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Three-dimensional porous microarray of gold modified electrode for ultrasensitive and simultaneous assay of various cancer biomarkers
Abstract
A three-dimensional (3D) microarray of a gold modified electrode with a nanoporous surface was successfully fabricated in this work. The special gold micro/nanostructure possessed an extremely high surface area (ca. 20 times its geometrical area), as well as highly stable and easily chemically modified properties, which could distinctly increase the binding sites for biological probes, facilitate the diffusion profile and enhance the electron transfer. Owing to these advantages, an ultrasensitive biosensor was designed on the porous microarray of the gold modified electrode (PMGE), which realized the simultaneous assay of cancer biomarkers of angiogenin (Ang) and thrombin (Tob). Under optimal experimental conditions, an impressively ultralow detection limit of 0.07 pM for Ang (a linear range from 0.2 pM to 10 nM) and a limit of 20 fM for Tob (a linear range from 50 fM to 5 nM) were obtained. Besides, the fabricated biosensor showed an excellent stability, good reproducibility and a high selectivity towards other biological proteins, which also exhibited promising potential for the application in a real serum sample analysis. Such a micro/nanostructure of gold could have widespread applications in biological sensing and quantitative biochemical analysis.
... There are two approaches used to achieve simultaneous electrochemical detection of biomarkers by aptasensors: multi-label [48][49][50][51][52][53] and multi-electrode methods [54][55][56][57][58][59]. The first approach uses a single measuring electrode, which is designed to show distinguishable multiple signals originating from respective electrochemical labels that allow the detection of individual biomarkers. ...
... Either decreased or increased oxidation/reduction peak current of electroactive labels can be observed with increasing biomarker concentration, meaning that the system is working in "signal-off" or "signal-on" (turn-off or turn-on type sensor) detection modes, respectively [30]. Both the "signal-on" [48,50,52] and "signal-off" [50,51,53] principles were used for the detection of cardiovascular biomarkers, suggesting that both types could be appropriate. These types are called structure-switching aptasensors and use square wave voltammetry (SWV) or differential pulse voltammetry (DPV) as detection methods. ...
... Either decreased or increased oxidation/reduction peak current of electroactive labels can be observed with increasing biomarker concentration, meaning that the system is working in "signal-off" or "signal-on" (turn-off or turn-on type sensor) detection modes, respectively [30]. Both the "signal-on" [48,50,52] and "signal-off" [50,51,53] principles were used for the detection of cardiovascular biomarkers, suggesting that both types could be appropriate. These types are called structure-switching aptasensors and use square wave voltammetry (SWV) or differential pulse voltammetry (DPV) as detection methods. ...
... (1) Structure-switching sensors [72], in which the redox-tag labelled aptamer alters the conformation upon binding a target molecule, resulting in changes in the rate of electron transfer between the redox tag and electrode surface [73][74][75][76][77][78][79][80][81][82][83], and (2) Sensors based on bioconjugates containing nanomaterials, such as gold nanoparticles (AuNPs), gold nanorods (AuNRs), or nano zirconium-metal organic framework (NMOF), modified further with aptamer and redox tag molecules [70,[84][85][86]. Depending on the sensor architecture, different signaling mechanisms are possible. ...
In recent years, the need for simple, fast, and economical detection of food and environmental contaminants, and the necessity to monitor biomarkers of different diseases have considerably accelerated the development of biosensor technology. However, designing biosensors capable of simultaneous determination of two or more analytes in a single measurement, for example on a single working electrode in single solution, is still a great challenge. On the other hand, such analysis offers many advantages compared to single analyte tests, such as cost per test, labor, throughput, and convenience. Because of the high sensitivity and scalability of the electrochemical detection systems on the one hand and the specificity of aptamers on the other, the electrochemical aptasensors are considered to be highly effective devices for simultaneous detection of multiple-target analytes. In this review, we describe and evaluate multi-label approaches based on (1) metal quantum dots and metal ions, (2) redox labels, and (3) enzyme labels. We focus on recently developed strategies for multiplex sensing using electrochemical aptasensors. Furthermore, we emphasize the use of different nanomaterials in the construction of these aptasensors. Based on examples from the existing literature, we highlight recent applications of multiplexed detection platforms in clinical diagnostics, food control, and environmental monitoring. Finally, we discuss the advantages and disadvantages of the aptasensors developed so far, and debate possible challenges and prospects.
... Building upon the variety of methods, many researchers have demonstrated integration of biological materials with threedimensional transducers in an effort to achieve enhanced bioelectronics performance at a miniaturized system scale [3], [4]. Three-dimensional structures at micro-and nano-scales offer a number of beneficial properties compared to their two-dimensional counterparts, allowing for an extensive range of applications including micro energy storage/harvesting device [5], micro/nano actuators [6], micro thermal management devices [7], waterrepellent surfaces [8], optical modulators [9], 3D VLSIs [10], etc. ...
Highly toxic pollutants, e.g. heavy metal ions, phenolics, toxins and pesticides, have posed major threats to ecosystem security and public health. It is imperative to develop simple, low cost, sensitive and reliable techniques for detecting these contaminants in the environment. Compared with traditional analytic techniques, photoelectrochemical (PEC) sensing as a newly emerged approach possesses a low background noise and high sensitivity, opening a new platform for rapid and accurate monitoring of the concerned pollutants. The performance of advanced PEC sensors is fundamentally related to the microstructures and configurations of semiconductor-based photoactive nanomaterials. Therefore, a multidisciplinary research effort focusing on the rational design and synthesis of innovative photoactive nanomaterials has recently emerged. This paper provides a comprehensive review on the engineered semiconductors (i.e. doped-semiconductors) and their heterojunctions (e.g. semiconductor-semiconductor, semiconductor-carbon, semiconductor-metal and multicomponent heterojunction) as well as their emerging applications in PEC sensing and monitoring. Particular attention has been paid to various morphologies, e.g. 0D quantum dots (QDs) and nanoparticles (NPs), 1D nanowires (NWs), nanotubes (NTs) and nanorods (NRs), 2D nanosheets (NSs) and 3D aligned arrays, and their effects on the sensing performances. Moreover, the signal response mechanisms and performance evaluations (e.g. sensitivity, linear range, limit of detection, selectivity and stability) of the constructed PEC sensors are discussed. At last, critical challenges and future research perspectives in the fields are proposed.
