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1.
Talanta ; 233: 122600, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34215088

RESUMO

Selective and sensitive detection of microRNA is crucial for early diagnosis and pathogenesis of disease. Here, we established a novel electrochemical biosensor for simple and accurate analysis of the tumor biomarker microRNA-141, which was based on in-situ catalytic hairpin assembly (CHA) actuated DNA tetrahedral (DTN) interfacial probes. Two hairpin structures used for CHA reaction were placed on the DTN, in which the hairpin H1 on the one vertex of DTN and hairpin H2 embedded in adjacent edge, respective. The target microRNA-141 could open the hairpin H1 and activated the in-situ CHA reaction between H1 and H2 to alter the conformational of DTN, increasing the chances of the direct interaction between methylene blue (MB) and the electrode surface, leading to an increase in the electrochemical signal. Meanwhile, the released miRNA-141 could unfold another H1, enabling the enzyme-free recycling of the target to obtain amplified electrochemical signals. Moreover, the in-situ catalytic hairpin assembly reaction on DTN could shorten the reaction time and enhance the sensitivity. The established biosensor exhibited a wide linear dynamic range of miRNA-141 from 1 fM to 100 pM with a detection limit of 0.32 fM. Besides, the approach can discriminate the target miRNA from mismatched ones with excellent selectivity and can be successfully applied in diluted serum samples, holding great potential for sensitive detection of various biomarkers clinically.


Assuntos
Técnicas Biossensoriais , DNA Catalítico , MicroRNAs , Sondas de DNA/genética , Técnicas Eletroquímicas , Limite de Detecção , MicroRNAs/genética
2.
Carbohydr Polym ; 268: 118259, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34127229

RESUMO

Nitrocellulose (NC) membrane can have value-added applications for lateral flow assay (LFA)-based diagnostic tools, which has great potential for the detection of pathogens, such as COVID-19, in different environments. However, poor sensitivity of the NC membrane based LFA limits its further application in many cases. Herein, we developed a facile method for LFA sensitivity enhancement, by incorporating two-sugar barrier into LFAs: one between the conjugation pad and the test line, and the other between the test line and the control line. ORF1ab nucleic acid of COVID-19 was used as the model target to demonstrate the concept on the HF120 membrane. Results show that at optimum conditions, the two sugar barrier LFAs have a detection limit of 0.5 nM, which is compared to that of 2.5 nM for the control LFA, achieving a 5-fold sensitivity increase. This low cost, easy-to-fabricate and easy-to-integrate LFA method may have potential applications in other cellulose paper-based platforms.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Colódio/química , RNA Mensageiro/análise , Açúcares/química , Proteínas Virais/genética , Teste de Ácido Nucleico para COVID-19/instrumentação , DNA/química , Sondas de DNA/química , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Poliproteínas/genética , SARS-CoV-2/química , Sensibilidade e Especificidade
3.
Anal Chem ; 93(24): 8381-8385, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34100608

RESUMO

Single-nanoparticle inductively coupled plasma mass spectrometry (SP-ICP-MS) has demonstrated unique advantages for the detection of biological samples. However, methods for enzyme activity detection based on SP-ICP-MS technology have been rarely explored. Here we report the development of a novel SP-ICP-MS assay for uracil-DNA glycosylase (UDG) activity detection based on its ability to specifically recognize and remove uracil to induce the cleavage of the DNA probe. Our design allows the generation of single gold nanoparticles correlated to the specific enzymatic reaction for a highly sensitive SP-ICP-MS measurement. The developed assay enables sensitive UDG activity detection with a detection limit of 0.0003 U/mL. The cell lysate analysis by the developed assay reveals its applicability for the detection of UDG activity in real samples. It is envisioned that our design may provide a new paradigm for developing the SP-ICP-MS assay for enzyme activity detection in biological samples.


