Personal glucose meters coupled with signal amplification technologies for quantitative detection of non-glucose targets:Recent progress and challenges in food safety hazards analysis / 药物分析学报

Ensuring food safety is paramount worldwide.Developing effective detection methods to ensure food safety can be challenging owing to trace hazards,long detection time,and resource-poor sites,in addition to the matrix effects of food.Personal glucose meter(PGM),a classic point-of-care testing device,possesses unique application advantages,demonstrating promise in food safety.Currently,many studies have used PGM-based biosensors and signal amplification technologies to achieve sensitive and specific detection of food hazards.Signal amplification technologies have the potential to greatly improve the analytical performance and integration of PGMs with biosensors,which is crucial for solving the challenges associated with the use of PGMs for food safety analysis.This review introduces the basic detection principle of a PGM-based sensing strategy,which consists of three key factors:target recog-nition,signal transduction,and signal output.Representative studies of existing PGM-based sensing strategies combined with various signal amplification technologies(nanomaterial-loaded multienzyme labeling,nucleic acid reaction,DNAzyme catalysis,responsive nanomaterial encapsulation,and others)in the field of food safety detection are reviewed.Future perspectives and potential opportunities and challenges associated with PGMs in the field of food safety are discussed.Despite the need for complex sample preparation and the lack of standardization in the field,using PGMs in combination with signal amplification technology shows promise as a rapid and cost-effective method for food safety hazard analysis.

Sensitive detection of microRNAs using polyadenine-mediated fluorescent spherical nucleic acids and a microfluidic electrokinetic signal amplification chip / 药物分析学报

The identification of tumor-related microRNAs(miRNAs)exhibits excellent promise for the early diag-nosis of cancer and other bioanalytical applications.Therefore,we developed a sensitive and efficient biosensor using polyadenine(polyA)-mediated fluorescent spherical nucleic acid(FSNA)for miRNA analysis based on strand displacement reactions on gold nanoparticle(AuNP)surfaces and electrokinetic signal amplification(ESA)on a microfluidic chip.In this FSNA,polyA-DNA biosensor was anchored on AuNP surfaces via intrinsic affinity between adenine and Au.The upright conformational polyA-DNA recognition block hybridized with 6-carboxyfluorescein-labeled reporter-DNA,resulting in fluores-cence quenching of FSNA probes induced by AuNP-based resonance energy transfer.Reporter DNA was replaced in the presence of target miRNA,leading to the recovery of reporter-DNA fluorescence.Sub-sequently,reporter-DNAs were accumulated and detected in the front of with Nafion membrane in the microchannel by ESA.Our method showed high selectivity and sensitivity with a limit of detection of 1.3 pM.This method could also be used to detect miRNA-21 in human serum and urine samples,with re-coveries of 104.0％-113.3％and 104.9％-108.0％,respectively.Furthermore,we constructed a chip with three parallel channels for the simultaneous detection of multiple tumor-related miRNAs(miRNA-21,miRNA-141,and miRNA-375),which increased the detection efficiency.Our universal method can be applied to other DNA/RNA analyses by altering recognition sequences.

Label-free Fluorescent DNA Detection Strategy Based on Hybridization Chain Reaction and Vacant Site-Binding Molecule / 分析化学

A label-free and enzyme-free fluorescent DNA sensor based on hybridization chain reaction ( HCR) and vacant site-binding molecule was developed. By rationally incorporating a cytosine in hairpin DNAs for HCR and utilizing vacant site-binding fluorophore ATMND, sensitive DNA detection was achieved, resulting from the large amount of vacant sites presented on the long DNA nanostructures upon HCR. Gel electrophoresis and atomic force microscopy were performed to characterize the hybridization chain reaction. Under the optimized conditions, the method could sensitively detect the target DNA with the low detection limit of 2. 0 nmol/L. The method shows great potential in signal amplification for DNA detection with advantages such as simple, label-free and low-cost.

