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Herein, a smartphone-based portable reader with integrated optics for standard microtiter plates (96 wells) has been designed and demonstrated for high-throughput quantitation of validated biomarkers in serum. The customized optical attachment was simply constructed with a convex lens and a light source, by which the transmitted light through a 96-well microtiter plate was converged for imaging with a smartphone, so that accurate and wide-range reading of the plate can be achieved. More importantly, relying on the digitized colorimetric analysis of the obtained images, concentrations of various biomarkers can be determined directly using the customized mobile app. A set of validated biomarkers for inflammation and infection, C-reactive protein (CRP), serum amyloid A (SAA), and procalcitonin (PCT) have been quantitated with this new system; both the response ranges and limits of detection meet the requirement of clinical tests. The consistency with the results obtained using a commercial microplate reader proves its reliability and precision, augments its potential as a point-of-care diagnostic device for on-site testing or resource-limited settings.
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Aplicaciones Móviles , Teléfono Inteligente , Reproducibilidad de los Resultados , Colorimetría/métodos , Sistemas de Atención de PuntoRESUMEN
To enable the optimal, biocompatible and non-destructive application of the highly useful copper (Cu+)-mediated alkyne-azide 'click' cycloaddition in water, we have isolated and characterized a 79-nucleotide DNA enzyme or DNAzyme, 'CLICK-17', that harnesses as low as sub-micromolar Cu+; or, surprisingly, Cu2+ (without added reductants such as ascorbate) to catalyze conjugation between a variety of alkyne and azide substrates, including small molecules, proteins and nucleic acids. CLICK-17's Cu+ catalysis is orders of magnitude faster than that of either Cu+ alone or of Cu+ complexed to PERMUT-17, a sequence-permuted DNA isomer of CLICK-17. With the less toxic Cu2+, CLICK-17 attains rates comparable to Cu+, under conditions where both Cu2+ alone and Cu2+ complexed with a classic accelerating ligand, THPTA, are wholly inactive. Cyclic voltammetry shows that CLICK-17, unlike PERMUT-17, powerfully perturbs the Cu(II)/Cu(I) redox potential. CLICK-17 thus provides a unique, DNA-derived ligand environment for catalytic copper within its active site. As a bona fide Cu2+-driven enzyme, with potential for being evolved to accept only designated substrates, CLICK-17 and future variants promise the fast, safe, and substrate-specific catalysis of 'click' bioconjugations, potentially on the surfaces of living cells.
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Cobre/metabolismo , Reacción de Cicloadición/métodos , ADN Catalítico/química , Alquinos/química , Azidas/química , Química Clic/métodos , Oxidación-Reducción , Agua/químicaRESUMEN
The determination of pH values is essential in many chemical, medical, and environmental monitoring processes, which has been relying on conventional pH meters (glass electrodes) for quantitation and pH test strips for qualitative (or semi-quantitative) assessment. In this work, we demonstrate a smartphone-based pH determination technique, which performs digital image analysis of commercial test strips, particularly the determination of either the dominant wavelength (λd) or complementary wavelength (λc) of the color image. In conjunction with a 3D-printed optical accessory (with a surface light source and a macro lens), the quality of captured images have been warranted, and the quantitation accuracy of 0.05 pH units has been achieved. More importantly, the performance of this smartphone-based pH reading system (namely "Smart-pH-Reader") was validated using multiple real-world samples, as the results are consistent with those determined with a standard pH meter. The Smart-pH-Reader is envisioned to be a simple, portable, and accurate tool for pH determination in the fields of environmental monitoring, medical diagnosis, and beyond.