Assuntos
Nanopartículas Metálicas , Uracila-DNA Glicosidase , Sondas de DNA , Ouro , Limite de Detecção
4.
Anal Chem ; 93(24): 8414-8422, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34114453

RESUMO

Sensitive, accurate, and nondestructive probing of endogenous messenger RNA (mRNA) in living cells places extremely high demands on nanocarriers and probes and is still a challenge. In the present study, we describe a target-triggered self-assembled DNA tree for amplified analysis of mRNA in intact living cells. The probes assembled into a DNA tree are transported into cells by exosomes, which is beneficial for reducing cell damage and realizing nondestructive analysis. The probes are l-configured single-stranded DNAs (LDNAs) that can resist the degradation of exonuclease and endonuclease, thus laying the foundation for accurate analysis. Under the induction of the target mRNA, the probes in the cells assemble into a small plantlet and eventually grow into a tree after a few rounds of self-cycling, achieving the exponential amplification of fluorescence signals. Compared with the signal amplification based on one-dimensional DNA trunk self-assembly, the three-dimensional DNA tree shows an excellent sensitivity both ex situ and in situ. In this way, favorable sensitivity, accuracy, and nondestructive analysis are integrated into one system. This DNA tree expands the analysis platform for analyzing more biomarkers on a genetic level in an intracellular, nondestructive, and hypersensitive manner and holds great potential in clinical diagnostic and research applications.


Assuntos
Exossomos , DNA/genética , Sondas de DNA , DNA de Cadeia Simples , Exossomos/genética , RNA Mensageiro/genética , Árvores
5.
Carbohydr Polym ; 268: 118259, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: covidwho-1242891

RESUMO

Nitrocellulose (NC) membrane can have value-added applications for lateral flow assay (LFA)-based diagnostic tools, which has great potential for the detection of pathogens, such as COVID-19, in different environments. However, poor sensitivity of the NC membrane based LFA limits its further application in many cases. Herein, we developed a facile method for LFA sensitivity enhancement, by incorporating two-sugar barrier into LFAs: one between the conjugation pad and the test line, and the other between the test line and the control line. ORF1ab nucleic acid of COVID-19 was used as the model target to demonstrate the concept on the HF120 membrane. Results show that at optimum conditions, the two sugar barrier LFAs have a detection limit of 0.5 nM, which is compared to that of 2.5 nM for the control LFA, achieving a 5-fold sensitivity increase. This low cost, easy-to-fabricate and easy-to-integrate LFA method may have potential applications in other cellulose paper-based platforms.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Colódio/química , RNA Mensageiro/análise , Açúcares/química , Proteínas Virais/genética , Teste de Ácido Nucleico para COVID-19/instrumentação , DNA/química , Sondas de DNA/química , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Poliproteínas/genética , SARS-CoV-2/química , Sensibilidade e Especificidade
6.
Analyst ; 146(13): 4340-4347, 2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: covidwho-1262015

RESUMO

Sensitive, reliable and cost-effective detection of pathogens has wide ranging applications in clinical diagnostics and therapeutics, water and food safety, environmental monitoring, biosafety and epidemiology. Nucleic acid amplification tests (NAATs) such as PCR and isothermal amplification methods provide excellent analytical performance and significantly faster turnaround times than conventional culture-based methods. However, the inherent cost and complexity of NAATs limit their application in resource-limited settings and the developing world. To help address this urgent need, we have developed a sensitive method for nucleic acid analysis based on padlock probe rolling circle amplification (PLRCA), nuclease protection (NP) and lateral flow detection (LFA), referred to as PLAN-LFA, that can be used in resource-limited settings. The assay involves solution-phase hybridization of a padlock probe to target, sequence-specific ligation of the probe to form a circular template that undergoes isothermal rolling circle amplification in the presence of a polymerase and a labeled probe DNA. The RCA product is a long, linear concatenated single-stranded DNA that contains binding sites for the labeled probe. The sample is then exposed to a nuclease which selectively cleaves single-stranded DNA, the double-stranded labeled probe is protected from nuclease digestion and detected in a lateral flow immunoassay format to provide a visual, colorimetric readout of results. We have developed specific assays targeting beta-lactamase resistance gene for monitoring of antimicrobial resistance and Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2, the novel coronavirus discovered in 2019) using the PLAN-LFA platform. The assay provides a limit of detection of 1.1 pM target DNA (or 1.3 × 106 copies per reaction). We also demonstrate the versatility and robustness of the method by performing analysis on DNA and RNA targets, and perform analysis in complex sample matrices like saliva, plant tissue extract and bacterial culture without any sample pretreatment steps.


