RESUMEN
Acoustofluidic technologies that integrate acoustic waves and microfluidic chips have been widely used in bioparticle manipulation. As a representative technology, acoustic tweezers have attracted significant attention due to their simple manufacturing, contact-free operation, and low energy consumption. Recently, acoustic tweezers have enabled the efficient and smart manipulation of biotargets with sizes covering millimeters (such as zebrafish) and nanometers (such as DNA). In addition to acoustic tweezers, other related acoustofluidic chips including acoustic separating, mixing, enriching, and transporting chips, have also emerged to be powerful platforms to manipulate micro/nano bioparticles (cells in blood, extracellular vesicles, liposomes, and so on). Accordingly, some interesting applications were also developed, such as smart sensing. In this review, we firstly introduce the principles of acoustic tweezers and various related technologies. Second, we compare and summarize recent applications of acoustofluidics in bioparticle manipulation and sensing. Finally, we outlook the future development direction from the perspectives such as device design and interdisciplinary.
RESUMEN
Excavating nucleic acid quantitative capabilities by combining clustered regularly interspaced short palindromic repeats (CRISPR) and isothermal amplification in one pot is of common interest. However, the mutual interference between CRISPR cleavage and isothermal amplification is the primary obstacle to quantitative detection. Though several works have demonstrated enhanced detection sensitivity by reducing the inhibition of CRISPR on amplification in one pot, few paid attention to the amplification process and even dynamic reaction processes between the two. Herein, we find that DNA quantification can be realized by regulating either recombinase polymerase amplification (RPA) efficiency or CRISPR/Cas12a cleaving efficiency (namely, tuning the dynamic reaction balance) in one pot. The sensitive quantification is realized by utilizing dual PAM-free crRNAs for CRISPR/Cas12a recognition. The varied RPA primer concentration with stabilized CRISPR systems significantly affects the amplification efficiency and quantitative performances. Alternatively, quantitative detection can also be achieved by stabilizing the amplification process while regulating the CRISPR/Cas12a concentration. The quantitative capability is proved by detecting DNA targets from Lactobacillus acetotolerans and SARS-CoV-2. The quantitative performance toward real samples is comparable to quantitative real-time PCR for detecting L. acetotolerans spiked in fermented food samples and SARS-CoV-2 clinical samples. We expect that the presented method will be a powerful tool for quantifying other nucleic acid targets.
Asunto(s)
Sistemas CRISPR-Cas , Técnicas de Amplificación de Ácido Nucleico , SARS-CoV-2 , Sistemas CRISPR-Cas/genética , SARS-CoV-2/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , COVID-19/diagnóstico , COVID-19/virología , Lactobacillus/genética , Humanos , Proteínas Asociadas a CRISPR/genética , Recombinasas/metabolismo , Endodesoxirribonucleasas/genética , Proteínas BacterianasRESUMEN
IMPORTANCE: Contact tracing is the process of identifying people who have recently been in contact with someone diagnosed with an infectious disease. During an outbreak, data collected from contact tracing can inform interventions to reduce the spread of infectious diseases. Understanding factors associated with completion rates of contact tracing surveys can help design improved interview protocols for ongoing and future programs. OBJECTIVE: To identify factors associated with completion rates of COVID-19 contact tracing surveys in New York City (NYC) and evaluate the utility of a predictive model to improve completion rates, we analyze laboratory-confirmed and probable COVID-19 cases and their self-reported contacts in NYC from October 1st 2020 to May 10th 2021. METHODS: We analyzed 742,807 case investigation calls made during the study period. Using a log-binomial regression model, we examined the impact of age, time of day of phone call, and zip code-level demographic and socioeconomic factors on interview completion rates. We further developed a random forest model to predict the best phone call time and performed a counterfactual analysis to evaluate the change of completion rates if the predicative model were used. RESULTS: The percentage of contact tracing surveys that were completed was 79.4%, with substantial variations across ZIP code areas. Using a log-binomial regression model, we found that the age of index case (an individual who has tested positive through PCR or antigen testing and is thus subjected to a case investigation) had a significant effect on the completion of case investigation - compared with young adults (the reference group,24 years old < age < = 65 years old), the completion rate for seniors (age > 65 years old) were lower by 12.1% (95%CI: 11.1% - 13.3%), and the completion rate for youth group (age < = 24 years old) were lower by 1.6% (95%CI: 0.6% -2.6%). In addition, phone calls made from 6 to 9 pm had a 4.1% (95% CI: 1.8% - 6.3%) higher completion rate compared with the reference group of phone calls attempted from 12 and 3 pm. We further used a random forest algorithm to assess its potential utility for selecting the time of day of phone call. In counterfactual simulations, the overall completion rate in NYC was marginally improved by 1.2%; however, certain ZIP code areas had improvements up to 7.8%. CONCLUSION: These findings suggest that age and time of day of phone call were associated with completion rates of case investigations. It is possible to develop predictive models to estimate better phone call time for improving completion rates in certain communities.