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Teléfono Inteligente , Electrodos , Concentración de Iones de HidrógenoRESUMEN
The design and testing of integrated colorimetric microarray immunochips (immuno-microarrays) are reported for the quantitation and direct visual determination of multiple illicit drugs (e.g., morphine, cocaine and amphetamine) in body fluids. Such an immuno-microarray platform utilizes a competitive immunoassay format, which is based on silver staining for quantitative detection and multicolor staining for direct visualization (i.e., qualitative identification) of analytes present in the sample. Under optimized conditions, the dynamic response ranges of 3.7-1000, 1.1-300 and 1.5-300 ng mL-1 were achieved for amphetamine, cocaine, and morphine, respectively, which are wider towards low concentrations than those of standard enzyme-linked immunosorbent assay (ELISA) tests. The limits of detection (LODs) for morphine, cocaine, and amphetamine were determined to be 1.5 ± 0.1, 1.1 ± 0.1 and 3.7 ± 0.2 ng mL-1, respectively in oral fluids, which meet government regulations for law enforcement. The obvious advantages of multiplexing, simultaneous visual recognition, and accurate quantitation make the on-site detection feasible, confirming that such a colorimetric immuno-microarray holds promise for practical applications.
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Líquidos Corporales/química , Colorimetría/instrumentación , Drogas Ilícitas/análisis , Inmunoensayo/instrumentación , Dispositivos Laboratorio en un Chip , HumanosRESUMEN
Herein we report a quantitative, multiplex assay for disease markers in plasma based on an integrated setup of a portable scanner and a disposable paper-based analytical device (PAD). The quantitative analysis relies on the digital colorimetric reading of the three-layer PAD with 30 assay sites for performing respective chromogenic reactions for plasma uric acid, glucose, and triglyceride, which are considered as important risk factors for cardiovascular diseases. A portable scanner with WiFi transmission capability was used to produce high-quality color images of the PADs and wirelessly transfer them to a smartphone or other mobile devices for data processing. The concentrations of biomarkers in both standard solutions and plasma samples can be directly obtained using a custom-designed smartphone app that is also capable of constructing calibration curves. The detection limits of uric acid, glucose, and triglyceride were determined to be 0.50 mg/dL, 0.84 mmol/L, and 14 mg/dL, respectively, which are below the normal limits and adequate for clinical validation. Owing to the distinct advantages-simple, portable, and cost-effective-this mobile assay protocol can be used for point-of-care (POC) settings or resource-limited situations, and potentially for the diagnosis and prevention of infectious diseases.
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Glucemia/análisis , Internet , Papel , Triglicéridos/sangre , Ácido Úrico/sangre , Biomarcadores/sangre , Colorimetría/instrumentación , Humanos , Sistemas de Atención de Punto , Reproducibilidad de los Resultados , Factores de Riesgo , Teléfono Inteligente , Tecnología InalámbricaRESUMEN
We report a new DNA nanostructure, an extended 1-dimensional composite built for the first time out of structurally robust yet conveniently disassembled DNA triple helices, interspersed with short stretches of G-quadruplexes. These "TQ Hybrid" 1-dimensional nanostructures require potassium ions and modestly acidic pH for their formation and are easily disassembled by changes to either of these requirements. We initially prepared and characterized a "monomeric" TQ Hybrid tile; followed by "sticky" TQs tiles, incorporating unique guanine-only sticky ends, that enable efficient self-assembly via G-quartet formation of nanostructures >150â nm in length, as seen with atomic force microscopy and transmission electron microscopy. We anticipate that such DNA TQ Hybrid structures will find unique and varied application as communication modules within larger nanostructures, and as sensors, logic gates, as well as in other aspects of DNA nanotechnology.