In this work, we introduce an electrowetting-assisted 3-D biofabrication process allowing both complete and localized functionalization of bionanoreceptors onto densely arranged 3-D microstructures. Integration of biomaterials with three-dimensional (3-D) microdevice components offers exciting opportunities for communities developing miniature bioelectronics with enhanced performances and advanced modes of operation. However, most biological materials are stable only in properly conditioned aqueous solutions, thus the water-repellent properties exhibited by densely arranged micro-/nano- structures (widely known as the Cassie-Baxter state) represent a significant challenge to biomaterial integration. Here, we first investigate such potential limitations using cysteine-modified Tobacco mosaic virus (TMV1cys) as a model bionanoreceptor and a set of Au-coated Si-micropillar arrays (µPAs) of varying densities. Further, we introduce a novel biofabrication system adopting electrowetting principles for the controlled localization of TMV1cys bionanoreptors on densely arraged µPAs. Contact angle analysis and SEM characterizations provide clear evidence to indicate structural hydrophobicity as a key limiting factor for 3-D biofunctionalization, and for electrowetting as an effective method to overcome this limitation. The successful 3-D biofabrication is confirmed using SEM and fluorescence microscopy that show spatially controlled and uniform assemblies of TMV1cys on µPAs. The increased density of TMV1cys per device foot-print produces a 7-fold increase in fluorescence intensity attributed to the µPAs when compared to similar assemblies on planar substrates. Combined, this work demonstrates the potential of electrowetting as a unique enabling solution for the controlled and efficient biofabrication of 3-D patterned micro/nano domains.
Herein, novel and versatile electrochemical aptasensors were constructed on a self-supported nanoporous gold (np-Au) microelectrode, integrating with an exonuclease III (Exo III) induced signal amplification strategy. Self-supported np-Au microelectrode with 3D bicontinuous nanoporous structures possesses tremendously large specific area, clean surface, high stability and biocompatibility, bringing about significant advantages in both molecular recognition and signal response. As paradigms, two analytes of bisphenol A (BPA) and ochratoxin A (OTA) were selected to demonstrate the superiority and versatility of designed aptasensors. Trace amounts of mDNA (associated with BPA or OTA concentration) hybridized with cDNA strands assembled on np-Au microelectrode, activating the cleavage reaction with Exo III. Thus, cDNA was digested and mDNA was released to undergo a new hybridization and cleavage cycle. Finally, residual cDNA strands were recognized by methylene blue labelled rDNA/AuNPs with the assistance of hDNA to generate the electrochemical signals, which were used to quantitatively monitor targets. Under the optimized conditions, prepared aptasensors exhibited wide linear ranges (25pg/mL to 2ng/mL for BPA, 10pg/mL to 5ng/mL for OTA) with ultralow detection limits (10pg/mL for BPA, 5pg/mL for OTA), excellent selectivity and stability, and reliable detection in real samples. This work opens a new horizon for constructing promising electrochemical aptasensors for environmental monitoring, medical diagnostics and food safety.
In this work, we have developed a new electrochemical aptasensor for IFN-γ assay, based on a hierarchical graphene/AuNPs modified electrode coupled with a dual enzyme-assisted signal amplification strategy. The graphene/AuNPs modified electrode with a large specific area, high conductivity, excellent stability and biocompatibility was used for the immobilization of plentiful duplex DNA strands of capture probes and IFN-γ aptamers. In the presence of IFN-γ, hybridized aptamers were released from the electrode surface due to the formation of aptamer/IFN-γ complexes. Meanwhile, aptamers were digested with RecJf exonuclease and IFN-γ was available for target recycling, creating numerous free capture probes on the electrode surface. Then the hybridization chain reaction was initiated with the help of linker probes and biotin-labeled reporter probes. Thus cascade duplex DNA polymers were produced on the electrode surface, providing lots of binding sites for streptavidin–alkaline phosphatase to generate robust enzyme-catalyzed signals. Due to this dual enzyme-assisted amplification strategy, the prepared aptasensor exhibited a wide linear range from 5 pM–5 nM with an ultralow detection limit of 2 pM (S/N = 3). Besides, the aptasensor showed an excellent selectivity, satisfactory reproducibility and stability, and a great potential in serum analysis. Importantly, the versatility of the designed sensing strategy makes it easily extended for analyzing other biomolecules.
A porous Au modified glassy carbon electrode (Autemp/GCE) is prepared by co-electrodeposition of Au and Au-based Prussian blue (PB) analogue template in an aqueous bath containing HAuCl4 and PB-precursors, followed by removal of the PB analogue template by alkali and then acidic treatments. The Autemp/GCE has large surface area (roughness factor = 64.6) and is employed to determine As(III) based on anodic stripping linear sweep voltammetry (ASLSV) in a dual-signal mode. Under optimal conditions, the ASLSV peak currents for oxidation of As(0) to As(III) and of As(III) to As(V) are linear with concentration of As(III) from 0.025 to 10 μM with a sensitivity of 1.13 mA μM⁻¹ and a limit of detection (LOD) of 6.6 nM (0.49 μg L⁻¹) (S/N = 3), and from 0.025 to 3.0 μM with a sensitivity of 0.88 mA μM⁻¹ and a LOD of 8.5 nM (0.63 μg L⁻¹) (S/N = 3), respectively. Determination of As(III) in real water samples on the Autemp/GCE yields satisfactory results.
Currently, the use of nano silicon in cancer therapy is limited as drug delivery vehicles and markers in imaging, not as manipulative/controlling agents. This is due to limited properties that native states of nano silicon and silicon oxides offers. We introduce nano-functionalized multi-phased silicon/silicon oxide biomaterials synthesized via ultrashort pulsed laser synthesis, with tunable properties that possess inherent cancer controlling properties that can passivate the progression of cancer. This nanostructured biomaterial is composed of individual functionalized nanoparticles made of a homogenous hybrid of multiple phases of silicon and silicon oxide in increasing concentration outwards from the core. The chemical properties of the proposed nanostructure such as number of phases, composition of phases and crystal orientation of each functionalized nanoparticle in the three dimensional nanostructure is defined based on precisely tuned ultrashort pulsed laser-material interaction mechanisms. The amorphous rich phased biomaterial shows a 30 fold (95%) reduction in number of cancer cells compared to bulk silicon in 48 hours. Further, the size of the cancer cells reduces by 76% from 24 to 48 hours. This method exposes untapped properties of combination of multiple phases of silicon oxides and its applications in cancer therapy.
