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The detection of biomarkers (such as DNA, RNA, and protein) plays a vital role in medical diagnosis. The CRISPR-based biosensors utilize the CRISPR/Cas system for biometric recognition of targets and use biosensor strategy to read out biological signals without the employment of professional operations. Consequently, the CRISPR-based biosensors demonstrate great potential for the detection of biomarkers with high sensitivity and specificity. However, the signal readout still relies on specialized detectors, limiting its application in on-site detection for medical diagnosis. In this review, we summarize the principles and advances of the CRISPR-based biosensors with a focus on medical diagnosis. Then, we review the advantages and progress of CRISPR-based naked eye biosensors, which can realize diagnosis without additional detectors for signal readout. Finally, we discuss the challenges and further prospects for the development of CRISPR-based biosensors.
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Técnicas Biosensibles , Sistemas CRISPR-Cas , Humanos , BiomarcadoresRESUMEN
Single-cell analysis provides an overwhelming strategy for revealing cellular heterogeneity and new perspectives for understanding the biological function and disease mechanism. Moreover, it promotes the basic and clinical research in many fields at a single-cell resolution. A digital polymerase chain reaction (dPCR) is an absolute quantitative analysis technology with high sensitivity and precision for DNA/RNA or protein. With the development of microfluidic technology, digital PCR has been used to achieve absolute quantification of single-cell gene expression and single-cell proteins. For single-cell specific-gene or -protein detection, digital PCR has shown great advantages. So, this review will introduce the significance and process of single-cell analysis, including single-cell isolation, single-cell lysis, and single-cell detection methods, mainly focusing on the microfluidic single-cell digital PCR technology and its biological application at a single-cell level. The challenges and opportunities for the development of single-cell digital PCR are also discussed.
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ADN , Microfluídica , Reacción en Cadena de la Polimerasa/métodos , ARN , Análisis de la Célula IndividualRESUMEN
Spatial profiling technologies fill the gap left by the loss of spatial information in traditional single-cell sequencing, showing great application prospects. After just a few years of quick development, spatial profiling technologies have made great progress in resolution and simplicity. This review introduces the development of spatial omics sequencing based on microfluidic array chips and describes barcoding strategies using various microfluidic designs with simplicity and efficiency. At the same time, the pros and cons of each strategy are compared. Moreover, commercialized solutions for spatial profiling are also introduced. In the end, the future perspective of spatial omics sequencing and research directions are discussed.
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MicrofluídicaRESUMEN
Diabetic periodontitis is a major complication of diabetes, which has a deep involvement in teeth loss and more serious systematic diseases, including Alzheimer's disease, atherosclerosis and cancers. Diabetic periodontitis is difficult to treat because of recalcitrant infection and hyperglycemia-induced tissue dysfunction. Current treatments fail to completely eliminate infection due to the diffusion-reaction inhibition of biofilm, and ignore the tissue dysfunction. Here, we design a glucose-driven transformable complex, composed of calcium alginate (CaAlg) hydrogel shell and Zeolitic imidazolate framework-8 (ZIF-8) core encapsulating Glucose oxidase (GOx)/Catalase (CAT) and Minocycline (MINO), named as CaAlg@MINO/GOx/CAT/ZIF-8 (CMGCZ). The reaction product of glucose-scavenging, gluconic acid, could dissolve ZIF-8 core and transform CMGCZ from inflexible to flexible, facilitating the complex to overcome the diffusion-reaction inhibition of biofilm. Meanwhile, reduced glucose concentration could ameliorate the pyroptosis of macrophages to decrease the secretion of pro-inflammatory factors, thereby reducing inflamm-aging to alleviate periodontal dysfunction.
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Synapses are crucial structures that mediate signal transmission between neurons in complex neural circuits and display considerable morphological and electrophysiological heterogeneity. So far we still lack a high-throughput method to profile the molecular heterogeneity among individual synapses. In the present study, we develop a droplet-based single-cell (sc) total-RNA-sequencing platform, called Multiple-Annealing-and-Tailing-based Quantitative scRNA-seq in Droplets, for transcriptome profiling of individual neurites, primarily composed of synaptosomes. In the synaptosome transcriptome, or 'synaptome', profiling of both mouse and human brain samples, we detect subclusters among synaptosomes that are associated with neuronal subtypes and characterize the landscape of transcript splicing that occurs within synapses. We extend synaptome profiling to synaptopathy in an Alzheimer's disease (AD) mouse model and discover AD-associated synaptic gene expression changes that cannot be detected by single-nucleus transcriptome profiling. Overall, our results show that this platform provides a high-throughput, single-synaptosome transcriptome profiling tool that will facilitate future discoveries in neuroscience.
