RESUMEN
Natural ligand-receptor interactions that play pivotal roles in biological events are ideal models for design and assembly of artificial recognition molecules. Herein, aiming at the structural characteristics of the spike trimer and infection mechanism of SARS-CoV-2, we have designed a DNA framework-guided spatial-patterned neutralizing aptamer trimer for SARS-CoV-2 neutralization. The â¼5.8 nm tetrahedral DNA framework affords precise spatial organization and matched valence as four neutralizing aptamers (MATCH-4), which matches with nanometer precision the topmost surface of SARS-CoV-2 spike trimer, enhancing the interaction between MATCH-4 and spike trimer. Moreover, the DNA framework provides a dimensionally complementary nanoscale barrier to prevent the spike trimer-ACE2 interaction and the conformational transition, thereby inhibiting SARS-CoV-2-host cell fusion and infection. As a result, the spatial- and valence-matched MATCH-4 ensures improved binding affinity and neutralizing activity against SARS-CoV-2 and its varied mutant strains, particularly the current Omicron variant, that are evasive of the majority of existing neutralizing antibodies. In addition, because neutralizing aptamers specific to other targets can be evolved and assembled, the present design has the potential to inhibit other wide-range and emerging pathogens.
Asunto(s)
COVID-19 , Nanoestructuras , Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes , Anticuerpos Antivirales , ADN , Humanos , Ligandos , Glicoproteínas de Membrana , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética , Proteínas del Envoltorio Viral/químicaRESUMEN
Simultaneous detection of multi-biomarkers not only enhances the accuracy of disease diagnosis but also improves detection efficiency and reduces cost. It is vital to achieve portable, simple, low-cost, and simultaneous detection of biomarkers for point-of-care (POC) diagnostics in a low-resource setting. Herein, a multichannel paper chip-based gas pressure bioassay was developed for the simultaneous detection of multiple biomarkers by combining multichannel paper chips with a portable gas pressure meter. Four DNA tetrahedral probes (DTPs) were used as capture probes and were immobilized in different detection zones of the paper chips to improve hybridization efficiency and reduce nonspecific adsorption. The formation of a sandwich structure between target microRNAs (miRNAs), the capture probe, and platinum nanoparticles (PtNPs)-modified complementary DNA (PtNPs-cDNA) transformed biomolecular recognition into quantitative detection of gas pressure. Four lung cancer-related miRNAs were detected simultaneously by a portable gas pressure meter. There is a good linear relationship between gas pressure and the logarithm of miRNA concentration in the range of 10 pM to 100 nM. Compared with single-stranded DNA capture probe, the signal-to-noise (S/N) of DNA tetrahedral probes improved more than 3 times. Using ring-oven washing, the unbound reagents in all channels of the paper chip were simultaneously and continuously washed away, leading to a more cheap, simple, and fast separation than magnetic separation. Therefore, it offers a promising multichannel paper chip-based gas pressure bioassay for portable and simultaneous detection of multiple biomarkers.
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Técnicas Biosensibles/instrumentación , MicroARNs/análisis , Papel , Pruebas en el Punto de Atención , Sondas de ADN/química , ADN Complementario/química , Diseño de Equipo , Humanos , Límite de Detección , MicroARNs/sangre , Platino (Metal)/químicaRESUMEN
The integration of one more gain media in droplet microlasers with morphology-dependent modes, which can be employed in optofluidic systems as multi-wavelength lasing sources, is highly attractive and demands new cavity design and fabrication approaches. Here, cholesteric liquid crystal (CLC) droplets with an integrative triple-emulsion cavity are fabricated via glass-capillary-based microfluidic technologies and dual-gain lasing with variable modes, flexibly configured by the combination and incorporation of gain dyes and CLCs into both the core and shell. The distributed feedback (DFB) mode, formed by the feedback from the self-assembled helix periodic structure of CLCs, the whispering gallery (WG) mode, and the hybrid, is selectively excited by controlling the spatial coupling between the pump beam and the droplet with gain. With the merits of dual-gain and controllable lasing, a prototype dual-wavelength-ratiometric thermometer with self-calibration capability is expected to be developed. Furthermore, the anisotropic CLC core is substituted with an isotropic fluid and the gain from the CLC shell is additionally removed, DFB lasings in both shell and core are absent, and only Bragg-shell reflection-based hybrid modes are excited for lasing. The CLC droplet microlasers with an integrative cavity are expected to provide a new route to future lab-on-chip (LOC) applications.
