensor for sensitive detection of acetamiprid, a kind of insecticide, was fabricated based on aptamer as a captured probe of acetamiprid and horseradish peroxidase (HRP) as a biocatalyst for signal amplification. The schematic diagram of fabrication of the biosensor was shown in Fig. 1. Fig. 1 The preparation of biosensor and the detection of acetamiprid Labeled aptamer of acetamiprid and a complementary DNA strand (cDNA) of the aptamer were synthesized by Shanghai Sangon Biological Engineering Technological Co. Ltd. (China). The base sequences are as-follows:[1] aptamer: 5′-NH2-(CH2)6-TGT AAT TTG TCT GCA GCG GTT CTT GAT CGC TGA CAC CAT ATT ATG AAG A-3′; cDNA: 5′- (SH) - (CH2)6 - T CTT CAT AAT ATG GTG TCA GCG ATC AAG AAC CGC TGC AGA CAA ATT ACA -3′. A 10 mL graphene oxide (GNO) suspension of 1.0 mg/mL was uniformly coated on the surface of a glassy carbon electrode (GCE) by drop-casting and allowed it to dry naturally in the air. The modified electrode (GNO/GCE) was immersed in 0.1 mol/L PBS solution containing of 0.4 mM xanthurenic acid (Xa) and was performed cyclic voltammetry scan from 0.65 V to -1.70 V (vs.SCE) for 10 cycles. GNO as the starting material was electrochemical reduction to ERGO at cathodic potential and the electropolymerization of xanthurenic acid (PXa) was achieved at anodic potential in a simply one-step process by cyclic voltammetry (CV), simultaneously. Because of the π–π* interaction between the conjugated GNO layers and aromatic rings of Xa, GNO can serve as a strong adsorbing platform for Xa monomer and promote the electropolymerization of Xa effectively. [2] After the PXa-ERGO composite film was synthesized, the PXa-ERGO/GCE electrode was immersed in 0.1 M phosphate buffer solution (PBS) of pH 7.0 containing 5.0 mM ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and 8.0 mM N-hydroxysuccinimide (NHS) to convert the terminal carboxylic group to active NHS ester. Double-helix DNA (dsDNA) was prepared by the hybridization between aptamer and cDNA. Then the dsDNA solution was coated on the surface of the PXa-ERGO/GCE electrode and incubated for 2 hours. The rich carboxylic groups of PXa-ERGO film were applied to immobilize the dsDNA with amino groups at 5’ end of cDNA by covalent bonding. The biosensor was denoted as dsDNA/PXa-ERGO/GCE. Gold nanoparticles (AuNPs) and HRP–AuNPs conjugates were prepared. Transmission electron microscope (TEM) images and ultraviolet-visible absorption spectra were used to characterize the AuNPs. AuNPs-HRP conjugates were modified onto the dsDNA/PXa-ERGO/GCE though Au–thiol interactions. Hydroquinone (HQ) and H2O2 in solution were catalyzed by HRP to generate an anodic voltammetric peak. The aptamer biosensor was successfully applied to determine acetamiprid by differential pulse voltammetry (DPV). The analysis was based on the change in the anodic peak current (i p) before and after sample solutions were applied to the surface of the working electrode at room temperature. When the background current stabilized, the anodic peak current response was recorded as i p0. In the presence of acetamiprid, aptamers bind with acetamiprid to produce a complex on the electrode surface. The HRP–AuNP–cDNA conjugates therefore fall off from the electrode surface. The anodic peak current decreased and was recorded as i p. The change in current was given by Δi p = i p0 - i p. A 25 mL aliquot of 0.1 M PBS (pH 7.4) was placed in a voltammetric cell and the required volumes of 3.0 mM HQ and 1.5 mM H2O2 solution were added. The DPV potential scan was performed from -0.4 V to 0.6 V (vs.SCE) at pulse amplitude of 20 mV, pulse frequency of 25 Hz and increment potential of 4 mV. Under the optimal conditions, the Δi p value highly and linearly increased with the increase in the logarithm of the acetamiprid concentration (c) over a range from 0.010 to 20 μg/L, with a detection limit of 0.005 μg/L. This aptamer sensor was applied in the determination of acetamiprid in river water with high specificity, sensitivity and selectivity.

Characterization of large deletions of the MECP2 gene in Rett syndrome patients by gene dosage analysis.