Assuntos
COVID-19 , SARS-CoV-2 , Sondas de DNA , Humanos , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico
7.
Talanta ; 232: 122429, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34074415

RESUMO

DNA glycosylase is an indispensable DNA damage repair enzyme which can recognize and excise the damaged bases in the DNA base excision-repair pathway. The dysregulation of DNA glycosylase activity will give rise to the dysfunction of base excision-repair and lead to abnormalities and diseases. The simultaneous detection of multiple DNA glycosylases can help to fully understand the normal physiological functions of cells, and determine whether the cells are abnormal in pre-disease. Regrettably, the synchronous detection of functionally similar DNA glycosylases is a great challenge. Herein, we developed a multifunctional dsDNA probe mediated exponential rolling circle amplification (E-RCA) method for the simultaneously sensitive detection of human alkyladenine DNA glycosylase (hAAG) and uracil-DNA glycosylase (UDG). The multifunctional dsDNA probe contains the hypoxanthine sites and the uracil sites which can be recognized by hAAG and UDG respectively to generate apyrimidinic (AP) sites in the dsDNA probe. Then the AP sites will be recognized and cut by endonuclease Ⅳ (Endo IV) to release corresponding single-stranded primer probes. Subsequently, two padlock DNA templates are added to initiate E-RCA to generate multitudinous G-quadruplexes and/or double-stranded dumbbell lock structures, which can combine N-methyl mesoporphyrin IX (NMM) and SYBR Green Ⅰ (SGI) for the generation of respective fluorescent signals. The detection limits are obtained as low as 0.0002 U mL-1 and 0.00001 U mL-1 for hAAG and UDG, respectively. Notably, this method can realize the simultaneous detection of two DNA glycosylases without the use of specially labeled probes. Finally, this method is successfully applied to detect hAAG and UDG activities in the lysates of HeLa cells and Endo1617 cells at single-cell level, and to detect the inhibitors of DNA glycosylases.


Assuntos
DNA Glicosilases , Técnicas de Amplificação de Ácido Nucleico , Uracila-DNA Glicosidase , Sondas de DNA , Reparo do DNA , Células HeLa , Humanos , Limite de Detecção , Uracila-DNA Glicosidase/metabolismo
8.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070753

RESUMO

In situ imaging of molecular markers on a physical chromosome is an indispensable tool for refining genetic maps and validation genome assembly at the chromosomal level. Despite the tremendous progress in genome sequencing, the plant genome assembly at the chromosome level remains a challenge. Recently developed optical and Hi-C mapping are aimed at assistance in genome assembly. For high confidence in the genome assembly at chromosome level, more independent approaches are required. The present study is aimed at refining an ultrasensitive Tyr-FISH technique and developing a reliable and simple method of in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH to find out the reasons behind the flaws of this technique. The accurate visualization of markers/genes appeared to be significantly dependent on the means of chromosome slide preparation, probe design and labeling, and high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.6 Kb with a frequency of 51.6%. Based on our results, we developed a more reliable and simple protocol for dual-color Tyr-FISH visualization of unique short DNA sequences on plant chromosomes. This new protocol can allow for more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.


Assuntos
Mapeamento Cromossômico/métodos , Cromossomos de Plantas/química , Genoma de Planta , Hibridização in Situ Fluorescente , Cebolas/genética , Coloração e Rotulagem/métodos , Cromossomos de Plantas/metabolismo , Sondas de DNA/síntese química , Sondas de DNA/metabolismo , DNA de Plantas/genética , DNA de Plantas/metabolismo , Ligação Genética , Marcadores Genéticos , Cebolas/metabolismo , Transcriptoma
9.
Analyst ; 146(13): 4226-4234, 2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34095908

RESUMO

DNA hybridization phenomena occurring on solid supports are not understood as clearly as aqueous phase hybridizations and mathematical models cannot predict some empirically obtained results. Ongoing research has identified important parameters but remains incomplete to accurately account for all interactions. It has previously been shown that the length of the overhanging (dangling) end of the target DNA strand following hybridization to the capture probe is correlated to interactions with the complementary strand in solution which can result in unbinding of the target and its release from the surface. We have developed an instrument for real-time monitoring of DNA hybridization on spherical particles functionalized with oligonucleotide capture probes and arranged in the form of a tightly packed monolayer bead bed inside a microfluidic cartridge. The instrument is equipped with a pneumatic module to mediate displacement of fluid on the cartridge. We compared this system to both conventional (passive) and centrifugally-driven (active) microfluidic microarray hybridization on glass slides to establish performance levels for the detection of single nucleotide polymorphisms. The system was also used to study the effect of the dangling end's length in real-time when the immobilized target DNA is exposed to the complementary strand in solution. Our findings indicate that increasing the length of the dangling end leads to desorption of target amplicons from bead-bound capture probes at a rate approaching that of the initial hybridization process. Finally, bead bed hybridization was performed with Streptococcus agalactiae cfb gene amplicons obtained from randomized clinical samples, which allowed for identification of group B streptococci within 5-15 min. The methodology presented here is useful for investigating competitive hybridization mechanisms on solid supports and to rapidly validate the suitability of microarray capture probes.