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Enfermedad de Alzheimer , Sinapsis , Humanos , Ratones , Animales , Sinapsis/genética , Sinapsis/metabolismo , Perfilación de la Expresión Génica/métodos , Sinaptosomas/metabolismo , Transcriptoma/genética , Enfermedad de Alzheimer/genética , Análisis de la Célula Individual/métodos , Análisis de Secuencia de ARN/métodosRESUMEN
Single-cell RNA sequencing (scRNA-seq) technology provides a powerful tool for understanding complex biosystems at the single-cell and single-molecule level. The past decade has been a golden period for the development of single-cell sequencing, with scRNA-seq undergoing a tremendous leap in sensitivity and throughput. The application of droplet- and microwell-based microfluidics in scRNA-seq has contributed greatly to improving sequencing throughput. This review introduces the history of development and important technical factors of scRNA-seq. We mainly focus on the role of microfluidics in facilitating the development of scRNA-seq technology. To end, we discuss the future directions for scRNA-seq.
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Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de la Célula Individual , Microfluídica , Análisis de Secuencia de ARNRESUMEN
Food poisoning and infectious diseases caused by Salmonella typhimurium (S. typhimurium) are serious public health concerns for human health and food safety. The diversity and complexity of food matrices pose great challenges for rapid and ultra-sensitive detection of S. typhimurium in food samples. A method capable of identification, detection, and quantification of S. typhimurium is essential for addressing these issues. In this study, aptamer-coated magnetic beads (Apt-MBs) are employed as capture bio-probes to specifically and selectively concentrate S. typhimurium in food samples. A self-priming chip-based digital PCR was then presented as another biosensor for on-site detection and quantification of S. typhimurium cells. The chip we developed was robust and did not require any external power for sample loading. The combination of Apt-MBs with an on-chip digital detection realized the integration into lab-on-a-chip-based biosensors for on-site monitoring of foodborne pathogens. It was possible to capture and detect S. typhimurium cells as low as 90 CFU/reaction with a capture efficiency of 94.5%. Additionally, the whole process only took about 2 h. This unique platform could also be used to monitor other target bacteria with high specificity and sensitivity by utilizing different aptamers. Furthermore, the platform has potential applications in point-of-care testing in the future.
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Aptámeros de Nucleótidos , Técnicas Biosensibles , Técnicas Biosensibles/métodos , Microbiología de Alimentos , Humanos , Separación Inmunomagnética/métodos , Reacción en Cadena de la Polimerasa , Salmonella typhimurium/genéticaRESUMEN
The rapid and accurate detection of viable bacteria is of great importance in food quality monitoring and clinical diagnosis. Escherichia coli (E. coli) is a major pathogenic bacterium, which causes potential threats to food safety and human health. Therefore, rapid and portable methods for preventing E. coli outbreaks are needed. Single cell analysis can be performed at the single-cell level, which has great advantages for analysis and diagnosis. Herein, we employed a thermosetting oil to generate a large-scale pico-droplet array for viable bacteria digital counting and dynamic tracking. In this array, the droplets can be solidified without any inducers due to the cross-linking reaction of the hydrosilation of vinyl silicone oil and hydrosilicone oil. Single E. coli cells were encapsulated in solidified droplets to form a microcolony. Resazurin was used as a fluorescent indicator to achieve amplification of bacterial growth signals. This method can achieve digital counting of viable E. coli cells in 4 h. We achieved real-time monitoring of E. coli cell growth and division in droplets. It is rapid, simple, and does not require a pre-enrichment process when compared to the traditional plate counting method. We successfully applied the method for the enumeration of E. coli in milk. In conclusion, the thermosetting oil enables the immobilization of droplets to achieve real-time monitoring and digital counting of bacterial growth without impairing the flexibility of droplet microfluidics, and it has the potential to provide dynamic information at high resolution in this process.