BackgroundRett syndrome (RTT) is a developmental disorder with an early onset and X‐linked dominant inheritance pattern. It is first recognized in infancy and is seen almost always in girls, but it may be seen in boys on rare occasions. Typical RTT is caused by de novo mutations of the gene MECP2 (OMIM*300005), and atypical forms of RTT can be caused by mutations of the CDKL5 (OMIM*300203) and FOXG1 (OMIM*164874) genes.MethodsApproximately 5% of the mutations detected in MECP2 are large rearrangements that range from exons to the entire gene. Here, we have characterized the deletions detected by multiplex ligation‐dependent probe amplification (MLPA) in the gene MECP2 of 21 RTT patients. Breakpoints were delineated by DNA‐qPCR until the amplification of the deleted allele by long‐PCR was possible.ResultsThis methodology enabled us to characterize deletions ranging from 1,235 bp to 85 kb, confirming the partial or total deletion of the MECP2 gene in all these patients. Additionally, our cases support the evidence claiming that most of these breakpoints occur in some restricted regions of the MECP2 gene.ConclusionThese molecular data together with the clinical information enable us to propose a genotype–phenotype correlation, which is essential for providing genetic counseling.

Identification of a novel large deletion and other copy number variations in the CFTR gene in patients with Cystic Fibrosis from a multiethnic population.

BackgroundCystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). There are over 2000 different pathogenic and non‐pathogenic variants described in association with a broad clinical heterogeneity. The most common types of mutations in this gene are single nucleotide substitutions or small deletions and insertions. However, large rearrangements, such as large duplications or deletions, are also a possible cause of CF; these variations are rarely tested in routine screenings, and much of them remain unidentified in some populations, especially those with high ethnic heterogeneity.MethodsThe present study utilized the Multiplex Ligation‐dependent Probe Amplification (MLPA) technique for the detection of duplications and deletions in 165 CF patients from the Rio de Janeiro State (Brazil), which after extensive mutational screening, still exhibited one or two unidentified CF alleles.ResultsFive patients with alterations in MLPA signals were detected. After validation, we identified three copy number variations, one large duplication (CFTRdup2‐3) and two large deletions (CFTRdel25‐26 and CFTRdel25‐27‐CTTNBP2). Two detected deletions were not validated. They were false positives caused by a small deletion of 18 base pairs (232del18) and a point mutation (S168L) in the probe binding site.ConclusionOur results highlight the importance of screening for large rearrangements in CF cases with no or only one CFTR mutation defined.

DNA alteration-based classification of uveal melanoma gives better prognostic stratification than immune infiltration, which has a neutral effect in high-risk group.

BackgroundIn uveal melanomas, immune infiltration is a marker of poor prognosis. This work intended to decipher the biological characteristics of intra‐tumor immune population, compare it to other established biomarkers and to patients' outcome.MethodsPrimary, untreated, and mainly large uveal melanomas with retinal detachment were analyzed using: transcriptomic profiling (n = 15), RT‐qPCR (n = 36), immunohistochemistry (n = 89), Multiplex Ligation‐dependent Probe Amplification (MLPA) for copy number alterations (CNA) analysis (n = 89), array‐CGH (n = 17), and survival statistics (n = 86).ResultsGene expression analysis divided uveal melanomas into two groups, according to the IFNγ/STAT1‐IRF1 pathway activation. Tumors with IFNγ‐signature had poorer prognosis and showed increased infiltration of CD8+ T lymphocytes and macrophages. Cox multivariate analyses of immune cell infiltration with MLPA data delineated better prognostic value for three prognostic groups (three‐tier stratification) than two (two‐tier stratification). CNA‐based model comprising monosomy 3, 8q amplification, and LZTS1and NBL1 deletions emerged as the best predictor for disease‐free survival. It outperformed immune cell infiltration in receiver operating characteristic curves. The model that combined CNA and immune infiltration defined risk‐groups according to the number of DNA alterations. Immune cell infiltration was increased in the high‐risk group (73.7%), where it did not correlate with patient survival, while it was associated with poorer outcome in the intermediate risk‐group.ConclusionsHigh degree of immune cell infiltration occurs in a subset of uveal melanomas, is interferon‐gamma‐related, and associated with poor survival. It allows for two‐tier stratification, which is prognostically less efficient than a three‐tier one. The best prognostic stratification is by CNA model with three risk‐groups where immune cell infiltration impacts only some subgroups.

Label-Free Telomerase Detection in Single Cell Using a Five-Base Telomerase Product-Triggered Exponential Rolling Circle Amplification Strategy.