Great challenges have remained in template-assisted fabrication of metal nanowire array on substrates, because enormous effort is prerequisite to address adhesion issue between substrates and adopted templates, e.g. anodic aluminum oxide (AAO). Therefore, novel and promising templates are highly desired for the construction of proposed structures. Here, vertical 1,5-diaminoanthraquinone (DAAQ) nanowires were in-situ prepared on substrates by a facile method, which were proved to be a reliable and alternative template for preparation of metal nanowire array. As an example, with the wet chemical reduction of HAuCl4, a three-dimensional gold nanowire array (3D GNA) was successfully obtained with DAAQ template. Proposed 3D GNA possesses an extremely large roughness factor of ca. 15, as well as high stability and favorable surface structure for diffusion profile of analytes, making it a suitable candidate in the construction of electrochemical biosensors with high performance. As a result, an ultrasensitive aptasensor was constructed for the detection of thrombin with a general sensing scheme. The fabricated aptasensor displays an impressively ultralow detection limit of 3 fM (S/N=3) with a wide linear response from 10 fM to 1 nM, as well as an excellent selectivity, good reproducibility and acceptable stability. Besides, the biosensor exhibits a potential application in the analysis of the real serum sample. The developed DAAQ nanowires is envisaged to become a general template for fabrication of more nanowire array and the as-synthesized 3D GNA would open up a wide horizon for potential applications in biological sensing.
Herein, a label-free and highly sensitive electrochemical aptasensor for the detection of angiogenin was proposed based on a conformational change of aptamer and amplification by poly(diallyldimethyl ammonium chloride) (PDDA)-functionalized graphene/gold nanoparticles (AuNPs) composites-modified electrode. PDDA-functionalized graphene (P-GR) nanosheets as the building block in the self-assembly of GR nanosheets/AuNPs heterostructure enhanced the electrochemical detection performance. The electrochemical aptasensor has an extraordinarily sensitive response to angiogenin in a linear range from 0.1pM to 5nM with a detection limit of 0.064pM. The developed sensor provides a promising strategy for the cancer diagnosis in medical application in the future.
Copyright © 2014 Elsevier B.V. All rights reserved.
In this work, novel three-dimensional graphene films (3D GFs) with controllable pore structures are directly fabricated on gold substrates through the hydrothermal reduction. An interfacial technique of the self-assembled monolayer is successfully introduced to address the binding issue between the graphene film and substrate. Adscititious silica spheres, serving as new connection centers, effectively regulate the dimensions of framework in graphene films, and secondary pore structures are produced once removing the spheres. Based on hierarchically porous 3D GFs with large surface area, excellent binding strength, high conductivity, and distinct interfacial micro-environments, selected examples of electrochemical aptasensors are constructed for the assay of adenosine triphosphate (ATP) and thrombin (Tob) respectively. Sensitive ATP and Tob aptasensors, with high selectivity, excellent stability, and promising potential in real serum sample analysis, are established on 3D GFs with different structures. The results demonstrate that the surface area, as well as interfacial micro-environments, plays a critical role in the molecular recognition. The developed reliable and scalable protocol is envisaged to become a general path for in situ fabrication of more graphene films and the as-synthesized 3D GFs would open up a wide horizon for potential applications in electronic and energy-related systems.
Due to their shape anisotropy, high aspect ratio magnetic nanoparticles offer many advantages in biomedical applications. For biocompatibility, it is essential to have full control over the dimensions and surface chemistry of the particles. The aim of this study was to make biocompatible nanowires with tuneable dimensions. This was achieved by electrodeposition of Ni in polycarbonate membranes. To assure biocompatibility, a continuous gold coating was deposited onto the Ni wires by a newly developed electroless deposition method. The coating was analysed using electron microscopy and X-ray diffraction. Magnetic properties, anisotropy and Au film thickness were studied using vibrating sample magnetometry. After biofunctionalization, no significant cytotoxic effects were found in studies involving a diverse range of primary and tumour cells exposed to increasing concentrations of nanowires for up to 7 days. These nanowires may thus be used for in-vivo applications such as magnetic drug delivery.
Highly oriented growth of a hybrid microarray was realized by a facile template-free method on gold substrates for the first time. The proposed formation mechanism involves an interfacial structure-directing force arising from self-assembled monolayers (SAMs) between gold substrates and hybrid crystals. Different SAMs and variable surface coverage of the assembled molecules play a critical role in the interfacial directing forces and influence the morphologies of hybrid films. A highly oriented hybrid microarray was formed on the highly aligned and vertical SAMs of 1,4-benzenedithiol molecules with rigid backbones, which afforded an intense structure-directing power for the oriented growth of hybrid crystals. Additionally, the density of the microarray could be adjusted by controlling the surface coverage of assembled molecules. Based on the hybrid microarray modified electrode with a large specific area (ca. 10 times its geometrical area), a label-free electrochemical DNA biosensor was constructed for the detection of an oligonucleotide fragment of the avian flu virus H5N1. The DNA biosensor displayed a significantly low detection limit of 5 pM (S/N = 3), a wide linear response from 10 pM to 10 nM, as well as excellent selectivity, good regeneration and high stability. We expect that the proposed template-free method can provide a new reference for the fabrication of a highly oriented hybrid array and the as-prepared microarray modified electrode will be a promising paradigm in constructing highly sensitive and selective biosensors.
We report a label-free and ultrasensitive aptasensor based on a fractal gold modified (FracAu) electrode for thrombin detection with a femtomolar detection limit. The FracAu electrode was prepared by electrodeposition of hydrogen tetrachloroaurate (HAuCl(4)) onto a bare indium tin oxide (ITO) electrode surface. After this process the electrode was characterized by SEM. A thiol-modified aptamer against thrombin was immobilized on the FracAu electrode through a self-assembling process. Upon thrombin binding, the interfacial electron transfer of the FracAu electrode was perturbed by the formation of an aptamer-thrombin complex. The concentration of thrombin in the sample solution was determined by measuring the change in the oxidation peak current of hydroxymethyl ferrocene (C(11)H(12)FeO) with differential pulse voltammetry (DPV). The current response (reduced peak current) had a linear relationship with the logarithm of thrombin concentrations in the range of 10(-15) to 10(-10) M with a detection limit of 5.7 fM. Furthermore, the as-prepared FracAu electrode exhibited high selectivity. The application of FracAu electrodes may be extended to prepare other types of biosensors, such as immunosensors, enzyme biosensors and DNA biosensors. These results show that FracAu electrodes have great promise for clinical diagnosis of disease-related biomarkers.