Asunto(s)
COVID-19 , Adolescente , Adulto Joven , Humanos , Adulto , Anciano , COVID-19/epidemiología , Trazado de Contacto/métodos , Ciudad de Nueva York/epidemiología , Encuestas y Cuestionarios , Brotes de EnfermedadesRESUMEN
Nucleic acid detection of pathogens in a point-of-need (PON) manner is of great significance yet remains challenging for sensitive and accurate visual discrimination. Here, we report a CRISPR-Cas12a-mediated lateral flow assay for PON detection of Salmonella typhimurium (S.ty) that is a prevailing pathogen disseminated through tainted food. The variation of the fluorescence color of the test line is exploited to interpret the results, enabling the discrimination between positive and negative samples on the basis of a hue-recognition mechanism. By leveraging the cleavage activity of Cas12a and hue-recognition readout, the assay facilitated by recombinase polymerase amplification can yield a visual detection limit of 1 copy µL-1 for S.ty genomic DNA within 1 h. The assay also displays a high specificity toward S.ty in fresh chicken samples, as well as a sensitivity 10-fold better than that of the commercial test strip. Moreover, a semiquantitative detection of S.ty ranging from 0 to 4 × 103 CFU/mL by the naked eye is made possible, thanks to the easily discernible color change of the test line. This approach provides an easy, rapid, accurate, and user-friendly solution for the PON detection of Salmonella and other pathogens.
Asunto(s)
Sistemas CRISPR-Cas , Salmonella typhimurium , Animales , Sistemas CRISPR-Cas/genética , Salmonella typhimurium/genética , Bioensayo , Pollos , Alimentos , Técnicas de Amplificación de Ácido NucleicoRESUMEN
Salmonella is one of four key global causes of diarrhea, and in humans, it is generally contracted through the consumption of contaminated food. It is necessary to develop an accurate, simple, and rapid method to monitor Salmonella in the early phase. Herein, we developed a sequence-specific visualization method based on loop-mediated isothermal amplification (LAMP) for the detection of Salmonella in milk. With restriction endonuclease and nicking endonuclease, amplicons were produced into single-stranded triggers, which further promoted the generation of a G-quadruplex by a DNA machine. The G-quadruplex DNAzyme possesses peroxidase-like activity and catalyzes the color development of 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) as the readouts. The feasibility for real samples analysis was also confirmed with Salmonella spiked milk, and the sensitivity was 800 CFU/mL when observed with the naked eye. Using this method, the detection of Salmonella in milk can be completed within 1.5 h. Without the involvement of any sophisticated instrument, this specific colorimetric method can be a useful tool in resource-limited areas.
Asunto(s)
ADN Catalítico , G-Cuádruplex , Humanos , ADN Catalítico/genética , ADN , Salmonella/genética , Técnicas de Amplificación de Ácido Nucleico/métodosRESUMEN
A novel covalent organic framework (COF) (Tp-BI-COF) with combined ketimine-type enol-imine and keto-enamine linkages was prepared through a cascade of ketimine condensation followed by aldimine condensation and characterized by XRD, solid state 13C NMR, IR, TGA and BET. Tp-BI-COF showed high stability toward acid, organic solvent, and boiling water. The 2D COF exhibited photochromic properties after being irradiated with a xenon lamp. The stable COF, with aligned one-dimensional nanochannels, provided nitrogen sites on pore walls, which confine and stabilize the H3PO4 in the channel via hydrogen-bonding interactions. After loading with H3PO4, the material showed excellent anhydrous proton conductivity.
RESUMEN
Circulating tumor cells (CTCs), as a type of tumor, have attracted wide attention because of their characteristics of shedding from the primary tumor and spreading to other tissues and organs through peripheral blood. The circulating tumor DNA (ctDNA), the DNA released by CTCs and other tumor cells into the peripheral blood, was considered as a promising detection substance for clinical application. By utilizing the biocompatibility of red blood cells to realize the attachment of tetrahedral DNA (TDN), as well as the specific target recognition ability of TDN to enable efficient recognition of targets, a biocompatible electrochemical biosensor for effective and rapid detection of ctDNA was developed using methylene blue (MB) as the signal probe. The current signal and the logarithm of ctDNA concentration were linearly correlated in the range from 1 fM to 100 pM with the detection limit of 0.66 fM. With high specificity, the TDN-based biosensor can detect ctDNA efficiently in the real biological environment such as serum, which provided a potential opportunity for the early clinical diagnosis.