Telomerase is a universal biomarker of malignant tumors. Sensitive and reliable analysis for telomerase activity is of vital importance for both early diagnosis and therapy of malignant tumors. Herein, a novel fluorescent strategy was proposed for sensitive and label-free detection of telomerase activity. One highlight of this strategy is that an exponential signal amplification can be triggered by a very short telomerase extension product (TEP). Without adding dATP, the designed telomerase primer can be easily controlled to extend five bases (GGGTT) to give short TEP with definite length. The resulting short TEP can then be constructed as a circular rolling circle amplification (RCA) template and thus initiate a nicking enzyme-mediated exponential RCA, producing G-rich amplification products that can be sensitively probed via specific binding between the fluorescent dye Thioflavin T (ThT) and the nucleic acid G-quadruplexes. Elevated telomerase translocation efficiency, combined with exponential signal amplification and specific probing of RCA products by ThT, endow the sensing platform with extraordinarily high detection sensitivity. The requirement for short TEP increases the possibility to analyze telomerase with low activity. The proposed sensing platform can achieve sensitive telomerase activity detection in individual cells, even with the interference of accumulated normal cells. It was also demonstrated to show excellent capability in screening for the inhibitors of telomerase. Therefore, the proposed sensing platform has great potential for not only clinical diagnosis but also anticancer drug development.

Enzyme-conjugated hybridization chain reaction for magneto-controlled immunoassay of squamous cell carcinoma antigen with pH meter

A nanoparticle-based potentiometric immunoassay was designed for the sensitive detection of squamous cell carcinoma antigen (SCCA; cervical carcinoma marker) on a portable pH meter coupling enzyme-labeled hybridization chain reaction (HCR) with two alternating hairpin DNA probes for the signal amplification. Initially, a sandwich-type immunoreaction was carried out between anti-SCCA capture antibody-conjugated magnetic bead and detection antibody/initiator strand-coated gold nanoparticle (AuNP). Then, the HCR reaction was readily executed between two glucose oxidase (GOx)-labeled hairpins through the initiator strand to form numerous GOx concatamers on the AuNP via the long nicked double-helix. The concatenated GOx oxidized glucose into gluconic acid and hydrogen peroxide, thus resulting in the pH change of the detection solution on a handheld pH meter. Several labeling protocols including GOx-antibody, GOx-AuNP-antibody and GOx-HCR-AuNP-antibody were investigated for detection of target SCCA, and improved analytical features were obtained with the immune-HCR assay. Under optimum conditions, the immune-HCR assay exhibited good pH responses for the determination of SCCA at a concentration as low as 5.7 pg/mL. Additionally, the immune-HCR assay had good precision and reproducibility, high specificity, and acceptable accuracy for analyzing human serum specimens.

MnO2 nanosheet-mediated ratiometric fluorescence biosensor for MicroRNA detection and imaging in living cells

MicroRNA (miRNA) plays significant roles in cell proliferation, differentiation and apoptosis, and has been considered to be valuable biomarker for cancer. Accurate and sensitive detection of miRNA is crucially significant for cancer diagnosis and treatment. Here, a MnO2 nanosheet-mediated ratiometric fluorescence biosensor was designed for miRNA detection and imaging in living cells. It contained MnO2 nanosheets acting as DNA carrier, and fluorescent donor (FAM)-labeled hairpin H1 (recognition probe) and fluorescent acceptor (TAMRA)-labeled hairpin H2 (amplification probe). When the biosensor entered cell by endocytosis, MnO2 nanosheets were degraded to Mn2+ via intracellular glutathione (GSH) and the adsorbed hairpins H1 and H2 were released. The intracellular target miRNA-21 hybridized with the recognition unit of H1 to initiate catalyzed hairpin assembly (CHA) and a large amount of H1-H2 duplexes were produced. This brought fluorescent donor FAM and fluorescent acceptor TAMRA into close proximity to produce fluorescence resonance energy transfer (FRET), inducing a ratiometric fluorescent response (donor signal decreased and acceptor signal enhanced) for miRNA-21 detection. Furthermore, this method could be applied to differentiate the expression levels of miRNA-21 in HeLa, HepG-2 and L02 cells. These results indicated that the proposed method possessed great potential in the early diagnosis of miRNA-related diseases.

Cellular microRNA detection with miRacles: microRNA- activated conditional looping of engineered switches.

MicroRNAs are short noncoding regulatory RNAs that are increasingly used as disease biomarkers. Detection of microRNAs can be arduous and expensive and often requires amplification, labeling, or radioactive probes. Here, we report a single-step, nonenzymatic microRNA detection assay using conformationally responsive DNA nanoswitches. Termed miRacles (microRNA-activated conditional looping of engineered switches), our assay has subattomole sensitivity and single-nucleotide specificity using an agarose gel electrophoresis readout. We detect cellular microRNAs from nanogram-scale RNA extracts of differentiating muscle cells and multiplex our detection for several microRNAs from one biological sample. We demonstrate 1-hour detection without expensive equipment or reagents, making this assay a compelling alternative to quantitative polymerase chain reaction and Northern blotting.