Assuntos
DNA , Microfluídica , DNA/genética , Sondas de DNA/genética , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos/genética
10.
Analyst ; 146(13): 4340-4347, 2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34106115

RESUMO

Sensitive, reliable and cost-effective detection of pathogens has wide ranging applications in clinical diagnostics and therapeutics, water and food safety, environmental monitoring, biosafety and epidemiology. Nucleic acid amplification tests (NAATs) such as PCR and isothermal amplification methods provide excellent analytical performance and significantly faster turnaround times than conventional culture-based methods. However, the inherent cost and complexity of NAATs limit their application in resource-limited settings and the developing world. To help address this urgent need, we have developed a sensitive method for nucleic acid analysis based on padlock probe rolling circle amplification (PLRCA), nuclease protection (NP) and lateral flow detection (LFA), referred to as PLAN-LFA, that can be used in resource-limited settings. The assay involves solution-phase hybridization of a padlock probe to target, sequence-specific ligation of the probe to form a circular template that undergoes isothermal rolling circle amplification in the presence of a polymerase and a labeled probe DNA. The RCA product is a long, linear concatenated single-stranded DNA that contains binding sites for the labeled probe. The sample is then exposed to a nuclease which selectively cleaves single-stranded DNA, the double-stranded labeled probe is protected from nuclease digestion and detected in a lateral flow immunoassay format to provide a visual, colorimetric readout of results. We have developed specific assays targeting beta-lactamase resistance gene for monitoring of antimicrobial resistance and Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2, the novel coronavirus discovered in 2019) using the PLAN-LFA platform. The assay provides a limit of detection of 1.1 pM target DNA (or 1.3 × 106 copies per reaction). We also demonstrate the versatility and robustness of the method by performing analysis on DNA and RNA targets, and perform analysis in complex sample matrices like saliva, plant tissue extract and bacterial culture without any sample pretreatment steps.


Assuntos
COVID-19 , SARS-CoV-2 , Sondas de DNA , Humanos , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico
11.
Anal Chem ; 93(22): 8084-8090, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34034482

RESUMO

The simultaneous sensing of endogenous wild and mutant proteins plays a critical role in disease diagnosis and drug screening, and this remains a major current challenge. Here, we present a new and highly specific target-triggered dual proximity ligation assay (dPLA) strategy for sensitive and simultaneous sensing of wild and mutant p53 proteins from cancer cells. Two proximity DNA probes bind the target protein to form the primer/circular DNA template complexes with two nicks in the presence of the hairpin and ssDNA connector sequences via the strand displacement reaction. Only when the two nicks are simultaneously ligated can the rolling circle amplification be triggered with high fidelity for yielding substantially enhanced fluorescence. By encoding the hairpin sequence, two distinct fluorescence signals can be generated for simultaneous detection of the wild and mutant p53 proteins. Importantly, our method significantly reduces the possibility of nonspecific ligation reactions by using two ligation nicks, which minimizes the background noise. With this dPLA method, the regulation transition of intracellular mutant p53 to wild p53 proteins upon anticancer drug treatment has also been demonstrated, highlighting its usefulness for potential early disease diagnosis and drug screening with high fidelity.


Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Sondas de DNA , Avaliação Pré-Clínica de Medicamentos , Detecção Precoce de Câncer , Humanos , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Neoplasias/genética , Técnicas de Amplificação de Ácido Nucleico , Proteína Supressora de Tumor p53/genética
12.
Analyst ; 146(9): 3034-3040, 2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-33949439

RESUMO

Monitoring ctDNA in blood is important for cancer management. Here, a one-step single particle counting approach was developed for directly quantifying ctDNA in plasma. Hairpin DNA containing a triple helix stem was immobilized onto QD 585 as a probe. The hairpin was opened by the target, and therefore hybridized with assistant DNA on QD 655, resulting in an aggregate of QD 585 and QD 655. The two-color QD aggregate was regarded as the target. Observed under a single particle transmission grating-based spectral microscope, the two-color QD aggregate was distinguished by a unique spectral pattern of two first-order streaks, and it was counted. The difference in the responses of the probes to perfect-match DNA, single-base mismatch DNA, and non-match DNA indicated that the probe had sufficient single-base discrimination capabilities. The success in plasma recovery tests demonstrated the feasibility of carrying out the direct detection of ctDNA in plasma.


Assuntos
Técnicas Biossensoriais , DNA Tumoral Circulante , Pontos Quânticos , DNA Tumoral Circulante/genética , DNA/genética , Sondas de DNA
13.
Anal Chim Acta ; 1166: 338545, 2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34023002

RESUMO

Single nucleotide variants (SNVs) have emerged as increasingly important biomarkers, particularly in the diagnosis and prognosis of cancers. However, most SNVs are rarely detected in blood samples from cancer patients as they are surrounded by abundant concomitant wild-type nucleic acids. Herein, we design a system that features a combination of competitive DNA probe system (CDPS) and duplex-specific nuclease (DSN) that we referred to as CAD. A theoretical model was established for the CAD system based on reaction networks. Guided by the theoretical model, we found that a minor loss in sensitivity significantly improved the specificity of the system, thus creating a theoretical discrimination factor (DF) > 100 for most conditions. This non-equivalent tradeoff between sensitivity and specificity provides a new concept for the analysis of rare DNA-sequence variants. As a demonstration of practicality, we applied as-proposed CAD system to identify low variant allele frequency (VAF) in a synthetic template (0.1% VAF) and human genomic DNA (1% VAF). This work promises complete guidance for the design of enzyme-based nucleic acid analysis.


Assuntos
DNA , Ácidos Nucleicos , DNA/genética , Sondas de DNA/genética , Endonucleases , Humanos , Nucleotídeos/genética , Polimorfismo de Nucleotídeo Único
14.
Sci Rep ; 11(1): 8988, 2021 04 26.
Artigo em Inglês | MEDLINE | ID: covidwho-1203449

RESUMO

Rapid tests for active SARS-CoV-2 infections rely on reverse transcription polymerase chain reaction (RT-PCR). RT-PCR uses reverse transcription of RNA into complementary DNA (cDNA) and amplification of specific DNA (primer and probe) targets using polymerase chain reaction (PCR). The technology makes rapid and specific identification of the virus possible based on sequence homology of nucleic acid sequence and is much faster than tissue culture or animal cell models. However the technique can lose sensitivity over time as the virus evolves and the target sequences diverge from the selective primer sequences. Different primer sequences have been adopted in different geographic regions. As we rely on these existing RT-PCR primers to track and manage the spread of the Coronavirus, it is imperative to understand how SARS-CoV-2 mutations, over time and geographically, diverge from existing primers used today. In this study, we analyze the performance of the SARS-CoV-2 primers in use today by measuring the number of mismatches between primer sequence and genome targets over time and spatially. We find that there is a growing number of mismatches, an increase by 2% per month, as well as a high specificity of virus based on geographic location.


Assuntos
Primers do DNA/genética , Sondas de DNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , Genoma Viral , Mutação
15.
ACS Nano ; 15(5): 8474-8483, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-33914524

RESUMO

The rapid and reliable recognition of nucleic acid sequences is essential to a broad range of fields including genotyping, gene expression analysis, and pathogen screening. For viral detection in particular, the capability is critical for optimal therapeutic response and preventing disease transmission. Here, we report an approach for detecting identifying sequence motifs within genome-scale single-strand DNA and RNA based on solid-state nanopores. By designing DNA oligonucleotide probes with complementarity to target sequences within a target genome, we establish a protocol to yield affinity-tagged duplex molecules the same length as the probe only if the target is present. The product can subsequently be bound to a protein chaperone and analyzed quantitatively with a selective solid-state nanopore assay. We first use a model DNA genome (M13mp18) to validate the approach, showing the successful isolation and detection of multiple target sequences simultaneously. We then demonstrate the protocol for the detection of RNA viruses by identifying and targeting a highly conserved sequence within human immunodeficiency virus (HIV-1B).