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The macrocyclic cucurbit[7]uril (CB[7]) host has exhibited great application potential as a pharmaceutical excipient due to its versatile abilities to modulate the chemical/physical properties of drug molecules (guests) and to control their in vivo delivery and release (upon complexation). The formation of stable CB[7]@drug complexes is the prerequisite for these promising applications; we report herein a general assay strategy to quantitate the complexation based on competitive binding with surface-immobilized redox guests in conjunction with conventional electrochemical techniques (e.g., cyclic voltammetry). Particularly, by incubating a mixture of CB[7] and a drug molecule with ferrocene (Fc)-terminated self-assembled monolayers (SAMs) on gold, the competitive host@guest binding between the CB[7]@drug complex formed in solution and the CB[7]@Fc complex formed on surface can be quantified with direct cyclic voltammetry measurements. On the basis of the known concentrations of CB[7]/drug and electrochemically determined surface densities of free/complexed Fc groups, the formation constant of CB[7]@drug complex can be determined. With several drug molecules as examples, we have demonstrated the capability of this method for quantitative studies of the formation of supramolecular excipient@drug complexes that are of interest in pharmaceutical and biomedical sciences. More importantly, this work promises a general assay strategy that allows electrochemical quantitation of a wide range of electro-inactive analytes based on the competitive supramolecular host@guest binding at redox-tagged molecular interfaces.
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Adamantano/análogos & derivados , Hidrocarburos Aromáticos con Puentes/análisis , Técnicas Electroquímicas , Imidazoles/análisis , Compuestos de Amonio Cuaternario/química , Tiazoles/química , Adamantano/química , Sitios de Unión , Compuestos Ferrosos/química , Sustancias Macromoleculares/análisis , Metalocenos/química , Estructura Molecular , Oxidación-Reducción , Propiedades de SuperficieRESUMEN
In terms of how the signal varies in response to increased concentration of an analyte, sensors can be classified as either "signal-on" or "signal-off" format. While both types hold potentials to be sensitive, selective, and reusable, in many situations "signal-on" sensors are preferred for their low background signal and better selectivity. In this study, with the detection of lysozyme using its DNA aptamer as a trial system, for the first time we demonstrated that such an aptamer-based electrochemical biosensor can be converted from intrinsically "signal-off" to "signal-on" with the aid of a DNA exonuclease. The fact that the stepwise cleavage of antilysozyme aptamer catalyzed by Exonuclease I (Exo I) is entirely inhibited upon binding lysozyme leads to the selective removal of unbound DNA probes (thiolate anti-lysozyme DNA aptamer strands immobilized on gold electrode) upon the introduction of Exo I to the sensor. With the aid of electrostatically bound redox cations ([Ru(NH3)6]3+), we were able to quantitate the number of aptamer strands that are bound with lysozymes via conventional cyclic voltammetry (CV) measurements. We demonstrated that Exo I-assisted signal-on conversion protocol not only improves the sensing performance (10 times better limit of detection) but also promises a versatile strategy for DNA-based biosensor design, i.e., it can be readily adapted to other aptamer-protein binding systems (thrombin, as another example).
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Aptámeros de Nucleótidos/metabolismo , Técnicas Biosensibles/métodos , Exodesoxirribonucleasas/metabolismo , Muramidasa/análisis , Aptámeros de Nucleótidos/química , Biocatálisis , Complejos de Coordinación/química , Sondas de ADN/química , Sondas de ADN/metabolismo , Técnicas Electroquímicas , Electrodos , Oro/química , Límite de Detección , Muramidasa/metabolismo , Rutenio/químicaRESUMEN
DNA self-assembled monolayers (SAMs) were prepared using potential-assisted deposition on clean gold single-crystal bead electrodes under a number of conditions (constant or square-wave potential perturbations in TRIS or phosphate immobilization buffers with and without Cl-). The local environment around the fluorophore-labeled DNA tethered to the electrode surface was characterized using in situ fluorescence microscopy during electrochemical measurements as a function of the underlying surface crystallography. Potential-assisted deposition from a TRIS buffer containing Cl- created DNA SAMs that were uniformly distributed on the surface with little preference to the underlying crystallography. A constant (+0.4 V/SCE) or a square-wave potential perturbation (+0.4 to -0.3 V/SCE, 50 Hz) resulted in similar DNA-modified surfaces in TRIS immobilization buffer. Deposition using a square-wave potential without Cl- resulted in lower DNA surface coverage. Despite this, the local environment around the DNA in the SAM appears to be densely packed. This implies the formation of clusters of densely packed DNA in the SAM. This effect was also demonstrated when depositing from a phosphate buffer. DNA clusters were significantly reduced when Cl- was present in the buffer. Clusters were most prevalent on the low-index plane surfaces (e.g., {111} and {100}) and less on the higher-index planes (e.g., {210} or {311}). A mechanism is proposed to rationalize the formation of DNA-clustered regions for deposition using a square-wave potential perturbation. The conditions for creating clusters of DNA in a SAM or for preventing these clusters from forming provide an approach for tailoring the surfaces used for biosensing.