Detection of sub-microscopic blood levels of Plasmodium falciparum using Tandem Oligonucleotide Repeat Cascade Amplification (TORCA) assay with an attomolar detection limit

Tandem Oligonucleotide Repeat Cascade Amplification (TORCA) based on signal rather than target amplification under isothermal conditions was developed for nucleic acid assays. The initial signal was generated by hybridization of single stranded DNA targets to immobilized recognition probes followed by hybrid cleavage with specific restriction endonuclease (REase), and release of trigger oligonucleotides (Tr1). The signal amplification chamber contained two bead types carrying single-stranded amplification probes and two amplification REases. The probes consisted of multiple tandem repeats of either Tr1 or another trigger Tr2, with the tandem-Tr1 anchored to the beads through the antisense Tr2 linker and vice versa. Addition of the recognition reaction solution and Tr1 hybridization to the anti-Tr1 linkers started cleavage and release of additional Tr1 and Tr2, resulting in exponential signal amplification. The cleavage cascade also released horseradish peroxidase (HRP) pre-attached to the amplification probes, and the resultant signal was measured colorimetrically. A TORCA assay was developed for detection of Plasmodium falciparum parasites in blood. It had the detection limit in the attomolar concentration range, successfully detecting sub-microscopic P. falciparum infections at less than 0.75 infected erythrocytes per microliter. Further TORCA optimization will likely produce the quantitative isothermal alternative to PCR at a fraction of its cost.

Cellular Microrna Detection using DNA Nanoswitches

MicroRNAs are short non-coding regulatory RNAs that are increasingly used as disease biomarkers. Detection of microRNAs can be arduous and expensive, and often requires amplification, labeling, or radioactive probes. Here we report a single-step, non-enzymatic microRNA detection assay using conformationally responsive DNA nanoswitches. Termed miRacles (microRNA activated conditional looping of engineered switches), our assay has sub-attomole sensitivity and single-nucleotide specificity using an agarose gel electrophoresis readout. We detect cellular microRNAs from nanogram-scale RNA extracts of differentiating muscle cells, and multiplex our detection for several microRNAs from one biological sample. We demonstrate one-hour detection without expensive equipment or reagents, making this assay a compelling alternative to qPCR and Northern blotting.

A fluorometric aptamer method for kanamycin by applying a dual amplification strategy and using double Y-shaped DNA probes on a gold bar and onmagnetite nanoparticles.

A simple and highly sensitive fluorometric method is described for the determination of the antibiotic kanamycin (Kana) in food. Dual signal amplification is accomplished by making use of double Y-shaped aptamer DNA probes acting as a capture probes and signal amplification probes. The DNA probes were immobilized on a gold bar and ona magnetic bar, respectively. On addition of Kana, the Y-shaped aptamer probe captures Kana and then is disassembled to release two single-stranded DNAs. These trigger target recycling and HCR between the two bars simultaneously. As a result, many long duplex DNA chains are formed in the supernatant. After pulling out the bars and adding the fluorescent intercalating probe SYBR Green I, strong fluorescence (with excitation/emission peaks at 497/525nm) is induced. The use of such double Y-shaped DNA probes obviously overcomes the unspecific signal amplification by HCR which increases selectivity and sensitivity. This is due to the fact that the hairpin of HCR is separated in being present in different arms of the Y-shaped probe. Under the optimal conditions, the assay has a limit of 0.45pg·mL-1 for Kana. It was applied to analyze spiked milk, fish and pork samples. Graphical abstract The scheme represents a sensitive fluorometric aptamer-based method to detect kanamycin (Kana). It is making use of a double stirring bar-assisted dual amplification strategy with zero background. Abbreviations: apt: aptamer, AuNPs: gold nanoparticles, HCR: hybridization chain reaction.