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Infecciones por Escherichia coli , Escherichia coli , Recuento de Células , Humanos , Microfluídica , Análisis de la Célula IndividualRESUMEN
Spontaneous DNA damage frequently occurs on the human genome, and it could alter gene expression by inducing mutagenesis or epigenetic changes. Therefore, it is highly desired to profile DNA damage distribution on the human genome and identify the genes that are prone to DNA damage. Here, we present a novel single-cell whole-genome amplification method which employs linear-copying followed by a split-amplification scheme, to efficiently remove amplification errors and achieve accurate detection of DNA damage in individual cells. In comparison to previous methods that measure DNA damage, our method uses a next-generation sequencing platform to detect misincorporated bases derived from spontaneous DNA damage with single-cell resolution.
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We report a novel single-cell whole-genome amplification method (LCS-WGA) that can efficiently capture spontaneous DNA damage existing in single cells. We refer to these damage-associated single-nucleotide variants as "damSNVs," and the whole-genome distribution of damSNVs as the damagenome. We observed that in single human neurons, the damagenome distribution was significantly correlated with three-dimensional genome structures. This nonuniform distribution indicates different degrees of DNA damage effects on different genes. Next, we identified the functionals that were significantly enriched in the high-damage genes. Similar functionals were also enriched in the differentially expressed genes (DEGs) detected by single-cell transcriptome of both Alzheimer's disease (AD) and autism spectrum disorder (ASD). This result can be explained by the significant enrichment of high-damage genes in the DEGs of neurons for both AD and ASD. The discovery of high-damage genes sheds new lights on the important roles of DNA damage in human diseases and disorders.
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Enfermedad de Alzheimer , Trastorno del Espectro Autista , Enfermedad de Alzheimer/genética , Trastorno del Espectro Autista/genética , Daño del ADN , Perfilación de la Expresión Génica , Genoma Humano , Humanos , TranscriptomaRESUMEN
Cancer stem-like cells (CSCs) displaying the properties of normal stem cells have become the main culprit associated with cancer transportation and recurrence. As of now, various CSC functions and marker genes have been identified due to the heterogeneity of cancer, such as aldehyde dehydrogenase (ALDH), the second member of the ABC transporter G-subfamily (ABCG2), activated leukocyte cell adhesion molecule (ALCAM) and CD133. To investigate these markers, most conventional approaches are bulk-based strategies, which may veil the disparity of single cells' gene expression. In this study, one-step digital RT-PCR at the single cell level was developed to co-determine the expression of ALDH1A1, ABCG2, ALCAM and CD133 genes in A549 cancer stem cells that perform high ALDH activities (ALDH+ A549 cells), as well as in ALDH- A549 cells and A549 cells, with 36, 20 and 20 cell samples each. The results demonstrated that, when compared to single ALDH- or A549 cells, the majority of single ALDH+ A549 cells displayed a 1.5- and 2.0-fold increase in the gene expression of ALDH1A1 and ALCAM (P < 0.001), respectively. However, for ABCG2 and CD133, there was no significant difference (P > 0.05), which means that they are not appropriate as co-indicated markers to identify ALDH+ A549 cells. Conclusively, as a single cell level approach, one-step digital RT-PCR has potential in exploring efficient co-detection markers for the classification and identification of CSCs.
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Antígeno AC133/análisis , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/análisis , Aldehído Deshidrogenasa/análisis , Antígenos CD/análisis , Moléculas de Adhesión Celular Neuronal/análisis , Proteínas Fetales/análisis , Proteínas de Neoplasias/análisis , Células Madre Neoplásicas/metabolismo , Células A549 , Familia de Aldehído Deshidrogenasa 1 , Adhesión Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Retinal-DeshidrogenasaRESUMEN
As an absolute quantification method at the single-molecule level, digital PCR has been widely used in many bioresearch fields, such as next generation sequencing, single cell analysis, gene editing detection and so on. However, existing digital PCR methods still have some disadvantages, including high cost, sample loss, and complicated operation. In this work, we develop an exquisite scalable self-priming fractal branching microchannel net digital PCR chip. This chip with a special design inspired by natural fractal-tree systems has an even distribution and 100% compartmentalization of the sample without any sample loss, which is not available in existing chip-based digital PCR methods. A special 10 nm nano-waterproof layer was created to prevent the solution from evaporating. A vacuum pre-packaging method called self-priming reagent introduction is used to passively drive the reagent flow into the microchannel nets, so that this chip can realize sequential reagent loading and isolation within a couple of minutes, which is very suitable for point-of-care detection. When the number of positive microwells stays in the range of 100 to 4000, the relative uncertainty is below 5%, which means that one panel can detect an average of 101 to 15 374 molecules by the Poisson distribution. This chip is proved to have an excellent ability for single molecule detection and quantification of low expression of hHF-MSC stem cell markers. Due to its potential for high throughput, high density, low cost, lack of sample and reagent loss, self-priming even compartmentalization and simple operation, we envision that this device will significantly expand and extend the application range of digital PCR involving rare samples, liquid biopsy detection and point-of-care detection with higher sensitivity and accuracy.