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ADN , Oro , ADN/genética , Electrodos , Microscopía Fluorescente , Propiedades de SuperficieRESUMEN
As the performance of hairpin DNA (hpDNA)-based biosensors is highly dependent on the yield of stem-loop (hairpin) conformations, we report herein a versatile fluorometric in situ hybridization protocol for examining hpDNA self-assembled monolayers (SAMs) on popularly used biochip substrates. Specifically, the ratio of fluorescence (FL) intensities of hpDNA SAMs (in an array format) before and after hybridization was adopted as the key parameter for performing such a determination. Upon confirming the existence of mixed and tunable DNA conformations in binary deposition solutions and efficient hybridization of the hairpin strands with the target DNA via gel electrophoresis assays, we tested the fluorometric protocol for determining the coverages of hpDNA in hpDNA/ssDNA SAMs prepared on gold; its accuracy was validated by Exonuclease I (Exo I)-assisted electrochemical quantitation. To further confirm its versatility, this FL protocol was adopted for quantifying hairpin conformations formed on glass and polycarbonate (PC) substrates. The molar ratios of surface-tethered hairpin conformations on the three different substrates were all found to be proportional to but less than those in the binary deposition solutions, and were dependent on the substrate morphology. The findings reported herein are beneficial for the construction of highly efficient DNA hairpin-based sensing surfaces, which essentially facilitates the creation of hpDNA-based biosensors with optimal detection performance.
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ADN/análisis , Fluorometría/métodos , Secuencias Invertidas Repetidas , Hibridación de Ácido Nucleico/métodos , ADN/química , ADN/genética , Exodesoxirribonucleasas/química , Vidrio/química , Oro/química , Ácidos Nucleicos Inmovilizados/análisis , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/genética , Cemento de Policarboxilato/químicaRESUMEN
Mechanoelectronic DNA nanoswitches refer to designed oligonucleotide constructs that are composed of conduction-interrupted duplex stems functionally coupled to ligand recognition motifs; they have been shown to undergo remarkable conduction switching upon binding molecular ligands/analytes. Herein we report a divergent pair of such mechanoelectronic DNA switches, the "signal-on" 3'AA-1 switch and the "signal-off" NB-1 switch, both activated by and responded to mercury ions (Hg2+) at nM levels. We first investigated their charge transport efficiency at a biochemical level, by studying how distinct base sequence at the switches' central three-way junction and at the recognition motif (capable of forming T-Hg2+-T metallo-base pairs) influences their overall conductivity. Gel electrophoresis assays revealed that the presence of two unpaired adenines (AA) at the junction led to "signal-on" behavior with increasing Hg2+ concentration; divergently, absence of these adenines led to a "signal-off" behavior. Upon immobilization on gold electrodes, both DNA switches, with enhanced and inhibited conductivity, respectively, showed excellent sensitivity as well as selectivity toward Hg2+ and can be regenerated for multicycle applications. The high performance of these devices, as both nanoswitches and biosensors with robust and reproducible properties, highlights their potential as an outstanding new class of DNA mechanoelectronic components with built-in biosensing capabilities.
