RESUMEN
PROTACs have emerged as a new class of drugs that can target the "undruggable" proteome by hijacking the ubiquitin proteasome system. Despite PROTACs' success, most current PROTACs interface with a limited number of E3 ligases, hindering their expansion to many challenging therapeutic uses. Currently, PROTAC drug discovery relies heavily on traditional Western blotting and reporter gene assays which are insensitive and prone to artifacts, respectively. New reliable methods to monitor true PROTAC function (i.e., ubiquitination and subsequent degradation of targets at physiological expression levels) without external tags are essential to accelerate the PROTAC discovery process and to address many unmet therapeutic areas. In this study, we developed a new high-throughput screening technology using "TUBEs" as ubiquitin-binding entities to monitor PROTAC-mediated poly-ubiquitination of native target proteins with exceptional sensitivity. As a proof of concept, targets including BRD3, Aurora A Kinase, and KRAS were used to demonstrate that ubiquitination kinetics can reliably establish the rank order potencies of PROTAC with variable ligands and linkers. PROTAC-treated cell lysates with the highest levels of endogenous target protein ubiquitination - termed "UbMax" - display excellent correlations with DC50 values obtained from traditional Western blots with the added benefits of being high throughput, providing improved sensitivity, and reducing technical errors.
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Aurora Quinasa A , Complejo de la Endopetidasa Proteasomal , Aurora Quinasa A/metabolismo , Descubrimiento de Drogas/métodos , Ligandos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteoma/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Ubiquitinas/metabolismoRESUMEN
The ubiquitin proteasome system (UPS) is a complex pathway that involves multiple enzymes and culminates in the formation of a polyubiquitin chain on a target protein. As its importance is becoming more evident in drug discovery, there is a renewed interest in understanding the role that polyubiquitin chains play. This has been a challenge, mostly due to the lack of experimental tools for detecting the polyubiquitinated forms of a protein of interest (POI). Tandem Ubiquitin Binding Entities (TUBEs) are engineered protein domains that bind specifically to polyubiquitin chains. These polyubiquitin affinity matrices are highly sensitive as they bind to polyubiquitin chains in the nanomolar range. They exist in two forms: pan-selective TUBEs and chain-selective TUBEs. The ability of TUBEs to be conjugated to different entities is truly what makes them unique. TUBEs are used in a wide variety of experiments such as in protein pulldowns to enrich for polyubiquitinated proteins. They are an alternative to ubiquitin antibodies in Western blots. Further, TUBEs are used as capture reagents for immobilizing polyubiquitinated proteins on a microtiter plate. The use of TUBEs as components of in vitro and cell-based assays presents the unique feature of confirming and assessing the polyubiquitination of a POI in response to inhibitors, activators, or PROTAC® molecules. Therefore, TUBEs not only play a big role in studying the UPS but also have a huge potential for speeding up the drug discovery process.
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Descubrimiento de Drogas , Complejo de la Endopetidasa Proteasomal , Ubiquitina , Poliubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Ubiquitina/metabolismoRESUMEN
Rapid and efficient biological sample preparation and pretreatment are crucial for highly sensitive, reliable and reproducible molecular detection of infectious diseases. Herein, we report a self-powered, integrated sample concentrator (SPISC) for rapid plasma separation, pathogen lysis, nucleic acid trapping and enrichment at the point of care. The proposed sample concentrator uses a combination of gravitational sedimentation of blood cells and capillary force for rapid, self-powered plasma separation. The pathogens (e.g., HIV virus) in separated plasma were directly lysed and pathogen nucleic acid was enriched by an integrated, flow-through FTA® membrane in the concentrator, enabling highly efficient nucleic acid preparation. The FTA® membrane of the SPISC is easy to store and transport at room temperature without need for uninterrupted cold chain, which is crucial for point of care sampling in resource-limited settings. The platform has been successfully applied to detect HIV virus in blood samples. Our experiments show that the sample concentrator can achieve a plasma separation efficiency as high as 95% and a detection sensitivity as low as 10 copies per 200 µL blood (â¼100 copies per mL plasma) with variability less than 7%. The sample concentrator described is fully compatible with downstream nucleic acid detection and has great potential for early diagnostics, monitoring and management of infectious diseases at the point of care.