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Técnicas Analíticas Microfluídicas/instrumentación , Reacción en Cadena de la Polimerasa/instrumentación , Reacción en Cadena de la Polimerasa/métodos , Línea Celular , Diseño de Equipo , Fractales , Humanos , Células Madre Mesenquimatosas/citología , Nanotecnología/instrumentaciónRESUMEN
Single cell analysis provides a new framework for understanding biology and disease, however, an absolute quantification of single cell gene expression still faces many challenges. Microfluidic digital polymerase chain reaction (PCR) provides a unique method to absolutely quantify the single cell gene expression, but only limited devices are developed to analyze a single cell with detection variation. This paper describes a self-priming compartmentalization (SPC) microfluidic digital polymerase chain reaction chip being capable of performing single molecule amplification from single cell. The chip can be used to detect four single cells simultaneously with 85% of sample digitization. With the optimized protocol for the SPC chip, we first tested the ability, precision, and sensitivity of our SPC digital PCR chip by assessing ß-actin DNA gene expression in 1, 10, 100, and 1000 cells. And the reproducibility of the SPC chip is evaluated by testing 18S rRNA of single cells with 1.6%-4.6% of coefficient of variation. At last, by detecting the lung cancer related genes, PLAU gene expression of A549 cells at the single cell level, the single cell heterogeneity was demonstrated. So, with the power-free, valve-free SPC chip, the gene copy number of single cells can be quantified absolutely with higher sensitivity, reduced labor time, and reagent. We expect that this chip will enable new studies for biology and disease.
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Visual detections based on fluorescence and the color changes under natural light are two promising product detections for isothermal nucleic acid amplifications (INAAs) such as the isothermal multiple-self-matching-initiated amplification (IMSA) as point-of-care testing techniques. However, the currently used approaches have shortcomings in application. For the former, fluorescence changes recognized by naked eye may be indistinguishable because of single fluorescence emitted and strong background noise, which requires empirical preset of cutoff intensity values. For the latter, visual detection sensitivity under natural light is not comparable to that based on fluorescence. Herein, hydroxyl naphthol blue (HNB) and SYBR Green I (SG) were coupled to acquire a label-free dual fluorescence for the visual product detection of IMSA. The mixed-dye-loaded off-chip (tube-based) and on-chip (microfluidic chip-based) IMSAs for the detection of hepatitis B virus were conducted. The results demonstrated that this dual fluorescence could realize distinguishable fluorescent color changes to improve visual detection sensitivity and avoid the preset of cutoff values. Moreover, the mixed dye is stable when kept at room temperature and compatible with the IMSA's reagents without a contamination-prone step of opening tubes after amplification. Also, this coupled dye inherits the advantages of achieving color changes under natural light from HNB and real-time detection from SG. In conclusion, the mixed-dye-based dual fluorescence has a potential in the point-of-care testing application for realizing off-chip and on-chip product detection of IMSA, loop-mediated isothermal amplification (LAMP), or other INAAs.