Asunto(s)
Colesterol/química , Cristales Líquidos/química , Técnicas Analíticas Microfluídicas , Técnicas Analíticas Microfluídicas/instrumentación , Tamaño de la PartículaRESUMEN
Currently, there are more than 200 fecal microbiota transplantation (FMT) clinical trials worldwide. However, our knowledge of this microbial therapy is still limited. Here we develop a strategy using sequential tagging with D-amino acid-based metabolic probes (STAMP) for assessing the viabilities of transplanted microbiotas. A fluorescent D-amino acid (FDAA) is first administered to donor mice to metabolically label the gut microbiotas in vivo. The labeled microbiotas are transplanted to recipient mice, which receive a second FDAA with a different color. The surviving transplants should incorporate both FDAAs and can be readily distinguished by presenting two colors simultaneously. Isolation of surviving bacteria and 16S rDNA sequencing identify several enriched genera, suggesting the importance of specific bacteria in FMT. In addition, using STAMP, we evaluate the effects on transplant survival of pre-treating recipients using different antibiotics. We propose STAMP as a versatile tool for deciphering the complex biology of FMT, and potentially improving its treatment efficacy.
Asunto(s)
Aminoácidos/administración & dosificación , Trasplante de Microbiota Fecal/métodos , Microbioma Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/microbiología , ARN Ribosómico 16S/genética , Coloración y Etiquetado/métodos , Aminoácidos/química , Animales , Antibacterianos/farmacología , Heces/microbiología , Colorantes Fluorescentes/química , Microbioma Gastrointestinal/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Viabilidad Microbiana/efectos de los fármacos , EstereoisomerismoRESUMEN
DNA hydrogels have received considerable attention in analytical science, however, some limitations still exist in the applications of intelligent hydrogels. In this paper, we describe a way to prepare gel film in a capillary tube based on the thermal reversible principle of DNA hydrogel and the principle of capillary action. Because of the slight change in the internal structure of gel, its permeability can be increased by the addition of some specific targets. The capillary behavior is thus changed due to the different permeability of the hydrogel film. The duration time of the target solution flowing through the capillary tube with a specified length is used to quantify this change. With this proposed method, ultra-trace DNA hydrogel (0.01 µL) is sufficient to realize the sensitive detection of cocaine without the aid of other instruments, which has a low detection limit (1.17 nM) and good selectivity.
Asunto(s)
Aptámeros de Nucleótidos , Tubo Capilar , Cocaína/análisis , Hidrogeles , Técnicas Biosensibles , Cocaína/orina , Diseño de Equipo , Humanos , Límite de Detección , PermeabilidadRESUMEN
To explore genome mutation meaningfully, it is in urgent need to develop an automated and inexpensive platform for DNA mutation analysis. Digital microfluidics is a powerful platform for a broad range of applications due to the advantages of high automatization and low reagent consumption. Pyrosequencing enables DNA sequencing based on non-electrophoresis bioluminescence, which is suitable for rapid and sensitive analysis of short sequences. Herein, we describe a palmtop sequencing platform for automatic, real-time and portable analysis of DNA mutations, which is based on the pyrosequencing principle and implemented by digital microfluidics. The portable system can sequence a DNA template with up to 53â¯bp with 100% accuracy within 2â¯h. Mutation in the KRAS gene can be detected within 30â¯min with a LOD as low as 5% mutant level. Portable and accurate gender identification was further demonstrated by sequencing a short amelogenin fragment. With the advantages of portability, ease of use, high accuracy, and low cost, the palmtop sequencing platform shows great potential for portable genetic testing in a variety of circumstances.