Electrochemical Luminescent DNA Sensor Based on Polymerase-assisted Signal Amplification / 分析化学

A novel polymerase-based electrochemiluminescence DNA sensor was constructed for messenger RNA (mRNA) detection by cyclic chain displacement polymerization,assisted by target mRNA cycle,and quantum dots signal amplification.Firstly,the mercapto-modified capture-type capture DNA (CP) was immobilized on the surface of a magneto-controlled glassy carbon electrode via Au-S bond.After adding the target mRNA,CP was opened and hybridized with mRNA to form dsDNA.After adding polymerase,primer chain (DNA1) and the base,the primer chain was extended to replace the target mRNA.After one cycle,the mRNA chain could open another hairpin in order to carry out next cycle of amplification.Finally,electrochemical luminescence detection was carried out by adding DNA2 labeled TGA-CdTe quantum dots.The amplification of the target mRNA by the addition of polymerase and the signal combined with the quantum dot mark improved the sensitivity of the sensor greatly.The result showed that the logarithm of target mRNA concentration had a good linear relationship with the corresponding ECL signal in the range of 1 × 10-15-1 × 10-11mol/L,with the detection limit of 3.4 × 10-16mol/L(S/N=3).Under the optimal conditions,the recoveries of mRNA spiked in human serum sample were 97.2% -102.3%.This sensor exhibited good selectivity,stability and reproducibility.

Differential Innervation of Secretory Coils and Ducts in Human Eccrine Sweat Glands / 中华医学杂志(英文版)

Background@#Previous studies demonstrate that eccrine sweat glands are innervated by both cholinergic and adrenergic nerves. However, it is still unknown whether the secretory coils and ducts of eccrine sweat glands are equally innervated by the sympathetic nerve fibers. To well understand the mechanisms on sweat secretion and reabsorption, the differential innervation of secretory coils and ducts in human eccrine sweat glands was investigated in the study.@*Methods@#From June 2016 to June 2017, six human skins were fixed, paraffin-embedded, and cut into 5 μm-thick sections, followed by costaining for nerve fiber markers protein gene product 9.5 (PGP 9.5), tyrosine hydroxylase (TH) and vasoactive intestinal peptide (VIP), and eccrine sweat gland markers K7, S100P, and K14 by combining standard immunofluorescence with tyramide signal amplification (IF-TSA). Stained sections were observed under the microscope, photographed, and analyzed.@*Results@#The fluorescent signals of PGP 9.5, TH, and VIP were easily visualized, by IF-TSA, as circular patterns surrounding eccrine sweat glands, but only PGP 9.5 could be observed by standard IF. The IF-TSA method is more sensitivity than standard IF in detecting antigens expressed at low levels. PGP 9.5, TH, and VIP appeared primarily surrounding the secretory coils and sparsely surrounding the sweat ducts.@*Conclusion@#Sweat secretion is mainly controlled by autonomic nerves whereas sweat reabsorption is less affected by nerve activity.

Use of fluorescent oligonucleotide probes for differentiation between Paracoccidioides brasiliensis and Paracoccidioides lutzii in yeast and mycelial phase

BACKGROUND Fluorescence in situ hybridisation (FISH) associated with Tyramide Signal Amplification (TSA) using oligonucleotides labeled with non-radioactive fluorophores is a promising technique for detection and differentiation of fungal species in environmental or clinical samples, being suitable for microorganisms which are difficult or even impossible to culture. OBJECTIVE In this study, we aimed to standardise an in situ hybridisation technique for the differentiation between the pathogenic species Paracoccidioides brasiliensis and Paracoccidioides lutzii, by using species-specific DNA probes targeting the internal transcribed spacer-1 (ITS-1) of the rRNA gene. METHODS Yeast and mycelial phase of each Paracoccidioides species, were tested by two different detection/differentiation techniques: TSA-FISH for P. brasiliensis with HRP (Horseradish Peroxidase) linked to the probe 5’ end; and FISH for P. lutzii with the fluorophore TEXAS RED-X® also linked to the probe 5’ end. After testing different protocols, the optimised procedure for both techniques was accomplished without cross-positivity with other pathogenic fungi. FINDINGS The in silico and in vitro tests show no reaction with controls, like Candida and Cryptococcus (in silico) and Histoplasma capsulatum and Aspergillus spp. (in vitro). For both phases (mycelial and yeast) the in situ hybridisation showed dots of hybridisation, with no cross-reaction between them, with a lower signal for Texas Red probe than HRP-TSA probe. The dots of hybridisation was confirmed with genetic material marked with 4’,6-diamidino-2-phenylindole (DAPI), visualised in a different filter (WU) on fluorescent microscopic. MAIN CONCLUSION Our results indicated that TSA-FISH and/or FISH are suitable for in situ detection and differentiation of Paracoccidioides species. This approach has the potential for future application in clinical samples for the improvement of paracoccidioidomycosis patients prognosis.