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A nanoliter self-priming compartmentalization (SPC) microfluidic chip suited for the digital polymerase chain reaction (dPCR) analysis in point-of-care testing (POCT) has been developed. This dPCR chip is fabricated of polydimethylsiloxane (PDMS). After the dPCR chip is evacuated, there will be a negative pressure environment in the chip because of the gas solubility of PDMS. The negative pressure environment can provide a self-priming power so that the sample solutions can be sucked into each reaction chamber sequentially. The whole sampling process requires no external power and is valve-free. Channels that contain water are designed around each sample panel to prevent the solvent (water) from evaporating during dPCR process. A glass coverslip is also used as a waterproof layer, which is more convenient and more efficient than other waterproof methods seen in literature. This dPCR chip allows three samples to be amplified at the same time. Each sample is distributed into 1040 reaction chambers, and each chamber is only 2.08 nL. Human ß-actin DNA solutions of known concentrations are used as the templates for the dPCR analyses to verify the sensitivity and accuracy of the method. Template DNA solutions diluted to concentrations of 300, 100 and 10 copies/µL are tested and shown that this simple, portable and self-priming dPCR chip can be used at any clinic as a real POCT technique.
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Técnicas Biosensibles/instrumentación , ADN/genética , Dispositivos Laboratorio en un Chip , Nanotecnología/instrumentación , Sistemas de Atención de Punto , Reacción en Cadena de la Polimerasa/instrumentación , Cartilla de ADN/genética , Diseño de Equipo , Análisis de Falla de Equipo , Microquímica/instrumentación , Microscopía Fluorescente/instrumentación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Procesamiento de Señales Asistido por Computador/instrumentaciónRESUMEN
A nanoliter self-priming compartmentalization (SPC) microfluidic chip suited for the digital polymerase chain reaction (dPCR) analysis in point-of-care testing (POCT) has been developed. This dPCR chip is fabricated of polydimethylsiloxane (PDMS). After the dPCR chip is evacuated, there will be a negative pressure environment in the chip because of the gas solubility of PDMS. The negative pressure environment can provide a self-priming power so that the sample solutions can be sucked into each reaction chamber sequentially. The whole sampling process requires no external power and is valve-free. Channels that contain water are designed around each sample panel to prevent the solvent (water) from evaporating during dPCR process. A glass coverslip is also used as a waterproof layer, which is more convenient and more efficient than other waterproof methods seen in literature. This dPCR chip allows three samples to be amplified at the same time. Each sample is distributed into 1040 reaction chambers, and each chamber is only 2.08 nL. Human ß-actin DNA solutions of known concentrations are used as the templates for the dPCR analyses to verify the sensitivity and accuracy of the method. Template DNA solutions diluted to concentrations of 300, 100 and 10 copies/µL are tested and shown that this simple, portable and self-priming dPCR chip can be used at any clinic as a real POCT technique.
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Técnicas Biosensibles/instrumentación , ADN/genética , Dispositivos Laboratorio en un Chip , Nanotecnología/instrumentación , Sistemas de Atención de Punto , Reacción en Cadena de la Polimerasa/instrumentación , Cartilla de ADN/genética , Diseño de Equipo , Análisis de Falla de Equipo , Microquímica/instrumentación , Microscopía Fluorescente/instrumentación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Procesamiento de Señales Asistido por Computador/instrumentaciónRESUMEN
An integrated on-chip valve-free and power-free microfluidic digital PCR device is for the first time developed by making use of a novel self-priming compartmentalization and simple dehydration control to realize 'divide and conquer' for single DNA molecule detection. The high gas solubility of PDMS is exploited to provide the built-in power of self-priming so that the sample and oil are sequentially sucked into the device to realize sample self-compartmentalization based on surface tension. The lifespan of its self-priming capability was about two weeks tested using an air-tight packaging bottle sealed with a small amount of petroleum jelly, which is significant for a practical platform. The SPC chip contains 5120 independent 5 nL microchambers, allowing the samples to be compartmentalized completely. Using this platform, three different abundances of lung cancer related genes are detected to demonstrate the feasibility and flexibility of the microchip for amplifying a single nucleic acid molecule. For maximal accuracy, within less than 5% of the measurement deviation, the optimal number of positive chambers is between 400 and 1250 evaluated by the Poisson distribution, which means one panel can detect an average of 480 to 4804 template molecules. This device without world-to-chip connections eliminates the constraint of the complex pipeline control, and is an integrated on-chip platform, which would be a significant improvement to digital PCR automation and more user-friendly.