Asunto(s)
Técnicas Biosensibles , ADN/aislamiento & purificación , Mediciones Luminiscentes , ADN/genética , Análisis Mutacional de ADN , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Microfluídica/métodos , MutaciónRESUMEN
The traditional Gram-staining method, which was invented more than a century ago for differentiating bacteria as Gram positive or Gram negative, is still widely practiced in microbiology. However, Gram staining suffers from several problems which can affect the accuracy of the diagnosis. Here, we report a new Gram-negative-specific fluorescent probe, which is based on a narrow-spectrum antibiotic, tridecaptin A1, and allows selective staining of Gram-negative bacteria in different fixed bacterial samples. Solid-phase peptide synthesis was used to prepare the tridecaptin A1-fluorophore conjugate with a single structure. Labeling selectivity of the probe toward Gram-negative bacteria was confirmed by testing against a panel of bacterial species. By combining the use of a previously reported Gram-positive-specific fluorescent probe, we then further showed the capability of the new probe in differential labeling of a number of complex bacterial samples, which included a mouse gut microbiota cultured in vitro, as well as microbiotas collected from the human oral cavity, soil, and crude oil. High labeling selectivity and coverage were observed in most samples. This method offers a new Gram-negative-specific probe with a defined structure, which allows facile fluorescence-based differentiation of Gram-positive and Gram-negative bacteria for further microbial studies.
Asunto(s)
Colorantes Fluorescentes/química , Violeta de Genciana/química , Bacterias Gramnegativas/aislamiento & purificación , Péptidos/química , Fenazinas/química , Citometría de Flujo , Bacterias Gramnegativas/clasificación , Bacterias Grampositivas/clasificación , Bacterias Grampositivas/aislamiento & purificaciónRESUMEN
It is of the significant importance to achieve facile and on-site detection of heavy metal ions due to the serious harm to environment and human health. Herein, a facile and portable strategy was developed for detection of Hg2+ via portable pressure meter. Biotinylated DNA1 was conjugated on the surface of streptavidin-coated magnetic beads (MBs) to form MBs-DNA1 complex. In the presence of Hg2+, MBs-DNA1 can hybridize with platinum nanoparticles (PtNPs)-functionalized DNA2 (DNA2-PtNPs) via T-Hg2+-T binding. Then, PtNPs effectively catalyzed the decomposition of H2O2 to generate oxygen, leading to an increase in pressure of sealed well of 96-well plate. The gas pressure was linearly related with the concentration of Hg2+ in the range between 10â¯pM and 100â¯nM with a detection limit of 2.79â¯pM, which is more sensitive than most of the previous reports. The specific T-Hg2+-T binding made it easy to selectively detect Hg2+ even when other metal ions co-existed with Hg2+. Therefore, it offers a cost-effective, rapid, facile and portable way to detect Hg2+ by combining gas-generation reaction with T-Hg2+-T binding, which holds great potential for detecting Hg2+ in water sample.
Asunto(s)
Técnicas Biosensibles/métodos , Ácidos Nucleicos Inmovilizados/química , Mercurio/análisis , Nanopartículas del Metal/química , Platino (Metal)/química , Contaminantes Químicos del Agua/análisis , Agua Potable/análisis , Humanos , Límite de Detección , Imanes/química , Presión , Ríos/químicaRESUMEN
A high serum HER-2 extracellular domain (sHER-2 ECD) level has a reverse association with tumor behaviors. In this study, a portable platform for the disease biomarker sHER-2 ECD detection has been established using a pressure-based bioassay. The pressure bioassay consists of a monoclonal antibody immobilized on an eight-well strip, the analyte HER-2, and another monoclonal antibody labeled with the Pt nanoparticles (PtNPs), which have the catalytic ability to decompose H2O2 into H2O and O2(g). The increased pressure due to O2(g) generation is measured by a hand-held pressure meter. A total of 34 serum samples were collected to validate the performance of the pressure bioassay. The results showed that the pressure bioassay platform of HER-2 had a dynamic range from 2 to 50 ng/mL with a limit of detection (LOD) of 2 ng/mL, which was consistent with the ELISA result. In the real serum samples, there was a significant correlation between sHER-2 ECD level and several clinicopathological parameters, especially tissue HER-2 status. Furthermore, the sHER-2 ECD level was found to decrease after targeted therapy in a patient with tHER-2 positive. Overall, this bioassay can facilitate breast cancer diagnosis and prognosis in clinical scenarios and resource-limited areas.