A sensitive electrochemical aptasensor for thrombin detection is presented based on the host-guest recognition technique. In this sensing protocol, a 15 based thrombin aptamer (ab. TBA) was dually labeled with a thiol at its 3' end and a 4-((4-(dimethylamino)phenyl)azo) benzoic acid (dabcyl) at its 5' end, respectively, which was previously immobilized on one Au electrode surface by AuS bond and used as the thrombin probe during the protein sensing procedure. One special electrochemical marker was prepared by modifying CdS nanoparticle with β-cyclodextrins (ab. CdS-CDs), which employed as electrochemical signal provider and would conjunct with the thrombin probe modified electrode through the host-guest recognition of CDs to dabcyl. In the absence of thrombin, the probe adopted linear structure to conjunct with CdS-CDs. In present of thrombin, the TBA bond with thrombin and transformed into its special G-quarter structure, which forced CdS-CDs into the solution. Therefore, the target-TBA binding event can be sensitively transduced via detecting the electrochemical oxidation current signal of Cd of CdS nanoparticles in the solution. Using this method, as low as 4.6 pM thrombin had been detected.
A novel and facile strategy for the fabrication of aptamer-based adenosine 5'-triphosphate (ATP) biosensor was developed by a quantum dot (QD) electrochemiluminescence (ECL) technique. Different from the existing strategies for the development of aptasensors based on electrochemical, fluorescent or other methods, the strategy proposed here is essentially based on the aptamer-ATP specific affinity and the rules of Watson-Crick base pairing. After the thiol modified anti-ATP probes were immobilized onto the pretreated Au electrode, the electrode was incubated in ATP solution to form aptamer-ATP bioaffinity complexes. The complementary DNA (cDNA) oligonucleotides were hybridized with the free probes. As a result, the avidin-modified QDs were bound to the aptasensor through the biotin-avidin system in the existence of biotin-modified cDNA. The ECL signal of the aptasensor was responsive to the amount of QDs bound to the cDNA oligonucleotides, which was inversely proportional to the combined target analyte ATP. The QDs were characterized by high resolution transmission electron microscopy (HRTEM), ultraviolet (UV) and photoluminescence (PL) spectra. The preparation process for the aptasensor was monitored by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Possible interference, such as from the pH value of the electrolyte, the incubation time and the concentration of coreactant K(2)S(2)O(8), on the aptasensor ECL response were investigated. The ATP concentration was measured through the decrease of ECL intensity. The ECL intensity of the aptasensor decreased with the increase of the logarithm of the ATP concentration over the 0.018-90.72 microM range. In addition, the aptasensor exhibited excellent selectivity responses toward the target analyte. This study may offer a new and relatively general approach to expand the application of QD ECL in the aptasensor field.
A multifunctional electrochemical strategy based on a dual-aptamer for the detection of adenosine and thrombin in one-pot was developed, by coupling the enhancement of bio-bar-code technology and anodic stripping voltammetry (ASV).
Human angiogenin is progressively up-regulated in the prostate epithelial cells during the development of prostate cancer from prostate intraepithelial neoplasia (PIN) to invasive adenocarcinoma. Mouse angiogenin is the most up-regulated gene in AKT-induced PIN in prostate-restricted AKT transgenic mice. These results prompted us to study the role that angiogenin plays in prostate cancer. Here, we report that, in addition to its well established role in mediating angiogenesis, angiogenin also directly stimulates prostate cancer cell proliferation. Angiogenin undergoes nuclear translocation in PC-3 human prostate cancer cells grown both in vitro and in mice. Thus, knocking down angiogenin expression in PC-3 human prostate adenocarcinoma cells inhibits ribosomal RNA transcription, in vitro cell proliferation, colony formation in soft agar, and xenograft growth in athymic mice. Blockade of nuclear translocation of angiogenin by the aminoglycoside antibiotic neomycin inhibited PC-3 cell tumor growth in athymic mice and was accompanied by a decrease in both cancer cell proliferation and angiogenesis. These results suggest that angiogenin has a dual effect, angiogenesis and cancer cell proliferation, in prostate cancer and may serve as a molecular target for drug development. Blocking nuclear translocation of angiogenin could have a combined benefit of antiangiogenesis and chemotherapy in treating prostate cancer.
• tumor therapy
• nuclear translocation
• ribosome biogenesis
This review introduces the basic concepts and terms associated with impedance and techniques of measuring impedance. The focus of this review is on the application of this transduction method for sensing purposes. Examples of its use in combination with enzymes, antibodies, DNA and with cells will be described. Important fields of application include immune and nucleic acid analysis. Special attention is devoted to the various electrode design and amplification schemes developed for sensitivity enhancement. Electrolyte insulator semiconductor (EIS) structures will be treated separately.
Figure
An alternating current which is forced to pass an interface is sensitive to surface changes and will detect impedance changes due to biomolecule immobilisation or formation of a recognition complex. This can be used for the construction of biosensor electrodes
We have developed a robust, nanobiotechnology-based electrochemical cytosensing approach with high sensitivity, selectivity, and reproducibility toward the simultaneous multiplex detection and classification of both acute myeloid leukemia and acute lymphocytic leukemia cells. The construction of the electrochemical cytosensor involves the hierarchical assembly of dual aptamer-functionalized, multilayered graphene-Au nanoparticle electrode interface and the utilization of hybrid electrochemical nanoprobes co-functionalized with redox tags, horseradish peroxidase, and cell-targeting nucleic acid aptamers. The hybrid nanoprobes are multifunctional, capable of specifically targeting the cells of interest, amplifying the electrochemical signals, and generating distinguishable signals for multiplex cytosensing. The as-assembled electrode interface not only greatly facilitates the interfacial electron transfer process due to its high conductivity and surface area but also exhibits excellent biocompatibility and specificity for cell recognition and adhesion. A superstructured sandwich-type sensor geometry is adopted for electrochemical cytosensing, with the cells of interest sandwiched between the nanoprobes and the electrode interface. Such an electrochemical sensing strategy allows for ultrasensitive, multiplex acute leukemia cytosensing with a detection limit as low as ∼350 cells per mL and a wide linear response range from 5 × 10(2) to 1 × 10(7) cells per mL for HL-60 and CEM cells, with minimal cross-reactivity and interference from non-targeting cells. This electrochemical cytosensing approach holds great promise as a new point-of-care diagnostic tool for early detection and classification of human acute leukemia and may be readily expanded to multiplex cytosensing of other cancer cells.