A Dual-signal Amplification Method for DNA Detection Based on Exonuclease Ⅲ and Fluorescence Quenching Ability of MoS2 Nanosheet / 分析化学

A highly sensitive and selective DNA biosensor is described based on the fluorescence quenching ability of MoS2 nanosheet and exonucleaseⅢ( ExoⅢ) assisted dual-signal amplification. In this sensor, the fluorescence probes ( HP1 and HP2 ) cannot be degraded by Exo Ⅲdue to the 3 '-termini protrusion and thus are adsorbed on the surface of MoS2 nanosheets, which will result in the quenching of MoS2 nanosheets toward the probes and induce a low fluorescent signal. The presence of the target DNA leads to the desorption of probes on the surface of MoS2 nanosheets due to the hybridization toward probes, generating many fluorescent fragments by Exo Ⅲdigestion and inducing dual-signal amplification. This method can improve the sensitivity and detection limit compared with single amplification method, and shows excellent selectivity in the discrimination of single base mismatched targets. On the basis of the significantly high sensitivity, the developed biosensor can be potentially extended to detect various DNA targets with excellent sequence selectivity.

Sensitive Detection of Telomerase Based on Hybridization Chain Reaction-assisted Multiple Signal Amplification / 分析化学

A new electrochemical method for telomerase activity assay was developed on the basis of hybridization chain reaction ( HCR)-assisted multiple signal amplification, aiming at improving the sensitivity and specificity of telomerase assay. The experiments utilized HeLa cells as original source of the telomerase in the electrochemical studies. The telomerase primer was firstly self-assembled on the surface of gold electrode. The telomerase catalyzed the elongation of the primer, producing the complementary sequences of hairpin probe H1. In this case, HCR was then initiated by interacting with two hairpin probes H1 and H2. Because both H1 and H2 were modified by biotin, horseradish peroxidase could be captured on the electrode surface through the high-affinity interaction between biotin and streptavidin, catalyzing the oxidation of o-phenylenediamine to produce 2,3-diaminophenazine. Therefore, the telomerase assay was realized by tracing the electrochemical signals with differential pulse voltammetry. This electrochemical method was of high efficiency and feasibility for detecting telomerase activity, and could trace the telomerase activity down to 10 cells/mL HeLa cells with a wide linear range. Besides, it could also easily distinguish the target enzyme from the control proteins with high specificity.

Progress of Metal Nanoclusters-based Electrochemiluminescent Analysis / 分析化学

In the past ten years, the development of electrochemiluminescent ( ECL ) analytical methods based on various types of nanostructures has become a research hotspot. Nanocluster, an intermediate between molecules and conventional nanoparticles, is renowned for its luminescent feature. The first report on ECL for nanoclusters can be traced back to 2009 . Here we summarized the main research progresses since 2011 . Firstly, the preparation of ECL-related nanoclusters was briefly introduced. Then, the mechanisms and applications of ECL by nanoclusters were described. To improve ECL performances, two main strategies, i. e. , nanostructure-based ECL enhancement and biological signal amplification were proposed. Besides, the nanoclusters as the energy transfer receptors in ECL systems were also discussed. In prospect part, the future development of ECL by nanoclusters was considered. We believed that the synthesis of high quality nanoclusters, the reveal of ECL structure-activity relationships, the rationale design and application of near-infrared ECL, and the role of ECL in the interdisciplinary research were the main problems we faced in the future.