Asunto(s)
Neoplasias de la Mama/sangre , Receptor ErbB-2/sangre , Anticuerpos Monoclonales , Bioensayo/métodos , Biomarcadores de Tumor/sangre , Femenino , Humanos , Presión , Receptor ErbB-2/químicaRESUMEN
Fetal aneuploidy and other chromosomal aberrations affect 9 in 1000 live births. Unlike the invasive diagnosis with high risk of miscarriage, non-invasive prenatal diagnosis (NIPD) sampling from maternal blood becomes a promising way for fetal genetic screening. However, fetal cell-based NIPD has a major challenge due to the small number of fetal cells present in maternal blood. We designed a frequency-enhanced transferrin receptor antibody-labelled microfluidic chip (FETAL-Chip) for efficient enrichment and identification of circulating fetal cells, i.e., circulating nucleated red blood cells (cNRBCs) from maternal blood. The FETAL-Chip can dramatically enhance the interaction of fetal cells with antibody-coated microposts to increase the capture efficiency while minimizing nonspecific adsorption. With the help of immunostaining, we can identify cNRBCs from as little as 2 milliliter maternal blood. Various numbers of cNRBCs were detected from volunteers as early as 7 weeks after conception and throughout the entire pregnancy. Gene analysis was also carried out to confirm the fetal origin of captured cells. With easy, non-invasive and highly efficient enrichment of cNRBCs, the method presented here offers great potential for non-invasive prenatal diagnosis.
Asunto(s)
Anticuerpos/inmunología , Separación Celular/instrumentación , Eritrocitos/citología , Dispositivos Laboratorio en un Chip , Diagnóstico Prenatal/instrumentación , Receptores de Transferrina/inmunología , Anticuerpos/química , Femenino , Humanos , EmbarazoRESUMEN
Substrate channeling, in which a metabolic intermediate is directly passed from one enzyme to the next enzyme in an enzyme cascade, accelerates the processing of metabolites and improves substrate selectivity. Synthetic design and precise control of channeling outside the cellular environment are of significance in areas such as synthetic biology, synthetic chemistry, and biomedicine. In particular, the precise control of synthetic substrate channeling in response to light is highly important, but remains a major challenge. Herein, we develop a photoresponsive molecule-based synthetic substrate channeling system on DNA origami to regulate enzyme cascade activity. The photoresponsive azobenzene molecules introduced into DNA strands enable reversible switching of the position of substrate channeling to selectively activate or inhibit the enzyme cascade activity. Moreover, DNA origami allows precise control of interenzyme distance and swinging range of the swing arm to optimize the regulation efficiency. By combining the accurate and addressable assembly ability of DNA origami and the clean, rapid, and reversible regulation of photoresponsive molecules, this light-driven substrate channeling system is expected to find important applications in synthetic biology and biomedicine.
Asunto(s)
Compuestos Azo/química , ADN/química , Nanoestructuras/química , Animales , Biocatálisis , Glucosafosfato Deshidrogenasa/química , L-Lactato Deshidrogenasa/química , Leuconostoc mesenteroides/enzimología , Luz , Oxigenasas de Función Mixta/química , Modelos Moleculares , Pediococcus/enzimología , Procesos Fotoquímicos , Conejos , Especificidad por Sustrato , Biología SintéticaRESUMEN
The upregulation of microRNA (miRNA) is highly related with some kinds of tumor, such as breast, prostate, lung, and pancreatic cancers. Therefore, for an important tumor biomarker, the point-of-care testing (POCT) of miRNA is of significant importance and is in great demand for disease diagnosis and clinical prognoses. Herein, a POCT assay for miRNA detection was developed via a portable pressure meter. Two hairpin DNA probes, H1 and H2, were ingeniously designed and functionalized with magnetic beads (MBs) and platinum nanoparticles (PtNPs), respectively, to form MBs-H1 and PtNPs-H2 complexes. In the presence of target microRNA 21 (miR-21), the cyclic strand displacement reaction (SDR) between MBs-H1 and PtNPs-H2 was triggered to continuously form the MBs-H1/PtNPs-H2 duplex. Owing to the amplification of cyclic SDR, numerous PtNPs were enriched onto the surface of MBs to catalytically decompose H2O2 for the generation of much O2. The gas pressure value has a linear relationship with the logarithmic value of miR-21 concentration in the range of 10 fM to 10 pM. The limit of detection is 7.6 fM, which is more sensitive than that in a number of previous reports. Hairpin DNA probes and magnetic separation highly ensured the specificity and reliability. Single-base mutation was easily discriminated, and the detection of miR-21 in the serum sample achieved satisfactory result. Therefore, it offers a reliable POCT strategy for the detection of miRNA, which is of great theoretical and practical importance for POCT clinical diagnostics.