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Enfermedades Transmisibles , Infecciones por VIH , Ácidos Nucleicos , Infecciones por VIH/diagnóstico , Humanos , Técnicas de Amplificación de Ácido Nucleico , Sistemas de Atención de Punto , Impresión TridimensionalRESUMEN
Detecting cancer at an early stage of disease progression promises better treatment outcomes and longer lifespans for cancer survivors. Research has been directed towards the development of accessible and highly sensitive cancer diagnostic tools, many of which rely on protein biomarkers and biomarker panels which are overexpressed in body fluids and associated with different types of cancer. Protein biomarker detection for point-of-care (POC) use requires the development of sensitive, noninvasive liquid biopsy cancer diagnostics that overcome the limitations and low sensitivities associated with current dependence upon imaging and invasive biopsies. Among many endeavors to produce user-friendly, semi-automated, and sensitive protein biomarker sensors, 3D printing is rapidly becoming an important contemporary tool for achieving these goals. Supported by the widely available selection of affordable desktop 3D printers and diverse printing options, 3D printing is becoming a standard tool for developing low-cost immunosensors that can also be used to make final commercial products. In the last few years, 3D printing platforms have been used to produce complex sensor devices with high resolution, tailored towards researchers' and clinicians' needs and limited only by their imagination. Unlike traditional subtractive manufacturing, 3D printing, also known as additive manufacturing, has drastically reduced the time of sensor and sensor array development while offering excellent sensitivity at a fraction of the cost of conventional technologies such as photolithography. In this review, we offer a comprehensive description of 3D printing techniques commonly used to develop immunosensors, arrays, and microfluidic arrays. In addition, recent applications utilizing 3D printing in immunosensors integrated with different signal transduction strategies are described. These applications include electrochemical, chemiluminescent (CL), and electrochemiluminescent (ECL) 3D-printed immunosensors. Finally, we discuss current challenges and limitations associated with available 3D printing technology and future directions of this field.
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Técnicas Biosensibles , Microfluídica , Neoplasias , Impresión Tridimensional , Neoplasias/diagnósticoRESUMEN
Widely accessible, inexpensive, easy-to-use consumer 3D printers, such as desktop stereolithography (SLA) and fused-deposition modeling (FDM) systems are increasingly employed in prototyping and customizing miniaturized fluidic systems for diagnostics and research. However, these 3D printers are generally limited to printing parts made of only one material type, which limits the functionality of the microfluidic devices without additional assembly and bonding steps. Moreover, mating of different materials requires good sealing in such microfluidic devices. Here, we report methods to print hybrid structures comprising a hard, rigid component (clear polymethacrylate polymer) printed by a low-cost SLA printer, and where the first printed part is accurately mated and adhered to a second, soft, flexible component (thermoplastic polyurethane elastomer) printed by an FDM printer. The prescribed mounting and alignment of the first-printed SLA-printed hard component, and its pre-treatment and heating during the second FDM step, can produce leak-free bonds at material interfaces. To demonstrate the utility of such hybrid 3D-printing, we prototype and test three components: i) finger-actuated pump, ii) quick-connect fluid coupler, and iii) nucleic acid amplification test device with screw-type twist sealing for sample introduction.