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ADN/metabolismo , Mercurio/metabolismo , Emparejamiento Base , Secuencia de Bases , Técnicas Biosensibles , ADN/química , Conductividad Eléctrica , ElectrodosRESUMEN
Graphene oxide (GO)-based aptasensors are currently one of the most popular sensing platforms for the simple and rapid detection of various targets. Unfortunately, the GO-based aptasensors with long aptamer strands typically show unsatisfactory performance resulting from insignificant structural transformations upon target binding. We report herein the utilization of an aptamer-truncating strategy to combat such a challenge. Taking a pre-selected anti-aflatoxin B1 (AFB1) aptamer (P-AFB1-50) as a trial system, we sequentially remove the extraneous nucleotides within the aptamer by means of circular dichroism (CD) spectroscopy and binding affinity analysis. Particularly, the ratio of the quenching constants between the GO sheets and the truncated aptamers (labelled with fluorophores) in the absence and presence of the target was determined for each of the truncated aptamers to evaluate the optimal sequence. As a result, the truncated aptamer comprising 40 nucleotides was confirmed to show the highest FL output and the best detection limit upon conjugation with GO sheets. More importantly, we demonstrated that this truncating strategy is versatile, i.e., it can be easily extended to other aptamer systems (anti-ochratoxin A (OTA) aptamer, P-OTA-61, as an example) for extraneous nucleotide identification. Impressively, the two optimal truncated aptamers can work together on GO sheets to achieve a simultaneous detection of two different mycotoxins (i.e., AFB1 and OTA) in one single test. Essentially, this research opens a new avenue for the design and testing of aptamer-/GO-based-sensing platforms for rapid, low-cost and multiplex quantification of analytical targets of interest.
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Aflatoxina B1/análisis , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , ADN/química , Grafito/química , Ocratoxinas/análisis , Aflatoxina B1/química , Secuencia de Bases , Fluorescencia , Colorantes Fluorescentes/química , Límite de Detección , Conformación de Ácido Nucleico , Ocratoxinas/químicaRESUMEN
The properties of DNA self-assembled monolayers (SAMs) have strong influences on the interfacial DNA-analyte binding behavior, which further affect the performance of biosensors built upon. In this work, we prepared binary thiolate DNA/6-ferrocenyl-1-hexanethiol (FcC6SH) SAMs on gold (DNA/FcC6S-Au) for convenient electrochemical characterization and subsequent data analysis. Our cyclic voltammetric (CV) studies confirmed that the redox responses of surface-tethered Fc and electrostatically bound [Ru(NH3)6]3+ are capable of providing quantitative information regarding the DNA film properties, including the surface density, structural heterogeneity, and molecular orientation under different preparation and measurement conditions. With the binary thiolate DNA/FcC6S-Au SAM prepared in the conventional post-assembly exchange protocol as a trial system, we are demonstrating the capability of introducing redox-active thiols as passivating and labeling reagents for preparing many other DNA-based biosensing interfaces via varied assembly steps and under different measurement conditions.
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ADN/química , Compuestos Ferrosos/química , Oro/química , Metalocenos/química , Sondas Moleculares/química , Compuestos de Sulfhidrilo/química , Técnicas Biosensibles , Técnicas Electroquímicas , Oxidación-Reducción , Electricidad EstáticaRESUMEN
The complete formation of stem-loop (i.e., hairpin) configuration on chip surface is of particular importance for the application of hairpin DNA (hpDNA) in building biosensors for various analytes with optimized performance. We report herein a convenient electrochemical protocol for evaluating the yield of hairpin DNA conformations upon self-assembly on electrode surface. As of the different hydrolysis capability of Exonuclease I (Exo I) toward single-stranded DNA (ssDNA) and hpDNA, we can selectively remove ssDNA from electrode but retain hpDNA strands; based on the changes in the cyclic voltammetric (CV) responses using [Ru(NH3)6]3+ as redox indicators, we can then determine the fraction of hairpin configurations in mixed DNA self-assembled monolayers (SAMs). It was discovered that the molar fraction of hairpin configuration formed on the surface is considerably lower than that in the binary deposition solution (containing both ssDNA and hpDNA). The accuracy of the Exo I-assisted electrochemical quantitative protocol has been validated by standard DNA hybridization experiments; the relationship between the overall DNA packing density and the yield of hairpin configurations was also evaluated. More importantly, taking HIV-1 gene detection as a trial system, the hpDNA-based biosensor shows significantly improved detection limit and broadened response range upon the background reduction by Exo I-catalyzed hydrolysis.