Expression and activity assay of recombinant flap endonuclease 1 / 生物工程学报

Flap endonuclease 1 (FEN1) is an endonuclease that catalyzes invasive reaction. It can be used in signal-amplification reaction-based nucleic acid assay. However, the application of FEN1 is hampered due to the lack of detailed protocols to express and purify the enzyme, and to quantify the enzyme activity. In this paper, the DNA fragment coding the gene of FEN1 from Archaeoglobus fulgidus was synthesized, and inserted into the plasmid of pET24a(+) to express recombinant FEN1 with His-tag. After optimizing the expression, detailed expression protocol of FEN1 was obtained by culturing the recombinant E. coli at 37 ℃ with 200 r/min of shaking for 8 h, followed by inducing with 0.05 mmol/L IPTG at 37 ℃ for 11 h. The purified recombinant FEN1 with the molecular mass of 38 kDa was obtained by Ni-affinity chromatography. Moreover, we developed a accurate quantification method with fluorescence-labelled probes. Finally, the recombinant FEN1 was used in real-time PCR coupled with high specific invader assay for aldh2 gene genotyping to obtain the correct typing results, indicating that the recombinant FEN1 can be used in gene polymorphism detection. We provide a reliable enzyme for developing invasive reaction-based nucleic acid assay.

Research progress in signal amplification-based methods for microRNA detection / 中华检验医学杂志

MicroRNA (miRNA) are a class of endogenous single -stranded non-coding RNA, which regulate gene expression post-transcriptionally by combining with the target mRNA and play a vital role in biological and pathological processes including the growth of organism , metabolic regulation, disease prediction and intervention.Thus miRNA detection is of considerable significance in disease diagnosis and the research of miRNA function.Because of the restriction factors about miRNA itself such as short sequence, low abundance and highly homologous , traditional methods for miRNA detection cannot meet the current demands due to the limitations like unsatisfactory sensitivity and complicated operation .This review summarizes the newly development about signal amplification -based methods for miRNA, including the advantages and limitations of all kinds of novel methods , and highlights the future trends as well.

Application Progress of Exonuclease-assisted Signal Amplification Strategies in Biochemical Analysis / 分析化学

As an important member of tool enzymes, exonuclease is a kind of hydrolytic enzymes without strict base sequence dependent. In recent years, by taking advantage of different hydrolysis ways of exonuclease and nanotechnology, cycle effect of enzyme digestion, aptamer, non Watson-Crick base pairing system by metal ions, fluorescent nucleic acid probes, electrochemical methods etc. , a series of exonuclease-assisted signal amplification strategies have been developed, which played a very key role in improving the sensitivity of detection methods. Therefore, exonuclease has been widely used in high sensitive detection of nucleic acids, proteins, ions, small molecules and so on. To understand it better and apply it well in the future, the application progress of exonuclease-assisted signal amplification strategies in biochemical analysis has been summarized in this review.

Distribution pattern of GPRC6A mRNA in mouse tissue by in situ hybridization / 中南大学学报(医学版)

Objective To explore the distribution pattern of G protein-coupled receptor family C, group 6, subtype A (GPRC6A) mRNA in adult mice. Methods The distribution of GPRC6A mRNA in paraffin embedded adult mouse tissues was determined by highly sensitive nonradioactive cRNA probe in situ hybridization (ISH). We compared ISH with and without addition of tyramide signal amplification (TSA). GPRC6A wild-type and littermate GPRC6A null mice tissue sections were investigated by ISH. Results TSA greatly increased the sensitivity of ISH to detect GPRC6A mRNA in wild type mouse tissues. There was no detection of GPRC6A mRNA in GPRC6A gene specific knockout tissue in paraffin embedded tissue section. The mRNA of GPRC6A was detectable in the digestive gland or accessory digestive gland including salivary gland and pancreas, as well as in the tissues including kidney, testis, brain, muscle, and fat. Conclusion The mRNA distribution pattern of GPRC6A gene is compatible with the phenotype of GPRC6A knockout mice.