Assuntos
Nanoporos , Ácidos Nucleicos , Sequência Conservada , DNA , Sondas de DNA , Humanos
16.
Analyst ; 146(11): 3526-3533, 2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-33881427

RESUMO

Since the application of RNA interference (RNAi) is rapidly developing in GMO technology, accurate and sensitive detection of functional RNA molecules was urgently needed, for the safety and functional assessment of RNAi crops. In this work, we developed an electrochemical biosensor for transgene-derived long RNA based on a poly-adenine (polyA) DNA capture probe. The polyA self-assembling monolayer (SAM) provided enhanced interface stability and optimized surface density for the subsequent hybridization of the long RNA molecule. A multiple reporter probe system (MRP) containing 12 reporter probes (RPs) and 2 spacers was applied to open the complex molecular secondary structure and hybridize with the long RNA, with the critical assistance of dimethyl sulfoxide (DMSO). By using 3 addressable RPs, structural recognition was performed among long stem-loop RNA, long dsRNA (no loop), and siRNA. Excellent selectivity was achieved when the extracted total RNA samples were directly analyzed. When reverse transcription recombinase polymerase amplification (RT-RPA) technology was combined, the sensitivity was improved to 10 aM. To the best of our knowledge, this is the first electrochemical biosensor with the excellent capability of quantification and structural analysis of the long RNA of the RNAi GMO. Our work shows great potential in a wide range of RNAi GMO samples.


Assuntos
Técnicas Biossensoriais , Zea mays , Sondas de DNA , Técnicas Eletroquímicas , Poli A , Interferência de RNA , Zea mays/genética
17.
Anal Chem ; 93(14): 5917-5923, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33798333

RESUMO

DNA self-assembly has created various nanostructured probes deployed in biosensors, whereas their direct charge contribution to sensitive bioassays remains elusive. Here, we report a supercharged tetrahedral DNA nanolabel-based electrochemical (eTDN) sensor for ultrasensitive detection of exosomal microRNAs (Exo-miRs). By using an "assembly before testing" strategy, there is high-efficiency recognition between the target Exo-miR and self-assembled TDN probe in a homogenous solution relative to surface-based hybridization. The TDN-miR complex can be further bridged specifically to form a stable sandwich construct with the surface-confined capture sequence via the base-stacking effect. The intrinsic supercharges of the TDN can adsorb numerous electroactive molecules in stoichiometry, which largely enhances the detection sensitivity, particularly by using electroneutral peptide nucleic acid instead of DNA probes to minimize the background signal. Using this approach, the eTDN sensor achieves a high sensitivity (34 aM), high specificity (against the single mismatch), and high selectivity (in serum). Furthermore, this ultrasensitive sensor provides a conjugation-free, non-enzymatic Exo-miR detection in blood and accurately distinguishes the breast cancer patients from normal individuals, showing to be a promising tool in the early diagnosis of malignant tumors.


Assuntos
Técnicas Biossensoriais , MicroRNAs , DNA/genética , Sondas de DNA/genética , Técnicas Eletroquímicas , Humanos , Limite de Detecção , MicroRNAs/genética , Hibridização de Ácido Nucleico
18.
Cytogenet Genome Res ; 161(1-2): 52-62, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33887732

RESUMO

With 82 species currently described, the genus Leptodactylus is the most diverse and representative one in the family Leptodactylidae. Concerning chromosomal organization, this genus represents an interesting and underexplored group since data from molecular cytogenetics are incipient, and little is known about the organization and distribution of repetitive DNA elements in the karyotypes. In this sense, this study aimed at providing a comparative analysis in 4 Leptodactylus species (L. macrosternum, L. pentadactylus, L. fuscus, and Leptodactylus cf. podicipinus), combining conventional cytogenetics (Giemsa staining, C-banding, and AgNOR staining) and mapping of molecular markers (18S rDNA, telomeric and microsatellite probes), to investigate mechanisms underlying their karyotype differentiation process. The results showed that all species had karyotypes with 2n = 22 and FN = 44, except for Leptodactylus cf. podicipinus which presented FN = 36. The 18S rDNA was observed in pair 8 of all analyzed species (corresponding to pair 4 in L. pentadactylus), coinciding with the secondary constrictions and AgNOR staining. FISH with microsatellite DNA probes demonstrated species-specific patterns, as well as an association of these repetitive sequences with constitutive heterochromatin blocks and ribosomal DNA clusters, revealing the dynamics of microsatellites in the genome of the analyzed species. In summary, our data demonstrate an ongoing process of genomic divergence inside species with almost similar karyotype, driven most likely by a series of pericentric inversions, followed by differential accumulation of repetitive sequences.