Asunto(s)
MicroARNs/química , Sondas de ADN , Peróxido de Hidrógeno , Reproducibilidad de los ResultadosRESUMEN
We have successfully developed a target-responsive aptamer cross-linked hydrogel for the visual detection of glucose, an important biomedical analyte. In this work, the glucose-responsive hydrogel was prepared using the target aptamer and its two short complementary DNA strands grafted onto a linear polyacrylamide chain as cross-linkers. Gold nanoparticles (AuNPs) modified with thiol-PEG were encapsulated in the gel and used as the output signal for visible detection. The complex of glucose and its ligand of boronic acid derivatives (Shinkai's receptor) can bind with the aptamer to disrupt the hydrogel, leading to the release of AuNPs with a distinct red colour in the supernatant. By this method glucose can be detected with the naked eye, and the sensor has a detection limit of 0.44 mM in buffer with the help of UV-Vis spectrophotometry. Furthermore, glucose spiked in 50% urine and 30% serum could also be detected respectively with the naked eye, and glucose was quantitatively detected in 50% urine. The hydrogel system provides a non-enzymatic and visual method for glucose detection, and offers promising applications in biotechnology and biomedicine.
Asunto(s)
Técnicas Biosensibles , ADN/química , Glucosa/análisis , Hidrogeles , Nanopartículas del Metal , Aptámeros de Nucleótidos/química , OroRESUMEN
A fluorescence strategy for alkaline phosphatase (ALP) assay in complicated samples with high sensitivity and strong stability is developed based on an allosteric probe (AP). This probe consists of two DNA strands, a streptavidin (SA) aptamer labeled by fluorophore and its totally complementary DNA (cDNA) with a phosphate group on the 5' end. Upon ALP introduction, the phosphate group on the cDNA is hydrolyzed, leaving the unhydrolyzed cDNA sequence for lambda exonuclease (λ exo) digestion and releasing SA aptamer for binding to SA beads, which results in fluorescence enhancement of SA beads that can be detected by flow cytometry or microscopy. We have achieved a detection limit of 0.012 U/mL with a detection range of 0.02~0.15 U/mL in buffer and human serum. These figures of merit are better than or comparable to those of other methods. Because the fluorescence signal is localized on the beads, they can be separated to remove fluorescence background from complicated biological systems. Notably, the new strategy not only applies to ALP detection with simple design, easy operation, high sensitivity, and good compatibility in complex solution, but also can be utilized in ALP-linked immunosorbent assays for the detection of a wide range of targets.
RESUMEN
As a vital enzyme in DNA phosphorylation and restoration, T4 polynucleotide kinase (T4 PNK) has aroused great interest in recent years. Therefore, numerous strategies have been established for highly sensitive detection of T4 PNK based on diverse signal amplification techniques. However, they often need sophisticated design, a variety of auxiliary reagents and enzymes, or cumbersome manipulations. We have designed a new kind of allosteric aptamer probe (AAP) consisting of streptavidin (SA) aptamer and the complementary DNA (cDNA) for simple detection of T4 PNK without signal amplification and with minimized interference in complex biological samples. When the 5'-terminus of the cDNA is phosphorylated by T4 PNK, the cDNA is degraded by lambda exonuclease to release the fluorescein amidite (FAM)-labeled SA aptamer, which subsequently binds to streptavidin beads. The enhancement of the fluorescence signal on SA beads can be detected precisely and easily by a microscope or flow cytometer. Our method performs well in complex biological samples as a result of the enrichment of the signaling molecules on beads, as well as simple manipulations to discard the background interference and nonbinding molecules. Without signal amplification techniques, our AAP method not only avoids complicated manipulations but also decreases the time required. With the advantages of ease of operation, reliability, and robustness for T4 PNK detection in buffer as well as real biological samples, the AAP has great potential for clinical diagnostics, inhibitor screening, and drug discovery.