Integrated Genomic Analysis of Chromosomal Alterations and Mutations in B-Cell Acute Lymphoblastic Leukemia Reveals Distinct Genetic Profiles at Relapse

Background: The clonal basis of relapse in acute lymphoblastic leukemia (ALL) is complex and not fully understood. Methods: Next-generation sequencing (NGS), array comparative genomic hybridization (aCGH), and multiplex-ligation-dependent probe amplification (MLPA) were carried out in matched diagnosis-relapse samples from 13 B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients to identify patterns of genetic evolution that could account for the phenotypic changes associated with disease relapse. Findings: The integrative genomic analysis of aCGH, MLPA and NGS revealed that 100% of BCP-ALL patients showed at least one genetic alteration at diagnosis and relapse. In addition, there was a significant increase in the frequency of chromosomal lesions at the time of relapse (median, 6 alterations per sample) relative to that at diagnosis (median, 47 alterations) (p = 0.019). The combination of MLPA and aCGH techniques showed that IKZF1 was the most frequently deleted gene. Notably, TP53 was the most frequently mutated gene at relapse (31%). Two TP53 mutations were detected only at relapse, whereas the three others showed an increase of their mutational burden at relapse. Interpretation: Clonal evolution patterns were heterogeneous, involving acquisition, loss and maintenance of lesions at relapse. Therefore, this study confirmed that BCP-ALL is a genetically dynamic disease with distinct genetic profiles at diagnosis and relapse. The combination of the NGS, aCGH, and MLPA approaches enables better molecular characterization of the genetic profile in ALL patients during the evolution from diagnosis to relapse. Funding Statement: This work was supported in part by a grant from the Consejeria de Educacion, Junta de Castilla y Leon, Fondos FEDER (SA085U16, SA271P18), Proyectos de Investigacion de la Gerencia Regional de Sanidad, SACYL, (GRS 1847/A/18), Fundacion Castellano Leonesa de Hematologia y Hemoterapia (FUCALHH 2017), a grant to AM from the Junta Provincial de Salamanca of the Asociacion Espanola Contra el Cancer (AECC), a grant to MFC from the Universidad Pedagogica y Tecnologica de Colombia – Vicerrectoria de Investigacion y Extension (Grupo de Investigacion en Ciencias Biomedicas UPTC – GICBUPTC, Escuela de Ciencias Biologicas). M. Hernandez-Sanchez is supported by FEHH-Janssen (Sociedad Espanola de Hematologia y Hemoterrapia), and a grant to JR and JMR from Instituto Carlos III (PI14/01971). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The study was approved by the local ethical committee, the Comite Etico de Investigacion Clinica, at the Hospital Universitario de Salamanca. Written informed consent was obtained from each patient or their legal guardian before entering the study.

Metal coordination polymer induced perylene probe excimer fluorescence and its application in acetylcholinesterase sensing and alpha-fetoprotein immunoassay.

A novel sensing strategy for acetylcholinesterase (AChE) and alpha-fetoprotein (AFP) is developed, based on the perylene probe monomer to excimer fluorescence transformation induced by the in situ generation of a metal coordination polymer. In the presence of AChE, acetylthiocholine chloride was hydrolyzed to thiocholine. Ag+ and the produced thiocholine formed a positively charged metal coordination polymer, which induced the aggregation of a negatively charged perylene probe and the formation of probe excimer emission. The intensity ratio of excimer to monomer emission was proportional to the AChE concentration. A sensing method for AChE was thus established with a detection limit of 0.02 mU mL-1. The excimer emission with a large Stokes shift could avoid the interference of background fluorescence from complex biological samples, and thus achieved selective and sensitive detection of AChE. In addition, a fluorescence immunoassay strategy for AFP was then developed. Gold nanoparticles (AuNPs) co-immobilized with acetylcholinesterase and the AFP antibody as the capture and amplification probe were first prepared. In the presence of AFP, the sandwich structure was formed by immunological recognition. The hydrolysis of acetylthiocholine was catalyzed by AChE on the AuNPs, and the metal coordination polymer was then formed which resulted in the aggregation of the perylene probe and the formation of the excimer emission. The proposed sensing method offers a new strategy for the detection of other biomarkers.

Chemiluminescence molecular probe with a linear chain reaction amplification mechanism.

A new signal amplification probe with a linear chain reaction amplification mechanism and distinct chemiluminescence output was developed. The probe is composed of a unique structural motif that combines a chemiexcitation mechanism with an intramolecular transesterification into a single molecular structure. As demonstrated with a probe designed to detect hydrogen peroxide, an auto-inductive chemiluminescence signal amplification was obtained through methanol release by an intramolecular transesterification of the generated 2-hydroxymethylbenzoate derivative. The methanol was then oxidized by alcohol oxidase to regenerate the analyte of interest, hydrogen peroxide. Our probe enabled direct measurement of light emission with a limit of detection of 2.5 μM, whilst the assay was rapidly completed within 14 to 150 minutes. Such molecular probes with chemiluminescence signal enhancement through analyte amplification could be used for the detection of other chemical and biological analytes.