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Técnicas Biosensibles/métodos , ADN Viral/química , Exodesoxirribonucleasas/metabolismo , VIH-1/genética , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/metabolismo , Secuencias Invertidas Repetidas , Secuencia de Bases , ADN Viral/genética , ADN Viral/metabolismo , Electroquímica , Hidrólisis , Ácidos Nucleicos Inmovilizados/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Propiedades de SuperficieRESUMEN
We demonstrate herein an integrated, smartphone-app-chip (SPAC) system for on-site quantitation of food toxins, as demonstrated with aflatoxin B1 (AFB1), at parts-per-billion (ppb) level in food products. The detection is based on an indirect competitive immunoassay fabricated on a transparent plastic chip with the assistance of a microfluidic channel plate. A 3D-printed optical accessory attached to a smartphone is adapted to align the assay chip and to provide uniform illumination for imaging, with which high-quality images of the assay chip are captured by the smartphone camera and directly processed using a custom-developed Android app. The performance of this smartphone-based detection system was tested using both spiked and moldy corn samples; consistent results with conventional enzyme-linked immunosorbent assay (ELISA) kits were obtained. The achieved detection limit (3 ± 1 ppb, equivalent to µg/kg) and dynamic response range (0.5-250 ppb) meet the requested testing standards set by authorities in China and North America. We envision that the integrated SPAC system promises to be a simple and accurate method of food toxin quantitation, bringing much benefit for rapid on-site screening.
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Aflatoxina B1/análisis , Colorimetría/métodos , Teléfono Inteligente , Aflatoxina B1/inmunología , Anticuerpos/inmunología , Análisis de los Alimentos , Inmunoensayo , Dispositivos Laboratorio en un Chip , Límite de Detección , Microfluídica/métodos , Impresión TridimensionalRESUMEN
Herein, we report a combined electrochemical and ESI-MS study of the enzymatic hydrolysis efficiency of DNA self-assembled monolayers (SAMs) on gold, platform systems for understanding nucleic acid surface chemistry, and for constructing DNA-based biosensors. Our electrochemical approach is based on the comparison of the amounts of surface-tethered DNA nucleotides before and after exonuclease I (Exo I) incubation using electrostatically bound [Ru(NH3)6]3+ as redox indicators. It is surprising to reveal that the hydrolysis efficiency of ssDNA SAMs does not depend on the packing density and base sequence, and that the cleavage ends with surface-bound shorter strands (9-13 mers). The ex-situ ESI-MS observations confirmed that the hydrolysis products for ssDNA SAMs (from 24 to 56 mers) are dominated with 10-15 mer fragments, in contrast to the complete digestion in solution. Such surface-restrained hydrolysis behavior is due to the steric hindrance of the underneath electrode to the Exo I/DNA binding, which is essential for the occurrence of Exo I-catalyzed processive cleavage. More importantly, we have shown that the hydrolysis efficiency of ssDNA SAMs can be remarkably improved by adopting long alkyl linkers (locating DNA strands further away from the substrates).