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Rapid diagnostics of infectious diseases and accurate identification of their causative pathogens play a crucial role in disease prevention, monitoring, and treatment. Conventional molecular detection of infectious pathogens requires expensive equipment and well-trained personnel, thus limiting its use in centralized clinical laboratories. To address this challenge, a portable smartphone-based quantitative molecular detection platform, termed "smart connected pathogen tracer" (SCPT), has been developed for pathogen monitoring and disease surveillance. The platform takes advantage of synergistically enhanced colorimetric loop-mediated isothermal amplification (LAMP) assay and smartphone-based color analysis, enabling simple, rapid and reliable nucleic acid quantification without need for expensive fluorescence detection equipment. The SCPT platform has been successfully applied to quantitatively detect: i) HPV DNA in saliva and clinical vaginal swab samples, and ii) HIV RNA in plasma samples with comparable sensitivity to state-of-art machine. It has also been demonstrated for disease spatiotemporal mapping and pathogen tracking by wireless connection and web-based surveillance. Such simple, cost-affordable, portable molecular detection platform has great potential for on-site early disease detection, remote healthcare monitoring, and epidemic surveillance.
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Prueba de VIH/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Infecciones por Papillomavirus/diagnóstico , Teléfono Inteligente , Colorimetría , ADN Viral/análisis , Femenino , Sistemas de Información Geográfica , Prueba de VIH/instrumentación , Humanos , Dispositivos Laboratorio en un Chip , Técnicas de Diagnóstico Molecular/instrumentación , Infecciones por Papillomavirus/virología , ARN Viral/sangre , Saliva/virología , Vagina/virologíaRESUMEN
We report herein a novel pipet-based "ELISA in a tip" as a new versatile diagnostic tool featuring better sensitivity, shorter incubation time, accessibility, and low sample and reagent volumes compared to traditional ELISA. Capture and analysis of data by a cell phone facilitates electronic delivery of results to health care providers. Pipette tips were designed and 3D printed as adapters to fit most commercial 50-200 µL pipettes. Capture antibodies (Ab1) are immobilized on the inner walls of the pipet tip, which serves as the assay compartment where samples and reagents are moved in and out by pipetting. Signals are generated using colorimetric or chemiluminescent (CL) reagents and can be quantified using a cell phone, CCD camera, or plate reader. We utilized pipet-tip ELISA to detect four cancer biomarker proteins with detection limits similar to or lower than microplate ELISAs at 25% assay cost and time. Recoveries of these proteins from spiked human serum were 85-115% or better, depending slightly on detection mode. Using CCD camera quantification of CL with femto-luminol reagent gave limits of detection (LOD) as low as 0.5 pg/mL. Patient samples (13) were assayed for 3 biomarker proteins with results well correlated to conventional ELISA and an established microfluidic electrochemical immunoassay.
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Biomarcadores de Tumor/análisis , Ensayo de Inmunoadsorción Enzimática , Impresión Tridimensional , Neoplasias de la Próstata/diagnóstico , Telemedicina , Anticuerpos/inmunología , Biomarcadores de Tumor/inmunología , Técnicas Biosensibles , Teléfono Celular , Técnicas Electroquímicas , Humanos , Factor I del Crecimiento Similar a la Insulina/análisis , Factor I del Crecimiento Similar a la Insulina/inmunología , Receptores de Lipopolisacáridos/análisis , Receptores de Lipopolisacáridos/inmunología , Masculino , Técnicas Analíticas Microfluídicas , Antígeno Prostático Específico/análisis , Antígeno Prostático Específico/inmunologíaRESUMEN
Rationale: Early and accurate detection of disease is crucial for its prevention, identification, and treatment. However, most of disease diagnostics is still limited in clinical laboratories due to the need of complicated instruments and professional personnel. Herein, we reported a smartphone-based synergistically enhanced colorimetric method for molecular diagnostics in our point of care (POC) smart cup platform. Methods: A disposable microfluidic chip was developed for colorimetric loop-mediated isothermal amplification (LAMP) detection of multiple HPV DNA in our POC smart cup platform. The colorimetric detection takes advantage of synergistic effect of PPi4- and H+ ions, two byproducts of LAMP reaction. Color signal of LAMP assay was recorded and analyzed by our custom Android app (dubbed "Hue Analyzer"). Results: Our method not only significantly improves colorimetric readout, but also provides a 10-fold increase in detection sensitivity. It has been successfully applied for HPV-associated cancer screening with spiked saliva and clinical swab samples. Conclusion: The proposed POC diagnostic platform is completely compatible with other nucleic acid biomarkers and has great potential for personalized health monitoring and disease prevention.