We have developed a highly sensitive SERS-based sandwich immunoassay by utilizing two derivatives of gold nanostars simultaneously. One is high-densely packed self-assembled substrates of gold nanostars and the other is labeled immune nanostar aggregates. Taking advantage of the electromagnetic field coupling between tips of adjacent individual nanostars, the self-assembled substrate of gold nanostars exhibited a better SERS performance than that of gold nanoparticles, which was also investigated by FDTD simulation. On the other hand, the immuno-aggregates made of gold stars also showed an improved SERS activity than those made of gold naoparticles. Thus, by combining the self-assembled substrates of gold nanostars and immuno-aggregates of gold nanostars, highly sensitive sandwich immunoassays were obtained. The experimental results show that there existed a linear correlation between the concentration of antigens and the prominent peak intensity of SERS signals. The limit of detection (LOD) is ~10 fg/mL, 5 to 6 orders smaller than that of the conventional ELISA protocols. Such a structure will have potential wide-range applications of biological sensing and quantitative biochemical analysis.
A novel resonance light scattering assay for neuron specific enolase (NSE) with high selectivity and sensitivity has been developed by using functionalized gold nanorods as an immunosensor probe.
We demonstrated a facile fabrication of high density Au nanostructures including nanothorns (NTs), nanocorals (NCs), nanoslices (NSs), and nanowires (NWs) which were electrochemically grown on flexible plastic substrates of polyethylene terephthalate (PET). A thrombin-binding aptamer was immobilized on the surfaces of the Au nanostructures to form highly sensitive electrochemical impedance spectroscopic (EIS) biosensors for thrombin recognition. The binding of thrombin to the aptamer sequence was monitored by EIS in the presence of [Fe(CN)6]3−/4−(aq). The protein (1–50 pM) was detected linearly by the Au nanostructures. Among them, the Au NWs exhibited excellent thrombin detection performances. The biosensor provided high sensitivity, selectivity, and stability due to its high surface area.
The development of aptamers and their use in biomarker discovery, detection, sensing, profiling, and characterizing of cells is discussed. Through the process of cell-SELEX (systematic evolution of ligands by exponential enrichment), aptamers may be selected with high selectivity and specificity toward a targeted cell line. Not only can aptamers bind to certain molecules with high affinity, they can also be chemically modified with relative ease. Cell-selected aptamers are able to recognize subtle differences among cellular membranes. These differences can be exploited for cell detection, capture, and imaging. Similar to aptamers selected against purified proteins, aptamers from cell-SELEX can also be analyzed to obtain the binding motif and be post engineered to enhance their affinity or functionality. Specifically, with their ability to distinguish cancer cells from normal cells, aptamers allow a comparative strategy to identify differences at the molecular level and promote the discovery of molecular features of cancer cells.
We developed a simple method for the detection of platelet-derived growth factors (PDGFs) based on base stacking effect coupled with an unmodified gold nanoparticle (AuNP) indicator. In the absence of a target, an aptamer probe and a capture probe stably co-exist in a solution, as it is difficult to sustain an interaction between both these probes due to the short 8bp duplex. However, when a target protein binds to the aptamer probe, the strong base stacking effect can lead to a favorable and stable interaction between the aptamer and capture probes. Hence, the capture probe dissociates from the AuNP surfaces, inducing AuNP aggregation. Compared with other AuNP-based aptasensors for PDGFs, using this base stacking effect can overcome a structured-aptamer method's limitation of requiring thiolated-aptamer-modified AuNPs. Under optimal detection conditions, this label-free colorimetric sensor could detect PDGFs down to 6nM with high selectivity in the presence of other interferring proteins. This simple detection approach provides viable methods for a structured-aptamer sensing protocol.
The construction of a sensitive electrochemical aptamer sensor (aptasensor) for thrombin detection is described. Among the advantages of using microelectrode-based devices are the possibility to work with small sample volumes and enjoying faster mass transport rates and lower interfacial capacitance than at macroelectrodes. Therefore, gold disk microelectrode arrays are an attractive transducer option for aptasensors. The morphology of the gold disk microelectrode arrays was inspected by scanning electron microscope. The interaction between a thrombin aptamer and thrombin on gold disk microelectrode arrays was demonstrated by differential pulse voltammetry using methylene blue (MB) as an electrochemical indicator. MB adsorbed to aptamers via their guanine base. When thrombin was introduced, it displaced the MB adsorbed to the aptamers and bound to them. This resulted in a decrease of MB peak current which correlated to the concentration of thrombin over a dynamic range spanning from 10(-5) to 10(-12)M. This method was able to linearly and selectively detect thrombin with a detection limit of 0.143pM.
We report a reagentless and reusable electrochemical DNA sensor that exploits competitive binding and target hybridization-induced change in the signalling probe conformation for robust detection of a target DNA sequence.
Nitrogen adsorption/desorption isotherms are used to investigate the Brunauer, Emmett, and Teller (BET) surface area and Barrett-Joyner-Halenda (BJH) pore size distribution of physically modified, thermally annealed, and octadecanethiol functionalized np-Au monoliths. We present the full adsorption-desorption isotherms for N(2) gas on np-Au, and observe type IV isotherms and type H1 hysteresis loops. The evolution of the np-Au under various thermal annealing treatments was examined using scanning electron microscopy (SEM). The images of both the exterior and interior of the thermally annealed np-Au show that the porosity of all free standing np-Au structures decreases as the heat treatment temperature increases. The modification of the np-Au surface with a self-assembled monolayer (SAM) of C(18)-SH (coverage of 2.94 × 10(14) molecules cm(-2) based from the decomposition of the C(18)-SH using thermogravimetric analysis (TGA)), was found to reduce the strength of the interaction of nitrogen gas with the np-Au surface, as reflected by a decrease in the 'C' parameter of the BET equation. From cyclic voltammetry studies, we found that the surface area of the np-Au monoliths annealed at elevated temperatures followed the same trend with annealing temperature as found in the BET surface area study and SEM morphology characterization. The study highlights the ability to control free-standing nanoporous gold monoliths with high surface area, and well-defined, tunable pore morphology.