Asunto(s)
Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , Regulación Alostérica , Técnicas Biosensibles , ADN , Reproducibilidad de los Resultados , Espectrometría de FluorescenciaRESUMEN
Detection of telomerase activity at the single-cell level is one of the central challenges in cancer diagnostics and therapy. Herein, we describe a facile and reliable point-of-care testing (POCT) strategy for detection of telomerase activity via a portable pressure meter. Telomerase primer (TS) was immobilized onto the surface of magnetic beads (MBs), and then was elongated to a long single-stranded DNA by telomerase. The elongated (TTAGGG)n repeat unit hybridized with several short PtNP-functionalized complementary DNA (PtNPs-cDNA), which specifically enriched PtNPs onto the surfaces of magnetic beads (MBs), which were separated using a magnet. Then, nanoparticle-catalyzed gas-generation reaction converted telomerase activity into significant change in gas pressure. Because of the self-amplification of telomerase and enrichment by magnetic separation, the diluted telomerase equivalent to a single HeLa cell was facilely detected. More importantly, the telomerase in the lysate of 1 HeLa cell can be reliably detected by monitoring change in gas pressure, indicating that it is feasible and possible to study differences between individual cells. The difference in relative activity between different kinds of cancer cells was easily and sensitively studied. Study of inhibition of telomerase activity demonstrated that our method has great potential in screening of telomerase-targeted antitumor drugs as well as in clinical diagnosis.
Asunto(s)
Pruebas de Enzimas/métodos , Análisis de la Célula Individual/métodos , Telomerasa/análisis , Técnicas Biosensibles/métodos , Línea Celular Tumoral , ADN/genética , Humanos , Límite de Detección , Nanopartículas del Metal/química , Hibridación de Ácido Nucleico , Oxígeno/química , Platino (Metal)/química , Sistemas de Atención de Punto , PresiónRESUMEN
Point-of-care testing (POCT) with the advantages of speed, simplicity, and low cost, as well as no need for instrumentation, is critical for the measurement of analytes in a variety of environments lacking access to laboratory infrastructure. In the present study, a hydrogel pressure-based assay for quantitative POCT was developed by integrating a target-responsive hydrogel with pressuremeter readout. The target-responsive hydrogels were constructed with DNA grafted linear polyacrylamide and the cross-linking DNA for selective target recognition. The hydrogel response to the target substance allows release of the preloaded Pt nanoparticles, which have good stability and excellent catalytic ability for decomposing H2O2 to O2. Then, the generated O2 in a sealed environment leads to significant pressure increase, which can be easily read out by a handheld pressuremeter. Using this target-responsive hydrogel pressure-based assay, portable and highly sensitive detection of cocaine, ochratoxin A, and lead ion were achieved with excellent accuracy and selectivity. With the advantages of portability, high sensitivity, and simple sample processing, the hydrogel pressure-based assay shows great potential for quantitative POCT of a broad range of targets in resource-limited settings.