Asunto(s)
Técnicas Biosensibles , ADN Tumoral Circulante , Nanoestructuras , Células Neoplásicas Circulantes , ADN/química , Técnicas Electroquímicas , Eritrocitos , Humanos , Límite de Detección , Azul de Metileno , Nanoestructuras/químicaRESUMEN
The development of highly specific biomimetic recognition material is a challenge for rapid detection of harmful residues in foodstuff. In this study, a paper-based boronate affinity metal-organic framework/molecularly imprinted polymer microfluidic chip (FZS-BA@MIP) was constructed based on the in situ construction strategy, which was also designed as a highly specific biomimetic recognition module. Here, the homogeneous zeolitic imidazole framework-8 (ZIF-8) membrane served as a great scaffold and enrichment layer. Besides, the recognition layer of MIP was prepared based on a highly oriented boronate affinity surface imprinting strategy. With the aid of the liquid flow channel, the highly specific enrichment and visual detection for antibiotic residues like kanamycin in actual products were achieved on the paper chip module of an integrated lateral flow platform. The whole analysis process could be accomplished within 30 min. In brief, this study offered a new integrated biomimetic recognition platform for visually detecting harmful veterinary residues containing cis-diols, which demonstrated promising commercial value in point-of-care testing of foodborne hazardous compounds.
Asunto(s)
Materiales Biomiméticos , Impresión Molecular , Biomimética , Kanamicina , Materiales Biomiméticos/químicaRESUMEN
Antibiotics are widely used for improving the living conditions of livestock. However, residual antibiotics present in animal products induce several human diseases. Therefore, a simple, rapid, and cost-effective system for detecting and monitoring the presence of antibiotics in foods is in great demand to alleviate safety concerns. In this study, a highly sensitive and selective aptameric electrochemical sensing platform was designed based on nanomaterial modification and DNA nanotechnology. Electrochemically reduced graphene oxide and gold nanoparticles were used to modify the working surface of a screen-printed electrode to enhance electrical conductivity and biocompatibility. The electrode surface was further modified with self-assembled tetrahedral DNA nanostructures (TDN) to improve the detection sensitivity. The TDN allowed controlling the nano-spacing of aptamers immobilized on the electrode surface and placing aptamers in a solution-phase-like detecting environment to improve the target-binding efficiency without signal amplification modules. Differential pulse voltammetry was employed to measure electrical signals in proportion to the amount of ampicillin, the target antibiotic, present in buffer and spiked milk samples. The designed aptasensor was able to detect and measure the target ampicillin in less than 30 min over a wide concentration range of 10 pM to 1 mM, with a limit of detection of 1 pM, which is 100 times better than when using the same sensing probe without TDN modification. The aptasensor was reusable by simply rinsing with deionized water, remained stable during 15-day storage, and yielded reproducible results.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Nanopartículas del Metal , Nanoestructuras , Ampicilina , Animales , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , ADN/química , Técnicas Electroquímicas/métodos , Oro/química , Límite de Detección , Nanopartículas del Metal/químicaRESUMEN
Aflatoxins (AFs) contamination in food and agricultural products poses a significant threat to human health. Sensitive and accurate detection of AFs provides a strong guarantee for ensuring food safety. Conventional chromatographic-based or mass spectrum methods, which rely on bulky instrument and skilled personnel, are not suitable for on-site surveillance. By contrast, visual detections which possess the merits of rapidity and sophisticated instrument-free present an excellent potential for the on-site detection of AFs. This review intends to summarize the latest development of visual methods for AFs detection, including paper-based tests, chromogenic reactions, and luminescent methods. Emerging technologies, like nanotechnology, DNAzymes, and aptamers combined with these visual methods are introduced. The basic principles, features, and application advantages of each type of visual methods are discussed. The biggest challenges and perspectives on their future trends are also addressed.