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Colorimetría/métodos , ADN Viral/genética , Detección Precoz del Cáncer/métodos , Papillomavirus Humano 16/aislamiento & purificación , Papillomavirus Humano 18/aislamiento & purificación , Papillomavirus Humano 31/aislamiento & purificación , Infecciones por Papillomavirus/diagnóstico , Cuello del Útero/virología , Colorimetría/normas , ADN Viral/clasificación , ADN Viral/aislamiento & purificación , Detección Precoz del Cáncer/instrumentación , Femenino , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Papillomavirus Humano 31/genética , Humanos , Dispositivos Laboratorio en un Chip , Técnicas de Diagnóstico Molecular/normas , Técnicas de Amplificación de Ácido Nucleico/normas , Prueba de Papanicolaou , Infecciones por Papillomavirus/virología , Sistemas de Atención de Punto , Saliva/virología , Sensibilidad y Especificidad , Teléfono InteligenteRESUMEN
Post-translational modifications (PTMs) by ubiquitin (Ub) are versatile, highly dynamic, and involved in nearly all aspects of eukaryote biological function. The reversibility and heterogeneity of Ub chains attached to protein substrates have complicated their isolation, quantification, and characterization. Strategies have emerged to isolate endogenous ubiquitylated targets, including technologies based on the use of Ub-binding peptides, such as tandem-repeated Ub-binding entities (TUBEs). TUBEs allow the identification and characterization of Ub chains, and novel substrates for deubiquitylases (DUBs) and Ub ligases (E3s). Here we review their impact on purification, analysis of pan or chain-selective polyubiquitylated proteins and underline the biological relevance of this information. Together with peptide aptamers and other Ub affinity-based approaches, TUBEs will contribute to unraveling the secrets of the Ub code.
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Ubiquitina/metabolismo , Ubiquitinación , Animales , Humanos , Unión Proteica , Procesamiento Proteico-PostraduccionalAsunto(s)
Bacterias/aislamiento & purificación , Técnicas de Química Analítica/métodos , Pruebas en el Punto de Atención , Teléfono Inteligente , Virus/aislamiento & purificación , Bacterias/química , Proteínas Bacterianas/análisis , Humanos , Plasmodium falciparum/química , Plasmodium falciparum/aislamiento & purificación , Proteínas Protozoarias/análisis , Proteínas Virales/análisis , Virus/químicaRESUMEN
Reactive metabolites of environmental chemicals and drugs can cause site specific damage to the p53 tumor suppressor gene in a major pathway for genotoxicity. We report here a high-throughput, cell-free, 96-well plate magnetic bead-enzyme system interfaced with LC-MS/MS sequencing for bioactivating test chemicals and identifying resulting adduction sites on genes. Bioactivated aflatoxin B1 was reacted with a 32 bp exon 7 fragment of the p53 gene using eight microsomal cytochrome (cyt) P450 enzymes from different organs coated on magnetic beads. All cyt P450s converted aflatoxin B1 to aflatoxin B1-8,9-epoxide that adducts guanine (G) in codon 249, with subsequent depurination to give abasic sites and then strand breaks. This is the first demonstration in a cell-free medium that the aflatoxin B1 metabolite selectively causes abasic site formation and strand breaks at codon 249 of the p53 probe, corresponding to the chemical pathway and mutations of p53 in human liver cells and tumors. Molecular modeling supports the view that binding of aflatoxin B1-8,9-epoxide to G in codon 249 precedes the SN2 adduction reaction. Among a range of metabolic enzymes characteristic of different organs, human liver microsomes and cyt P450 3A5 supersomes showed the highest bioactivation rate for p53 exon 7 damage. This method of identifying metabolite-related gene damage sites may facilitate predictions of organ specific cancers for test chemicals via correlations with mutation sites.