Assuntos
Anuros/genética , Cromossomos/ultraestrutura , DNA Ribossômico/genética , Cariotipagem , Repetições de Microssatélites , Animais , Bandeamento Cromossômico , Inversão Cromossômica , Análise Citogenética , Citogenética , Sondas de DNA , Feminino , Geografia , Heterocromatina/metabolismo , Hibridização in Situ Fluorescente , Cariótipo , Masculino , Meiose , Mitose , Região Organizadora do Nucléolo , Filogenia , Especificidade da Espécie
19.
Methods Mol Biol ; 2300: 41-58, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33792870

RESUMO

Successful detection of very small RNAs (tiny RNAs, ~8-15 nt in length) by northern blotting depends on tailored protocols with respect to transfer and immobilization on membranes as well as design of sensitive detection probes. For RNA crosslinking to positively charged membranes, we compared UV light with chemical RNA crosslinking by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), using either denaturing or native polyacrylamide gels. We show that northern blot detection of tiny RNAs with 5'-digoxigenin-labeled DNA/LNA mixmer probes is a highly sensitive and specific method and, in our hands, more sensitive than using a corresponding DNA/LNA mixmer probe with a 5'-32P-end-label. Furthermore, we provide a robust protocol for northern blot analysis of noncoding RNAs of intermediate size (~50-400 nt).


Assuntos
Reagentes para Ligações Cruzadas/química , Sondas de DNA/metabolismo , Etildimetilaminopropil Carbodi-Imida/química , RNA/análise , Northern Blotting , Sondas de DNA/química , Eletroforese em Gel de Gradiente Desnaturante , Digoxigenina/química , Eletroforese em Gel de Poliacrilamida Nativa , RNA/química
20.
Sensors (Basel) ; 21(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810389

RESUMO

The pH drop in the hindgut of the horse is caused by lactic acid-producing bacteria which are abundant when a horse's feeding regime is excessively carbohydrate rich. This drop in pH below six causes hindgut acidosis and may lead to laminitis. Lactic acid-producing bacteria Streptococcus equinus and Mitsuokella jalaludinii have been found to produce high amounts of L-lactate and D-lactate, respectively. Early detection of increased levels of these bacteria could allow the horse owner to tailor the horse's diet to avoid hindgut acidosis and subsequent laminitis. Therefore, 16s ribosomal ribonucleic acid (rRNA) sequences were identified and modified to obtain target single stranded deoxyribonucleic acid (DNA) from these bacteria. Complementary single stranded DNAs were designed from the modified target sequences to form capture probes. Binding between capture probe and target single stranded deoxyribonucleic acid (ssDNA) in solution has been studied by gel electrophoresis. Among pairs of different capture probes and target single stranded DNA, hybridization of Streptococcus equinus capture probe 1 (SECP1) and Streptococcus equinus target 1 (SET1) was portrayed as gel electrophoresis. Adsorptive stripping voltammetry was utilized to study the binding of thiol modified SECP1 over gold on glass substrates and these studies showed a consistent binding signal of thiol modified SECP1 and their hybridization with SET1 over the gold working electrode. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to examine the binding of thiol modified SECP1 on the gold working electrode and hybridization of thiol modified SECP1 with the target single stranded DNA. Both demonstrated the gold working electrode surface was modified with a capture probe layer and hybridization of the thiol bound ssDNA probe with target DNA was indicated. Therefore, the proposed electrochemical biosensor has the potential to be used for the detection of the non-synthetic bacterial DNA target responsible for equine hindgut acidosis.


Assuntos
Acidose , Técnicas Biossensoriais , Animais , DNA , Sondas de DNA , Técnicas Eletroquímicas , Eletrodos , Firmicutes , Ouro , Cavalos , Hibridização de Ácido Nucleico , Streptococcus bovis
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