Asunto(s)
Aflatoxinas , ADN Catalítico , Aflatoxinas/análisis , Contaminación de Alimentos/análisis , Inocuidad de los Alimentos , Humanos , NanotecnologíaRESUMEN
Colorimetric and fluorescent methods for Ochratoxin A (OTA) detection are convenient and well received. However, the pigments and autofluorescence originated from food matrices often interfere with detection signals. We have developed a strategy with colorimetric and fluorescent dual modes to solve this challenge. In the colorimetric mode, OTA aptamer (AP9) was assembled into a DNA triple-helix switch with a specially designed signal-amplifying sequence. The OTA-induced G-quadruplex (G4) of AP9 would open the switch and release the signal-amplifying sequence for colorimetric signal amplification. The G4 structures of AP9 were further utilized to combine with the fluorogenic dye ThT for fluorescent mode. By skillfully engineering DNA G4 assembly for signal amplification, there was no need for any DNA amplification or nanomaterials labeling. Detections could be carried out in a wide temperature range (22-37 â) and finished rapidly (colorimetric mode, 60 min; fluorescent mode, 15 min). Broad linear ranges (colorimetric mode, 10-1.5 ×103 µg/kg; fluorescent mode, 0.05-1.0 ×103 µg/kg) were achieved. The limit of detection for colorimetric and fluorescent modes were 4 µg/kg and 0.01 µg/kg, respectively. The two modes have been successfully applied to detect OTA in samples with intrinsic pigments and autofluorescence, showing their applicability and reliability.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Colorimetría , ADN , Límite de Detección , Ocratoxinas , Reproducibilidad de los ResultadosRESUMEN
Vibrio parahaemolyticus (V. parahaemolyticus), which may cause gastrointestinal disorders in humans, is a pathogen commonly found in seafood. There are many methods for detecting V. parahaemolyticus, yet they have some shortcomings, such as high cost, labor-intensiveness, and complicated operation, which are impractical for resource-limited settings. Herein, we present a sequence-specific, label-free, and colorimetric method for visual detection of V. parahaemolyticus. This method utilizes CRISPR/Cas12a to specifically recognize the loop-mediated isothermal amplification (LAMP) products for further trans-cleaving the G-quadruplex DNAzyme and depriving its peroxidase-mimicking activity. In this way, the results can be directly observed with the naked eyes via the color development of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS2-), which displays colorless for positive samples while green for target-free samples. We term such Cas12a-crRNA preventing ABTS2- from developing color by trimming the G-quadruplex DNAzyme as Cascade. The proposed method can detect 9.8 CFU (per reaction) of pure cultured V. parahaemolyticus, and the sensitivity is comparable to real-time LAMP. It has been applied for practical use and showed the capability to detect 6.1 × 102 CFU/mL V. parahaemolyticus in shrimp samples. Based on this, the newly established Cascade method can be employed as a universal biosensing strategy for pathogenic bacterial testing in the field.
Asunto(s)
ADN Catalítico , Vibrio parahaemolyticus , Sistemas CRISPR-Cas , Colorimetría , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Vibrio parahaemolyticus/genéticaRESUMEN
The convenient and effective detection of toxins is urgently demanded for food security and human health. Herein, based on the catalytic activity of mimetic peroxidase from the Cu2O@Fe(OH)3 yolk-shell nanocages, a dual-modal multi-colorimetric and ratiometric fluorescence immunosensor for the sensitive detection of ochratoxin A (OTA) was successfully developed. For the multi-colorimetric detection, H2O2 can be effectively decomposed by Cu2O@Fe(OH)3 to form ·OH groups, thus Au nanorods (Au NRs) can be etched to exhibit vivid color variations and localized surface plasmon resonance (LSPR) shifts. For the ratiometric fluorescence detection, o-phenylenediamine was oxidized by Cu2O@Fe(OH)3 to form 2,3-diaminophenazine (DAP) in the presence of H2O2. Interestingly, the exogenous fluorescence signal source of carbon dots can be quenched by DAP via inner filter effect, while a new emission peak at 563 nm can be discovered, forming a ratiometric fluorescence signal. Due to the independent signals and mutual confirmation, the performance of the dual-modal immunosensor for the detection of OTA was significantly improved, where a broad linear range from 1 ng/L to 10 µg/L with a detection limit of 0.56 ng/L (S/N = 3) was achieved. The sensing strategy was also used to monitor OTA in millet and lake water samples with a satisfied performance.