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Exones , Campos Magnéticos , Espectrometría de Masas , Oligonucleótidos/química , Análisis de Secuencia de ADN/métodos , Proteína p53 Supresora de Tumor/genética , Aflatoxina B1/análogos & derivados , Aflatoxina B1/química , Reactores Biológicos , Cromatografía Liquida , Sistema Enzimático del Citocromo P-450/química , HumanosRESUMEN
We report here the fabrication and validation of a novel 3D-printed, automated immunoarray to detect multiple proteins with ultralow detection limits. This low cost, miniature immunoarray employs electrochemiluminescent (ECL) detection measured with a CCD camera and employs touch-screen control of a micropump to facilitate automated use. The miniaturized array features prefilled reservoirs to deliver sample and reagents to a paper-thin pyrolytic graphite microwell detection chip to complete sandwich immunoassays. The detection chip achieves high sensitivity by using single-wall carbon nanotube-antibody conjugates in the microwells and employing massively labeled antibody-decorated RuBPY-silica nanoparticles to generate ECL. The total cost of an array is $0.65, and an eight-protein assay can be done in duplicate for $0.14 per protein with limits of detection (LOD) as low as 78-110 fg mL-1 in diluted serum. The electronic control system costs $210 in components. Utility of the automated immunoarray was demonstrated by detecting an eight-protein prostate cancer biomarker panel in human serum samples in 25 min. The system is well suited to future clinical and point-of-care diagnostic testing and could be used in resource-limited environments.
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Automatización , Biomarcadores de Tumor/sangre , Técnicas Analíticas Microfluídicas , Nanoestructuras/química , Proteínas de Neoplasias/sangre , Impresión Tridimensional , Neoplasias de la Próstata/sangre , Línea Celular Tumoral , Humanos , Masculino , Técnicas Analíticas Microfluídicas/instrumentación , Impresión Tridimensional/instrumentación , Neoplasias de la Próstata/diagnósticoRESUMEN
Molecular diagnostics that involve nucleic acid amplification tests (NAATs) are crucial for prevention and treatment of infectious diseases. In this study, we developed a simple, inexpensive, disposable, fully 3D printed microfluidic reactor array that is capable of carrying out extraction, concentration and isothermal amplification of nucleic acids in variety of body fluids. The method allows rapid molecular diagnostic tests for infectious diseases at point of care. A simple leak-proof polymerization strategy was developed to integrate flow-through nucleic acid isolation membranes into microfluidic devices, yielding a multifunctional diagnostic platform. Static coating technology was adopted to improve the biocompatibility of our 3D printed device. We demonstrated the suitability of our device for both end-point colorimetric qualitative detection and real-time fluorescence quantitative detection. We applied our diagnostic device to detection of Plasmodium falciparum in plasma samples and Neisseria meningitides in cerebrospinal fluid (CSF) samples by loop-mediated, isothermal amplification (LAMP) within 50â¯min. The detection limits were 100 fg for P. falciparum and 50 colony-forming unit (CFU) for N. meningitidis per reaction, which are comparable to that of benchtop instruments. This rapid and inexpensive 3D printed device has great potential for point-of-care molecular diagnosis of infectious disease in resource-limited settings.