Asunto(s)
Hidrogeles/química , Cocaína , Hidrogel de Polietilenoglicol-Dimetacrilato , Peróxido de Hidrógeno , Pruebas en el Punto de AtenciónRESUMEN
Heterogeneity among individual molecules and cells has posed significant challenges to traditional bulk assays, due to the assumption of average behavior, which would lose important biological information in heterogeneity and result in a misleading interpretation. Single molecule/cell analysis has become an important and emerging field in biological and biomedical research for insights into heterogeneity between large populations at high resolution. Compared with the ensemble bulk method, single molecule/cell analysis explores the information on time trajectories, conformational states, and interactions of individual molecules/cells, all key factors in the study of chemical and biological reaction pathways. Various powerful techniques have been developed for single molecule/cell analysis, including flow cytometry, atomic force microscopy, optical and magnetic tweezers, single-molecule fluorescence spectroscopy, and so forth. However, some of them have the low-throughput issue that has to analyze single molecules/cells one by one. Flow cytometry is a widely used high-throughput technique for single cell analysis but lacks the ability for intercellular interaction study and local environment control. Droplet microfluidics becomes attractive for single molecule/cell manipulation because single molecules/cells can be individually encased in monodisperse microdroplets, allowing high-throughput analysis and manipulation with precise control of the local environment. Moreover, hydrogels, cross-linked polymer networks that swell in the presence of water, have been introduced into droplet microfluidic systems as hydrogel droplet microfluidics. By replacing an aqueous phase with a monomer or polymer solution, hydrogel droplets can be generated on microfluidic chips for encapsulation of single molecules/cells according to the Poisson distribution. The sol-gel transition property endows the hydrogel droplets with new functionalities and diversified applications in single molecule/cell analysis. The hydrogel can act as a 3D cell culture matrix to mimic the extracellular environment for long-term single cell culture, which allows further heterogeneity study in proliferation, drug screening, and metastasis at the single-cell level. The sol-gel transition allows reactions in solution to be performed rapidly and efficiently with product storage in the gel for flexible downstream manipulation and analysis. More importantly, controllable sol-gel regulation provides a new way to maintain phenotype-genotype linkages in the hydrogel matrix for high throughput molecular evolution. In this Account, we will review the hydrogel droplet generation on microfluidics, single molecule/cell encapsulation in hydrogel droplets, as well as the progress made by our group and others in the application of hydrogel droplet microfluidics for single molecule/cell analysis, including single cell culture, single molecule/cell detection, single cell sequencing, and molecular evolution.
Asunto(s)
Hidrogeles , Microfluídica/métodos , Análisis de la Célula Individual , Evolución Molecular , Citometría de Flujo , Microscopía de Fuerza Atómica , Pinzas Ópticas , Espectrometría de FluorescenciaRESUMEN
Aflatoxin B1 (AFB1), as the secondary metabolite of molds, is the most predominant and toxic mycotoxin that seriously threatens the health of humans and animals. In this work, an AFB1-responsive hydrogel was synthesized for highly sensitive and portable detection of AFB1. The AFB1-responsive hydrogel was prepared using an AFB1 aptamer and its two short complementary DNA strands as cross-linkers. For visual detection of AFB1, the hydrogel is preloaded with gold nanoparticles (AuNPs). Upon introduction of AFB1, the AFB1 aptamer binds with AFB1, leading to the disruption of the hydrogel and release of the AuNPs with a distinct color change of the supernatant from colorless to red. In order to lower the detection limit and extend the method to quantitative analysis, a distance-readout volumetric bar chart chip (V-chip) was combined with an AFB1-responsive hydrogel preloaded with platinum nanoparticles (PtNPs). In the presence of AFB1, the hydrogel collapses and releases PtNPs which can catalyze the decomposition of H2O2 to generate O2. The increasing gas pressure moves a red ink bar in the V-chip and provides a quantitative relationship between the distance and the concentration of AFB1. The method was applied for detection of AFB1 in beer, with a detection limit of 1.77 nM (0.55 ppb) where an immunoaffinity column (IAC) of AFB1 was used to cleanup and pre-concentrate the sample, which satisfies the testing requirement of 2.0 ppb set by the European Union. The combination of an AFB1-responsive hydrogel with a distance-based readout V-chip offers a user-friendly POCT device, which has great potential for rapid, portable, selective, and quantitative detection of AFB1 in real samples to ensure food safety and avoid subsequent economic losses.
Asunto(s)
Aflatoxina B1/análisis , Carcinógenos Ambientales/análisis , Hidrogeles/química , Dispositivos Laboratorio en un Chip , Modelos Químicos , Aptámeros de Nucleótidos/química , ADN Complementario/química , Estudios de Factibilidad , Inspección de Alimentos/instrumentación , Oro/química , Ácidos Nucleicos Inmovilizados/química , Límite de Detección , Nanopartículas del Metal/química , Platino (Metal)/químicaRESUMEN
Artificial multi-enzyme systems with precise and dynamic control over the enzyme pathway activity are of great significance in bionanotechnology and synthetic biology. Herein, we exploit a spatially addressable DNA nanoplatform for the directional regulation of two enzyme pathways (G6pDH-MDH and G6pDH-LDH) through the control of NAD(+) substrate channeling by specifically shifting NAD(+) between the two enzyme pairs. We believe that this concept will be useful for the design of regulatory biological circuits for synthetic biology and biomedicine.