Application of protein-based, direct electron communication in bioelectronic devices, biosensors, or biofuel cells usually requires high stability and function density of the immobilized proteins or enzymes. Traditional methods have been used to increase the function density using multilayer immobilization techniques at the expense of losing stability and electron-communication rate, that is, generally only protein molecules near the electrode surface are electroactive. In order to overcome the above problems, a three-dimensional, ordered, macroporous gold film electrode is synthesized electrochemically by an inverted colloidal crystal template technique. The uniform, three-dimensional macroporous gold provides superior conductivity, high stability, and large surface area. Its interconnected macroporous structure, containing gold nanoparticles, significantly enhances the amount of adsorbed hemoglobin (Hb) molecules at the monolayer level and also provides a good microenvironment for retaining the biological activity of the adsorbed protein, as confirmed by electrochemical and attenuated total reflection Fourier-transform infrared spectroscopy. Therefore, direct electron transfer between the adsorbed Hb and the electrode is achieved. Adsorption of Hb on the macroporous gold film electrode is monitored using electrochemical impedance spectroscopy. ne saturated adsorption amount, T, of the Hb is determined to be 6.55x10(-10) mol cm(-2) with a surface coverage of 88.1 %. The electrochemical behavior and the adsorption mechanism of Hb on the macroporous gold film electrode are discussed on the basis of the experimental results.
We report here a graphene oxide (GO)-based fluorescent aptasensor for adenosine detection by employing exonuclease III (Exo III) as a signal amplifying element. In the absence of adenosine, the adenosine aptamers hybridized with the complementary DNA (cDNA), and the Exo III could not cleave the single-strand signal probes labeled with carboxylfluorescein (FAM) at its 5' ends. When the graphene oxide was finally added, it could strongly adsorb the single-strand signal probes and quenched the fluorophore effectively. In the presence of adenosine, the aptamers associated with the targets, which led to the formation of duplex DNAs between the cDNAs and the signal probes. The Exo III thereafter could digest the duplex DNAs from 3' blunt terminus of signal probes, liberating the fluorophore. Upon adding the GO, the fluorophore could not be adsorbed and quenched. By coupling cyclic enzymatic cleavage, a remarkable fluorescent increase was obtained. Due to the specific recognition ability of the aptamer for the target and the powerful quenching property of GO for signal probe, this proposed approach has a good selectivity and high sensitivity for adenosine. In the optimum conditions described, >100% signal enhancement was achieved and a limit of detection as low as 1 nM was obtained, which is lower than those of commonly used fluorescent aptamer sensors. Moreover, the biosensor exhibited an ultrahigh sensitivity and held a versatile platform for clinical diagnostics, molecular biology and drug developments.
The relationship between cancer and thrombosis has been recognized for nearly 150 years. Although the mechanisms underlying this association are not completely understood, there are increasing evidences suggesting a pivotal role of thrombin in cancer biology. This review will focus on the most important pathways by which thrombin may affect cancer growth and dissemination. In addition, the potential role of congenital (i.e., hemophilia) and pharmaceutical (i.e., antithrombotic agents) anticoagulation in cancer incidence and survival will be investigated through the analysis of the published experimental and clinical studies.
A study was conducted to demonstrate the use of gold nanoparticles (AuNP) in chemical and biological sensing. AuNPs possessed distinct physical and chemical attributes that made them excellent scaffolds for the fabrication of novel chemical and biological sensors. AuNPs were synthesized in a straightforward manner and made highly stable, while possessing unique optoelectronic properties and providing high surface-to-volume ratio with excellent biocompatibility using appropriate ligands. These synthetic routes and properties of AuNPs made them excellent probes for different sensing strategies. A variety of other ligands had been used to passivate and functionalize AuNPs, while most AuNP functionalization had been done using thiol or thiolated ligands.
An aptamer is an artificial functional oligonucleic acid, which can interact with its target molecule with high affinity and specificity. Enzyme linked aptamer assay (ELAA) is developed to detect cocaine using aptamer fragment/cocaine configuration based on the affinity interaction between aptamer fragments with cocaine. The aptasensor was constructed by cleaving anticocaine aptamer into two fragments: one was assembled on a gold electrode surface, while the other was modified with biotin at 3'-end, which could be further labelled with streptavidin-horseradish peroxidase (SA-HRP). Upon binding with cocaine, the HRP-labelled aptamer fragment/cocaine complex formed on the electrode would increase the reduction current of hydroquinone (HQ) in the presence of H(2)O(2). The sensitivity and the specificity of the proposed electrochemical aptasensor were investigated by differential pulse voltammetry (DPV). The results indicated that the DPV signal change could be used to sensitively detect cocaine with the dynamic range from 0.1 μM to 50 μM and the detection limit down to 20 nM (S/N=3). The proposed aptasensor has the advantages of high sensitivity and low background current. Furthermore, a new configuration for ELAA requiring only a single aptamer sequence is constructed, which can be generalized for detecting different kinds of targets by cleaving the aptamers into two suitable segments.
In this work, a sandwich-type electrochemical aptasensor for simultaneous sensitive detection of platelet-derived growth factor (PDGF) and thrombin is fabricated. Reduced graphene oxide sheets (rGS) are used as matrices to immobilize the redox probes, which are subsequently coated with platinum nanoparticles (PtNPs) to form the PtNPs-redox probes-rGS nanocomposites. With the employment of the as prepared nanocomposites, a signal amplification strategy was described based on bienzyme (glucose oxidase and horseradish peroxidase) modified PtNPs-redox probes-rGS nanocomposites as the tracer labels for secondary aptamers (Apt II) through sandwiched assay. Gold nanoparticles functionalized single-walled carbon nanotubes (AuNPs@SWCNTs) as the biosensor platform enhance the surface area to capture a large amount of primary aptamers (Apt I), thus amplifying the detection response. The experiment results show that the multi-labeled PtNPs-redox probes-rGS nanocomposites display satisfying electrochemical redox activity and highly electrocatalytic activity of PtNPs and bienzyme, which exhibit high sensitivity for detection of proteins. The linear range of PDGF is 0.01-35 nM with a detection limit of 8 pM, while the linear ranges from 0.02 to 45 nM and a detection limit of 11 pM for thrombin are obtained.