Asunto(s)
Técnicas Biosensibles , Colorimetría , Ocratoxinas/análisis , Biomimética , Peróxido de Hidrógeno , Inmunoensayo , Límite de Detección , PeroxidasaRESUMEN
The rapid growth of in vitro nucleic acid replication has offered a powerful tool for clinical diagnosis, food safety detection and environmental monitorning. Successful implementation of various isothermal nucleic acid amplification methods enables rapid replication of target sequences without the participant of a thermal cycler. Point-of-need analysis possesses great superiorities in user-friendly, instant results analysis, low manufacturing, and consumable costs. To meet the great challenge of point-of-need analysis, developing simple and rapid visual methods becomes crucial. Submicro- and nanomaterials possess unique surface properties, which enables their rapid response to DNA amplicons. Their unique optical, magnetic, catalytic, and other physical/chemical properties have been frequently employed for the visual detection of in vitro nucleic acid replications. Herein, we aim to review the submicro- and nanomaterials-based visual methods for detection of nucleic acid amplification. The visual methods are classified according to the designing strategies (e.g. LSPR, bridging flocculation, luminescence, catalytic reaction, separation, etc.). The basic principles, merits and drawbacks of each strategy are described. The application in analysis of nucleic acid targets and non-nucleic acid targets are discussed. The main challenges and future research directions are also highlighted in this rapidly emerging field.
Asunto(s)
Técnicas Biosensibles , Ácidos Nucleicos , ADN/genética , Humanos , Magnetismo , Técnicas de Amplificación de Ácido Nucleico , Ácidos Nucleicos/genéticaRESUMEN
In this work, a smart analytical strategy that combines excitation-emission matrix (EEM) fluorescence detection with alternating trilinear decomposition (ATLD) algorithm was developed for fast, on-line and interference-free study on the photodegradation kinetics of aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) in rice and wheat under UV-Vis light (λâ¯=â¯250-500â¯nm) treatment. With the aid of prominent "second-order advantage" of ATLD method, pure fluorescence signals of two targeted analytes can be directly resolved out from heavily overlapping spectral environment and accurately quantified even in the presence of unknown matrix interferences. Cereal samples in kinetic processing of photodegradation were detected without complex pretreatment steps except for a simple extraction using methanol/water solution (4:1, v/v), which solves the problem facing varied matrix interferences in the case of on-line monitoring of aflatoxins. The kinetic signals of analytes of interest were directly extracted regardless of varied matrix backgrounds of various cereals. The kinetic curves and degradation speeds of AFB1 and AFG1 can be estimated by resolved quantitative data, optimal radiation conditions including 365â¯nm wavelength and 35â¯Jâ¯m-2 density were discussed for high-efficiency detoxification control of aflatoxins in rice and wheat. This strategy was promising to be as an alternative tool for eco-friendly photodegradation kinetic study of mycotoxins or other hazards in complex foodstuff matrixes.
Asunto(s)
Aflatoxinas/análisis , Contaminación de Alimentos/análisis , Espectrometría de Fluorescencia , Algoritmos , Calibración , Cromatografía Líquida de Alta Presión , Grano Comestible , Fluorescencia , Cinética , Metanol/química , Modelos Teóricos , Oryza/química , Fotólisis , Programas Informáticos , Triticum/química , Agua/químicaRESUMEN
Metal-organic frameworks (MOFs) have shown considerable prospects for sensing pesticide residues. However, the low stability of MOFs in water hinders them from testing food and environmental samples. Herein, we report an easy and cost-efficient synthesis of a water-stable zirconium luminescent MOF (Zr-LMOF) and its application for rapid, sensitive, and in situ detection of organophosphorous pesticides (OPPs). The Zr-MOF is prepared using Zr(IV) and 1,2,4,5-tetrakis(4-carboxyphenyl)benzene. The synthesized Zr-LMOF rapidly absorbs trace amounts of OPP parathion-methyl and indicates its presence. A low limit of detection of 0.115 µg kg-1 (0.438 nM) with a wide linear range from 70 µg kg-1 to 5.0 mg kg-1 was achieved. Satisfactory recoveries ranging from 78% to 107% were obtained for spiked food and environmental samples. Further, the Zr-LMOF was applied to imitate rapid and in situ imaging detection of pesticide residue on fresh produce nondestructively; visual signals appeared under ultraviolet light within 5 min. These results suggest that the Zr-LMOF has the potential for low-cost, rapid, and in situ imaging detection of OPPs contamination via easy-to-read visual signal.