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Técnicas Biosensibles , Neisseria meningitidis/aislamiento & purificación , Plasmodium falciparum/aislamiento & purificación , Líquido Cefalorraquídeo/microbiología , Colorimetría , Humanos , Límite de Detección , Malaria Falciparum/sangre , Malaria Falciparum/parasitología , Infecciones Meningocócicas/líquido cefalorraquídeo , Infecciones Meningocócicas/microbiología , Infecciones Meningocócicas/patología , Microfluídica , Neisseria meningitidis/patogenicidad , Ácidos Nucleicos/química , Ácidos Nucleicos/aislamiento & purificación , Patología Molecular , Plasmodium falciparum/patogenicidad , Sistemas de Atención de Punto , Impresión TridimensionalRESUMEN
Low cost, miniaturized assay platforms that work with small sample volumes, high sensitivity and rapid detection will have high value in future biomolecular diagnostics. Herein we report an automated, 3D printed electrochemiluminescent (ECL) immunoarray integrated with a nanostructured pyrolytic graphite sheet (PGS) microwell chip configured to detect 2 proteins simultaneously from complex liquid samples with high sensitivity and selectivity. Assays are done in 18 min at cost of < $1.00 using 1-2 microliters of sample. 3D printed microfluidic array design integrates reagent and sample chambers with rapid ECL detection. A commercial programmable syringe pump used with a preset program allows pump to pause and resume reagent delivery as required for completion of the sandwich immunoassays. Nanostructured surfaces feature antibody-decorated single wall carbon nanotube forests on PGS chip microwells, and sensitivity is amplified via massively labeled RuBPY-silica nanoparticles for detection. Prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA) were measured simultaneously from human serum on the immunoarray with detection limits 150 fg mL-1 for PSA and 230 fg mL-1 for PSMA, with dynamic ranges up to 5 ng mL-1. Validation of the immunoarray by measuring these proteins in human serum showed good correlation with single protein ELISA. These 3D printed platforms can be easily adapted to multiple applications and configurable CAD files for the immunoarray can be downloaded from our lab's website.
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Nearly all implantable bioelectronics are powered by bulky batteries which limit device miniaturization and lifespan. Moreover, batteries contain toxic materials and electrolytes that can be dangerous if leakage occurs. Herein, an approach to fabricate implantable protein-based bioelectrochemical capacitors (bECs) employing new nanocomposite heterostructures in which 2D reduced graphene oxide sheets are interlayered with chemically modified mammalian proteins, while utilizing biological fluids as electrolytes is described. This protein-modified reduced graphene oxide nanocomposite material shows no toxicity to mouse embryo fibroblasts and COS-7 cell cultures at a high concentration of 1600 µg mL-1 which is 160 times higher than those used in bECs, unlike the unmodified graphene oxide which caused toxic cell damage even at low doses of 10 µg mL-1. The bEC devices are 1 µm thick, fully flexible, and have high energy density comparable to that of lithium thin film batteries. COS-7 cell culture is not affected by long-term exposure to encapsulated bECs over 4 d of continuous charge/discharge cycles. These bECs are unique, protein-based devices, use serum as electrolyte, and have the potential to power a new generation of long-life, miniaturized implantable devices.
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Selective removal of albumin from human serum is an essential step prior to proteomic analyses, especially when using mass spectrometry. Here we report stable synthetic nanopockets on magnetic nanoparticle surfaces that bind to human serum albumin (HSA) with high affinity and specificity. The nanopockets are created by templating HSA on 200 nm silica-coated paramagnetic nanoparticles using polymer layers made using 4 organo-silane monomers. These monomers have amino acid-like side chains providing hydrophobic, hydrophilic and H-bonding interactions that closely mimic features of binding sites on antibodies. The binding capacity of the material was 21 mg HSA g-1, and consistently removed â¼88% albumin from human serum in multiple repeated use.
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Nanopartículas de Magnetita/química , Albúmina Sérica/química , Albúmina Sérica/aislamiento & purificación , Dióxido de Silicio/química , Humanos , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
A novel, automated, low cost, three-dimensional (3-D) printed microfluidic array was developed to detect DNA damage from metabolites of chemicals in environmental samples. The electrochemiluminescent (ECL) detection platform incorporates layer-by-layer (LbL) assembled films of microsomal enzymes, DNA and an ECL-emitting ruthenium metallopolymer in â¼10 nm deep microwells. Liquid samples are introduced into the array, metabolized by the human enzymes, products react with DNA if possible, and DNA damage is detected by ECL with a camera. Measurements of relative DNA damage by the array assess the genotoxic potential of the samples. The array analyzes three samples simultaneously in 5 min. Measurement of cigarette and e-cigarette smoke extracts and polluted water samples was used to establish proof of concept. Potentially genotoxic reactions from e-cigarette vapor similar to smoke from conventional cigarettes were demonstrated. Untreated wastewater showed a high genotoxic potential compared to negligible values for treated wastewater from a pollution control treatment plant. Reactivity of chemicals known to produce high rates of metabolite-related DNA damage were measured, and array results for environmental samples were expressed in terms of equivalent responses from these standards to assess severity of possible DNA damage. Genotoxic assessment of wastewater samples during processing also highlighted future on-site monitoring applications.