Angiogenin (Ang), one of the most potent angiogenic factor, is related with the growth and metastasis of numerous tumors. This paper presents a very simple and label-free square-wave voltammetry (SWV) aptasensor to detect angiogenin, in which an anti-angiogenin-aptamer was used as a molecular recognition element, and the couple ferro/ferricyanide as a redox probe. At the bare gold electrode, the redox couple (K(4)[Fe(CN)(6)]/K(3)[Fe(CN)(6)]) can be very easily accessed to the electrode surface to give a very strong SWV signal. At the anti-angiogenin/Au electrode surface, when angiogenin was added to the electrochemical cell, the binding of the analyte results in less availability for a redox reaction, which led to smaller SWV current. To quantify the amount of angiogenin, current suppressions of SWV peak were monitored using the redox couple of an [Fe(CN)(6)](4-/3-) probe. The plot of signal suppression against the logarithm of angiogenin concentration is linear with over the range from 0.01 nM to 30 nM with a detection limit of 1 pM. The aptasensor also showed very good selectivity for angiogenin without being affected by the presence of other proteins in serum. It is the first time to use a very simple method to detect the cancer marker. Such an aptasensor opens a rapid, selective and sensitive route for angiogenin detection and provides a promising strategy for other protein detections.
Nanoporous gold (NPG), made by dealloying low carat gold alloys, is a relatively new nanomaterial finding application in catalysis, sensing, and as a support for biomolecules. NPG has attracted considerable interest due to its open bicontinuous structure, high surface-to-volume ratio, tunable porosity, chemical stability and biocompatibility. NPG also has the attractive feature of being able to be modified by self-assembled monolayers. Here we use scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize a highly efficient approach for protein immobilization on NPG using N-hydroxysuccinimide (NHS) ester functionalized self-assembled monolayers on NPG with pore sizes in the range of tens of nanometres. Comparison of coupling under static versus flow conditions suggests that BSA (Bovine Serum Albumin) and IgG (Immunoglobulin G) can only be immobilized onto the interior surfaces of free standing NPG monoliths with good coverage under flow conditions. AFM is used to examine protein coverage on both the exterior and interior of protein modified NPG. Access to the interior surface of NPG for AFM imaging is achieved using a special procedure for cleaving NPG. AFM is also used to examine BSA immobilized on rough gold surfaces as a comparative study. In principle, the general approach described should be applicable to many enzymes, proteins and protein complexes since both pore sizes and functional groups present on the NPG surfaces are controllable.
Aptamers, which are in vitro selected functional oligonucleotides, have been employed to design novel biosensors (i.e., aptasensors) due to their inherent selectivity, affinity, and their multifarious advantages over traditional recognition elements. In this work, we reported a multifunctional reusable label-free electrochemical biosensor based on an integrated aptamer for parallel detection of adenosine triphosphate (ATP) and alpha-thrombin, by using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). A Au electrode as the sensing surface was modified with a part DNA duplex which contained a 5'-thiolated partly complementary strand (PCS) and a mixed aptamer (MBA). The unimolecular MBA contained small-molecule ATP binding aptamer (ABA) and also protein alpha-thrombin binding aptamer (TBA). Thus, the aptasensor could be used for detection of ATP and alpha-thrombin both. The detection limit of ATP was 1 x 10(-8) M, and its detection range could extend up to 10(-4) M, whereas the detection limit of alpha-thrombin was 1 x 10(-11) M, and its detection range was from 1 x 10(-11) to 1 x 10(-7) M. Meanwhile, after detecting alpha-thrombin, the sensing interface could be used for ATP recognition as well. The aptasensor regeneration could be realized by rehybridizing of the MBA strand with the partly complementary strand immobilized on the Au surface after ATP detection or by treating with a large amount of ATP and then rehybridizing the MBA strand with the partly complementary strand immobilized on the Au surface after alpha-thrombin detection. The aptasensor fabricated exhibited several advantages such as label-free detection, high sensitivity, regeneration, and multifunctional recognition. It also showed the detectability in biological fluid. Therein it held promising potential for integration of the sensing ability such as the simultaneous detection for multianalysis in the future.
The construction of a reagentless, sensitive, disposable and multiplexed electronic sensing platform for one-spot simultaneous determination of biomolecules with significant difference in size (proteins and small molecules) is described. The sensing surface is fabricated by the hybridization of two types of redox-tags conjugated aptamers with the corresponding complementary DNAs, which are self-assembled on a gold nanoparticle-modified screen printed carbon electrode. The presence of the target analytes leads to the release of the tagged signaling aptamers from the sensing surface, and the surface-remained tags exhibit well-resolved peaks, whose positions and sizes reflect the identities and concentrations of the target analytes, respectively. The application of the proposed sensing platform for molecular logic gate operations is also demonstrated.
Urchin-like gold submicrostructures (UGS) were successfully synthesized by a seed-mediated method which is quite facile and does not need any template or surfactant agent. The effect of the added silver seeds on the morphology and size of final products were investigated, and a possible growth mechanism of crystals was proposed. Electrochemical characterization indicated that these UGS have better catalytic activity for the glucose oxidation compared with flower-like gold submicrostructures (FGS), which could be ascribed to its higher surface to volume ratio. An electrochemical nonenzymatic glucose sensor was fabricated simply by casting the UGS and Nafion solution onto glass carbon electrode. This sensor displays a wide linear range from 0.2 to 13.2mM with a high sensitivity of 16.8 μA mM(-1)cm(-2), and a detection limit of 10 μM. The unique properties of this sensor, such as fast response and well stability reveal the potential application of the UGS based materials in nonenzymatic detection of glucose.
Based on the "nicking endonuclease assisted" detection strategy, a novel electrochemical biosensor for the sequence-specific detection of DNA with ultrahigh sensitivity and selectivity is developed. By employing the above strategy, this DNA biosensor can detect as low as 0.068 fM target DNA and exhibits high discrimination ability even against a single-base mismatch.
Biomolecular recognition is versatile, specific, and high affinity, qualities that have motivated decades of research aimed at adapting biomolecules into a general platform for molecular sensing. Despite significant effort, however, so-called “biosensors” have almost entirely failed to achieve their potential as reagentless, real-time analytical devices; the only quantitative, reagentless biosensor to achieve commercial success so far is the home glucose monitor, employed by millions of diabetics.