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ADN de Neoplasias/química , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Reacción en Cadena de la Polimerasa/instrumentación , Reacción en Cadena de la Polimerasa/métodos , Línea Celular Tumoral , HumanosRESUMEN
Digital nucleic acid amplification provides unprecedented opportunities for absolute nucleic acid quantification by counting of single molecules. This technique is useful for molecular genetic analysis in cancer, stem cell, bacterial, non-invasive prenatal diagnosis in which many biologists are interested. This paper describes a self-priming compartmentalization (SPC) microfluidic chip platform for performing digital loop-mediated amplification (LAMP). The energy for the pumping is pre-stored in the degassed bulk PDMS by exploiting the high gas solubility of PDMS; therefore, no additional structures other than channels and reservoirs are required. The sample and oil are sequentially sucked into the channels, and the pressure difference of gas dissolved in PDMS allows sample self-compartmentalization without the need for further chip manipulation such as with pneumatic microvalves and control systems, and so on. The SPC digital LAMP chip can be used like a 384-well plate, so, the world-to-chip fluidic interconnections are avoided. The microfluidic chip contains 4 separate panels, each panel contains 1200 independent 6 nL chambers and can be used to detect 4 samples simultaneously. Digital LAMP on the microfluidic chip was tested quantitatively by using ß-actin DNA from humans. The self-priming compartmentalization behavior is roughly predictable using a two-dimensional model. The uniformity of compartmentalization was analyzed by fluorescent intensity and fraction of volume. The results showed that the feasibility and flexibility of the microfluidic chip platform for amplifying single nucleic acid molecules in different chambers made by diluting and distributing sample solutions. The SPC chip has the potential to meet the requirements of a general laboratory: power-free, valve-free, operating at isothermal temperature, inexpensive, sensitive, economizing labour time and reagents. The disposable analytical devices with appropriate air-tight packaging should be useful for point-of-care, and enabling it to become one of the common tools for biology research, especially, in point-of-care testing.
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Técnicas Analíticas Microfluídicas/instrumentación , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Sistemas de Atención de Punto , Dimetilpolisiloxanos/química , Diseño de EquipoRESUMEN
Near-infrared gold-doped CdHgTe quantum dots (QDs) with improved photoluminescence and biocompatibility were developed using an aqueous solution route with L-glutathione and L-cysteine as stabilizers. As-prepared Au:CdHgTe QDs were covalently linked to arginine-glycine-aspartic acid (RGD) peptide, anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb), and anti- carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) MAb separately. Three Au:CdHgTe QD bioconjugates (QD800-RGD, QD820-anti-CEACAM1, and QD840-anti-EGFR) were successfully used as probes for in vivo tumor-targeted multispectral fluorescence imaging of xenografts. Fluorescence signals from the QD bioconjugates used to detect three tumor markers were spectrally unmixed, and their co-localization was analyzed. The results indicate that multiple tumor markers could be simultaneously detected by multispectral fluorescence imaging in vivo using QD bioconjugates as probes. This approach has excellent potential as an imaging method for the noninvasive exploration and detection of multiple tumor markers in vivo, thereby substantially aiding the diagnosis of cancer.
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Compuestos de Cadmio/química , Compuestos de Mercurio/química , Imagen Molecular/métodos , Neoplasias/diagnóstico , Animales , Línea Celular Tumoral , Femenino , Fluorescencia , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Neoplasias/química , Puntos CuánticosRESUMEN
A microdevice made of glass for genetic analysis has been fabricated, for the first time, for integration of extraction of nucleic acids and loop-mediated isothermal amplification (LAMP), followed by online fluorescence detection of amplification products on a single chip. The nucleic acid (NA) extraction region consists of a microfabricated serpentine channel in which micropillars were etched to increase the channel surface area and the capture efficiency of NAs. Nucleic acid molecules were bound to these pillars and channel surface in the presence of the chaotropic salt guanidine hydrochloride and eluted into a downstream amplification chamber with low ionic strength buffer where loop-mediated isothermal amplification was efficiently performed. Amplification can be detected online by the increase of fluorescence intensity at 540 nm when a low concentration of SYBR Green I, a fluorescent dsDNA intercalating dye, is employed. Flow control was accomplished by using laminar flow and differential channel flow resistances. Through passivation of the LAMP chamber and the channel between the extraction region and amplification domain, effective nucleic acid extraction and amplification were performed by just using a double-channel syringe pump and a heating block. By using this integrated microdevice, the purification of nucleic acids from complex biological matrixes and their subsequent amplification and detection online could be finished within 2 h.