A Novel Electrochemical Enzyme-linked Immunosensor Based on Tyramine Signal Amplification / 分析化学

A novel and highly sensitive voltammetric enzyme-linked immunosensor was developed based on tyramine) oxidation deposition. It was shown that gold nano-particles(colloid Au) could be used as a platform to immobilize antibodies by adsorption. By a sandwich immunossary format with goat-anti-human IgG labled Horseradish peroxidase(HRP) as the second antibody and catalytic amplification by biotin-tyramine, the immunosensor′s) catalytic ability to hydrogen peroxide increased nearly 20 times), the sensor exhibitd a linear response to human IgG in the concentration range from 1.5 μg/L-22 mg/L, and the detection limit was 0.1 μg/L), the regression equation could be expressed as Δi_p(μA) =2.8859c(mg/L)+17.152 with a correlation) coefficient of 0.9872. The immunosensor can be used to quantitatively determine hIgG in the sample) of human serum).

Modification of catalyzed signal amplification method / 第二军医大学学报

Objective: To verify whether hot PBS washing after the enzymatic reaction between tyramine and horseradish peroxidase can improve the sensitivity and specificity of catalyzed signal amplification (CSA). Methods: Using 7 different types of primary antibodies and 2 tumor microarray system, we carried out En Vision, LSAB, Standard CSA, and modified CSA staining and compared their sensitivities and specificities. Results: The modified CSA method was the most sensitive one among the 4 methods, followed by standard CSA, En Vision, and LsAB method. The sensitivity of modified CSA method was 2-3 times higher than that of standard CSA. The 4 methods had similar background staining and had exact localization of cells. Conclusion: The modified CSA is more stable and sensitive than traditional CSA staining method.

Multiply Labeled Primers Amplifying Fluorescent Signal on Oligonucleotide Microarray / 生物化学与生物物理进展

Oligonucleotide microarray technology is a powerful data-mining platform and has been widely applied in biosciences. To improve the performance of assays on the oligonucleotide microarray, the factors that influence the hybridization effects such as surface chemistry, probe size, spacer length, hybridization conditions etc were intensely studied and optimized. However, it is a key problem with DNA microarrays how to generate higher fluorescent signals to improve the detection sensitivity. Two types of multiply labeled primers, termed multiply labeled linear primer and multiply labeled branched primer, were used to enhance the fluorescent signal obtained from two-dimensional DNA microarrays.The signal was intensified by increasing the number of fluorophores labeled on the target DNA segment. It was indicated that the detection limit (minimum template amoumt for detection) of the multiply labeled primers is about 1% of that of the singly labeled primer. Multiple labeling is an effective signal amplification method to increase the detection sensitivity of the probes in a miniaturized array format.

Piezoelectric Gene Biosensors for Detection of Pseudomonas aeruginosa Using Signal Amplification Method with Cationic Conjugated Polymers / 中华医院感染学杂志

OBJECTIVE To discuss the feasibility of signal amplification method with cationic conjugated polymers(liposome) applied during the detection of Pseudomonas aeruginosa using piezoelectric gene biosensors.(METHODS) Oligonucleotide probe for P.aeruginosa was immobilized on the surface of gene sensor array and(hybridized) by PCR production of P.aeruginosa.After hybridization,liposome was added.The frequency shifts were recorded and compared with those ones of the control groups.RESULTS The frequency shifts were(significantly) increasing when adding liposome and the compatible concentration of liposome was 0.8?g/?l.(CONCLUSIONS) Liposome signal amplification is proved to be an effective method to amplify the piezoelectric(signal).

Modification of catalyzed signal amplification method / 第二军医大学学报

Objective:To verify whether hot PBS washing after the enzymatic reaction between tyramine and horseradish peroxidase can improve the sensitivity and specificity of catalyzed signal amplification (CSA). Methods: Using 7 different types of primary antibodies and 2 tumor microarray system, we carried out EnVision, LSAB, Standard CSA, and modified CSA staining and compared their sensitivities and specificities. Results: The modified CSA method was the most sensitive one among the 4 methods, followed by standard CSA, EnVision, and LsAB method. The sensitivity of modified CSA method was 2-3 times higher than that of standard CSA. The 4 methods had similar background staining and had exact localization of cells. Conclusion: The modified CSA is more stable and sensitive than traditional CSA staining method.