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Methylation of cytosine (C) at C-phosphate-guanine (CpG) sites enhances reactivity of DNA towards electrophiles. Mutations at CpG sites on the p53 tumor suppressor gene that can result from these adductions are in turn correlated with specific cancers. Here we describe the first restriction-enzyme-assisted LC-MS/MS sequencing study of the influence of methyl cytosines (MeC) on kinetics of p53 gene adduction by model metabolite benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), using methodology applicable to correlate gene damage sites for drug and pollutant metabolites with mutation sites. This method allows direct kinetic measurements by LC-MS/MS sequencing for oligonucleotides longer than 20 base pairs (bp). We used MeC and non-MeC (C) versions of a 32 bp exon 7 fragment of the p53 gene. Methylation of 19 cytosines increased the rate constant 3-fold for adduction on G at the major reactive CpG in codon 248 vs. the non-MeC fragment. Rate constants for non-CpG codons 244 and 243 were not influenced significantly by MeC. Conformational and hydrophobicity changes in the MeC-p53 exon 7 fragment revealed by CD spectra and molecular modeling increase the BPDE binding constant to G in codon 248 consistent with a pathway in which preceding reactant binding greatly facilitates the rate of covalent SN2 coupling.
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7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/química , Citosina/química , Aductos de ADN/análisis , Proteína p53 Supresora de Tumor/genética , Sitios de Unión , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Islas de CpG , Citosina/análogos & derivados , Exones , Humanos , Cinética , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico , Espectrometría de Masas en Tándem , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
While 3D printing technologies first appeared in the 1980s, prohibitive costs, limited materials, and the relatively small number of commercially available printers confined applications mainly to prototyping for manufacturing purposes. As technologies, printer cost, materials, and accessibility continue to improve, 3D printing has found widespread implementation in research and development in many disciplines due to ease-of-use and relatively fast design-to-object workflow. Several 3D printing techniques have been used to prepare devices such as milli- and microfluidic flow cells for analyses of cells and biomolecules as well as interfaces that enable bioanalytical measurements using cellphones. This review focuses on preparation and applications of 3D-printed bioanalytical devices.
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Impresión Tridimensional , MicrofluídicaRESUMEN
Herein we report a low cost, sensitive, supercapacitor-powered electrochemiluminescent (ECL) protein immunoarray fabricated by an inexpensive 3-dimensional (3D) printer. The immunosensor detects three cancer biomarker proteins in serum within 35 min. The 3D-printed device employs hand screen printed carbon sensors with gravity flow for sample/reagent delivery and washing. Prostate cancer biomarker proteins, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA) and platelet factor-4 (PF-4) in serum were captured on the antibody-coated carbon sensors followed by delivery of detection-antibody-coated Ru(bpy)3(2+) (RuBPY)-doped silica nanoparticles in a sandwich immunoassay. ECL light was initiated from RuBPY in the silica nanoparticles by electrochemical oxidation with tripropylamine (TPrA) co-reactant using supercapacitor power and ECL was captured with a CCD camera. The supercapacitor was rapidly photo-recharged between assays using an inexpensive solar cell. Detection limits were 300-500f gmL(-1) for the 3 proteins in undiluted calf serum. Assays of 6 prostate cancer patient serum samples gave good correlation with conventional single protein ELISAs. This technology could provide sensitive onsite cancer diagnostic tests in resource-limited settings with the need for only moderate-level training.