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ADN de Cadena Simple/análisis , Técnicas Electroquímicas/métodos , Exodesoxirribonucleasas/metabolismo , Oro/química , Espectrometría de Masa por Ionización de Electrospray , Complejos de Coordinación/química , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Electrodos , Hidrólisis , Oxidación-Reducción , Rutenio/químicaRESUMEN
Acute myocardial infarction (AMI) is the leading cause of mortality and morbidity globally. To reduce the number of mortalities, reliable and rapid point-of-care (POC) diagnosis of AMI is extremely critical. We herein present a Blu-ray technology-based assay platform for multiplex cardiac biomarker detection; not only off-the-shelf Blu-ray discs (BDs) were adapted as substrates to prepare standard immunoassays and DNA aptamer/antibody hybrid assays for the three key cardiac marker proteins (myoglobin, troponin I, and C-creative protein) but also an unmodified optical drive was directly employed to read the assay results digitally. In particular, we have shown that all three cardiac markers can be quantitated in their respective physiological ranges of interest, and the detection limits achieved are comparable with conventional enzyme-linked immunosorbent assay (ELISA) kits. The Blu-ray assay platform was further validated by measuring real-world samples and establishing a linear correlation with the simultaneously obtained ELISA data. Without the need to modify either the hardware (Blu-ray discs and optical drives) or the software driver, this assay-on-a-BD technique promises to be a low-cost user-friendly quantitative tool for on-site chemical analysis and POC medical diagnosis.
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Biomarcadores/análisis , Inmunoensayo/métodos , Infarto del Miocardio/diagnóstico , Sistemas de Atención de Punto , Anticuerpos/química , Anticuerpos/inmunología , Aptámeros de Nucleótidos/química , Proteína C-Reactiva/análisis , Humanos , Inmunoensayo/instrumentación , Límite de Detección , Mioglobina/análisis , Troponina I/análisisRESUMEN
Beyond their essential applications in portable data storage for the past 30 years, optical discs and corresponding recording/reading technologies have been extensively explored with the ultimate goal of creating novel analytical tools for on-site chemical analysis and point-of-care (POC) medical diagnosis. In particular, the disc media (CD, DVD, and BD) are proven to be inexpensive and versatile substrate materials for the preparation of various biochips and microfluidic systems; conventional computer drives and disc players are widely adapted for biochip signal reading and microscopic imaging. Herein we provide an overview of such optical disc technology-enabled analytical devices, e.g., integrated systems developed from specifically fabricated analog disks, modified optical drives, or adapted software algorithms.
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A sensitive and cost-effective method for the simultaneous quantitation of trace amounts of Hg(2+) and Pb(2+) in real-world samples has been developed using DNA molecular beacon probes bound to bona fide digital video discs (DVDs). With specially designed T-rich or G-rich loop sequences, the detection is based on the strong T-Hg(2+)-T coordination chemistry of Hg(2+) and the formation of G-quadruplexes induced by Pb(2+), respectively. In particular, the presence of metal cations leads to hairpin opening and exposure of the terminal biotin moiety for binding nanogold-streptavidin conjugates. The recognition signal was subsequently enhanced by gold nanoparticle-promoted silver deposition, which leads to quantifiable digital signals upon reading with a standard computer drive. This method exhibits a wide response range and low detection limits for both Hg(2+) and Pb(2+). In addition, the quantitative determination of heavy metals in food products (e.g., rice samples) has been demonstrated and the method compares favorably with other optical sensors developed recently.
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Técnicas Biosensibles/instrumentación , Discos Compactos , Sondas de ADN/química , Contaminantes Ambientales/análisis , Plomo/análisis , Mercurio/análisis , Secuencias Invertidas RepetidasRESUMEN
On-site oral fluid testing for drugs of abuse has become prominent in order to take immediate administrative action in an enforcement process. Herein, we report a DVD technology-based indirect competitive immunoassay platform for the quantitative detection of drugs of abuse. A microfluidic approach was adapted to prepare multiplex immunoassays on a standard DVD-R, an unmodified multimode DVD/Blu-Ray drive to read signal, and a free disc-quality analysis software program to process the data. The DVD assay platform was successfully demonstrated for the simultaneous, quantitative detection of drug candidates (morphine and cocaine) in oral fluids with high selectivity. The detection limit achieved was as low as 1.0 ppb for morphine and 5.0 ppb for cocaine, comparable with that of standard mass spectrometry and ELISA methods.