RESUMEN
Calcium carbonate is wildly used in cementitious composites at different scales and can affect the properties of cementitious composites through physical effects (such as the filler effect, dilution effect and nucleation effect) and chemical effects. The effects of macro (>1 mm)-, micro (1 µmâ»1 mm)- and nano (<1 µm)-sizes of calcium carbonate on the hydration process, workability, mechanical properties and durability are reviewed. Macro-calcium carbonate mainly acts as an inert filler and can be involved in building the skeletons of hardened cementitious composites to provide part of the strength. Micro-calcium carbonate not only fills the voids between cement grains, but also accelerates the hydration process and affects the workability, mechanical properties and durability through the dilution, nucleation and even chemical effects. Nano-calcium carbonate also has both physical and chemical effects on the properties of cementitious composites, and these effects behave even more effectively than those of micro-calcium carbonate. However, agglomeration of nano-calcium carbonate reduces its enhancement effects remarkably.
RESUMEN
We have developed an effective competitive electrochemical immunosensor assay based on hapten-grafted programmed probe (HGPP) as a corecognition element for highly sensitive and selective detection of acetamiprid. Starting with the synthesis of hapten, HGPP was prepared using carboxyl group in the hapten and amino group in the 5' end of the programmed probe through covalent conjugation. Acetamiprid present in samples competes with HGPP to bind with capture antibody on the electrodes by specific recognition interaction. Methylene blue probe (MBP) was used as the electrochemical redox probe to capture the hybridized HGPP on the electrodes. The competitive reaction changes in accordance with the quantity of the target acetamiprid in the sample, as the amounts of the hybridized HGPP and the immobilized antibody are constant, i.e., the more acetamiprid samples are added, the less MBP is combined on the electrodes. In the optimal conditions, thus, biosensor output showed a linear relationship from 5 to 105 ng L-1 for the acetamiprid assay with a detecting limit of 3.2 ng L-1. The biosensor was successful in quantifying the amount of acetamiprid in spiked strawberry and cabbage extracts. This competitive immunosensor assay represents a rapid and sensitive technology for acetamiprid assay or other small molecule targets in food.
Asunto(s)
Técnicas Biosensibles/métodos , Productos Agrícolas/química , Inmunoensayo/métodos , Neonicotinoides/análisis , Residuos de Plaguicidas/análisis , Técnicas Biosensibles/instrumentación , Brassica/química , Contaminación de Alimentos/análisis , Fragaria/química , Frutas/química , Haptenos/química , Inmunoensayo/instrumentación , Límite de DetecciónRESUMEN
DNA G-quadruplexes are special three-dimensional (3D) DNA nanostructures formed by specific G-rich DNA sequences. These 3D DNA nanostructures can bind with hemin and significantly improve the intrinsic peroxidase activity of hemin. Besides this function, they also enhance the fluorescence intensity of some G-quadruplex-specific dyes. Owing to these features, G-quadruplexes possess several superiorities in the detection of enzymes involved in nucleic acid metabolism, including facile probe fabrication without labeling, simple detection process without washing or separation steps, rapid observation by naked eyes, and easy integration with nucleic acid amplification strategies to amplify signals. Herein, we describe two strategies for label-free detection of enzyme activity based on DNA G-quadruplexes. To increase sensitivity, template-dependent and template-independent DNA amplifications were introduced for the amplification of G-rich DNA sequences. DNA methyltransferase and terminal deoxynucleotidyl transferase were detected as two model analytes, respectively.
Asunto(s)
Bioensayo/métodos , ADN/química , Nanoestructuras/química , Secuencia de Bases , Técnicas Biosensibles/métodos , Colorimetría/métodos , Metilasas de Modificación del ADN/química , Fluorescencia , G-Cuádruplex , Hemina/química , Técnicas de Amplificación de Ácido Nucleico/métodos , Peroxidasa/químicaRESUMEN
In heterozygous patients with a diabetic syndrome called mutant INS gene-induced diabetes of youth (MIDY), there is decreased insulin secretion when mutant proinsulin expression prevents wild-type (WT) proinsulin from exiting the endoplasmic reticulum (ER), which is essential for insulin production. Our previous results revealed that mutant Akita proinsulin is triaged by ER-associated degradation (ERAD). We now find that the ER chaperone Grp170 participates in the degradation process by shifting Akita proinsulin from high-molecular weight (MW) complexes toward smaller oligomeric species that are competent to undergo ERAD. Strikingly, overexpressing Grp170 also liberates WT proinsulin, which is no longer trapped in these high-MW complexes, enhancing ERAD of Akita proinsulin and restoring WT insulin secretion. Our data reveal that Grp170 participates in preparing mutant proinsulin for degradation while enabling WT proinsulin escape from the ER. In principle, selective destruction of mutant proinsulin offers a rational approach to rectify the insulin secretion problem in MIDY.