A label-free and sensitive faradic impedance spectroscopy (FIS) aptasensor based on target-induced aptamer displacement was developed for the determination of lysozyme as a model system. The aptasensor was fabricated by self-assembling the partial complementary single strand DNA (pcDNA)-lysozyme binding aptamer (LBA) duplex on the surface of a gold electrode. To measure lysozyme, the change in interfacial electron transfer resistance of the aptasensor using a redox couple of [Fe(CN)(6)](3-/4-) as the probe was monitored. The introduction of target lysozyme induced the displacement of the LBA from the pcDNA-LBA duplex on the electrode into the solution, decreasing the electron transfer resistance of the aptasensor. The decrease in the FIS signal is linear with the concentration of lysozyme in the range from 0.2 nM to 4.0 nM, with a detection limit of 0.07 nM. The fabricated aptasensor shows a high sensitivity, good selectivity and satisfactory regeneration. This work demonstrates that a high sensitivity of the fabricated aptasensor can be obtained using a relatively short pcDNA. This work also demonstrates that the target-induced aptamer displacement strategy is promising in the design of an electrochemical aptasensor for the determination of lysozyme with good selectivity and high sensitivity.
Functional DNA and RNA sensors, the nucleic acid enzyme (NAE) based sensors, are reviewed. Selection of a UO 2 ion-specific RNA-cleaving DNAzyme is used to illustrate the principle of selection of DNAzymes. It is found that during selection for RNA-cleaving DNAzymes, if a fluorophore and a quencher is incorporated immediately adjacent to the cleavage site, the resulting NAEs are found as sensors after selection. An investigation of Pb 2+-induced disassembly of AuNP aggregates show that the DNAzyme is inactive in head-to-tail aligned aggregates and active in tail-to-tail aligned aggregates. AuNPs are employed as quenchers for designing structure-switching aptamer sensors for protein detection. The performance of the aptamer-based system is comparable to traditional all antibody-based Enzyme-linked immunosorbent assays (ELISA) detections, and a detection limit of 25 ρg/mL is reported.
Aptamers are double-stranded DNA or single-stranded RNA molecules that bind specific molecular targets. Large randomly generated populations can be enriched in aptamers by in vitro selection and polymerase chain reaction. But so far single-stranded DNA has not been investigated for aptamer properties, nor has a target protein been considered that does not interact physiologically with nucleic acid. Here we describe the isolation of single-stranded DNA aptamers to the protease thrombin of the blood coagulation cascade and report binding affinities in the range 25-200 nM. Sequence data from 32 thrombin aptamers, selected from a pool of DNA containing 60 nucleotides of random sequence, displayed a highly conserved 14-17-base region. Several of these aptamers at nanomolar concentrations inhibited thrombin-catalysed fibrin-clot formation in vitro using either purified fibrinogen or human plasma.
We have developed an electrochemical method to quantify the surface density of DNA immobilized on gold. The surface density of DNA, more specifically the number of nucleotide phosphate residues, is calculated from the amount of cationic redox marker measured at the electrode surface. DNA was immobilized on gold by forming mixed monolayers of thiol-derivitized, single-stranded oligonucleotide and 6-mercapto-1-hexanol. The saturated amount of charge-compensation redox marker in the DNA monolayer, determined using chronocoulometry, is directly proportional to the number of phosphate residues and thereby the surface density of DNA. This method permits quantitative determination of both single- and double-stranded DNA at electrodes. Surface densities of single-stranded DNA were precisely varied in the range of (1-10) x 10(12) molecules/cm2, as determined by the electrochemical method, using mixed monolayers. We measured the hybridization efficiency of immobilized single-stranded DNA to complementary strands as a function of the immobilized DNA surface density and found that it exhibits a maximum with increasing surface density.
Electrochemical DNA detection systems are an attractive approach to the development of multiplexed, high-throughput DNA analysis systems for clinical and research applications. We have engineered a new class of nanoelectrode ensembles (NEEs) that constitute a useful platform for biomolecular electrochemical sensing. High-sensitivity DNA detection was achieved at oligonucleotide-functionalized NEEs using a label-free electrocatalytic assay. Attomole levels of DNA were detected using the NEEs, validating the promise of nanoarchitectures for ultrasensitive biosensing.
(Figure Presented) Blue, gold, and DNA: A methylene blue (MB) tagged, thrombin-binding DNA aptamer immobilized on a gold surface undergoes a large conformational change upon target binding (see schematic representation; eT: electron transfer). This folding produces a large, readily measurable change in redox current and allows the electrochemical detection of thrombin in blood serum.
Apt to change color: DNA aptamers have been used to assemble DNA-functionalized gold nanoparticles to produce highly sensitive and selective colorimetric sensors with an instantaneous color response on addition of a substrate. The general method has been shown for adenosine and cocaine (see picture), but should be applicable to any aptamer of choice. (Figure Presented).
Thrombin binding stabilizes the alternative G-quadruplex conformation of the aptamer, liberating the methylene blue (MB)-tagged oligonucleotide to produce a flexible, single-stranded DNA element. This allows the MB tag to collide with the gold electrode surface, producing a readily detectable Faradaic current at thrombin concentrations as low as approximately 3 nM.
The coupling of aptamers with the coding and amplification features of inorganic nanocrystals is shown for the first time to offer a highly sensitive and selective simultaneous bioelectronic detection of several protein targets. This is accomplished in a single-step displacement assay in connection to a self-assembled monolayer of several thiolated aptamers conjugated to proteins carrying different inorganic nanocrystals. Electrochemical stripping detection of the nondisplaced nanocrystal tracers results in a remarkably low (attomole) detection limit, that is, significantly lower than those of existing aptamer biosensors. The new device offers great promise for measuring a large panel of disease markers present at ultralow levels during early stages of the disease progress.
Advanced cancer is associated with a hypercoagulable state that is triggered by tissue factor (TF). TF-initiated thrombin generation is crucial for metastasis through fibrin and platelet deposition, as well as thrombin-dependent protease-activated receptor (PAR) 1 signaling. Surprisingly, PAR2, which is not cleaved by thrombin, appears to cosignal with PAR1 to elicit thrombin effects in metastatic tumor cells. In contrast to TF-driven thrombin pathways in metastasis, direct TF signaling plays a role in angiogenesis-dependent tumor growth. In TF cytoplasmic-domain-deleted mice, PAR2-dependent angiogenesis and tumor growth is enhanced, demonstrating a role for host cell TF signaling. In tumor cells, TF-factor VIIa (FVIIa) activates PAR2 and thereby regulates proangiogenic growth factor expression as well as integrins involving crosstalk with the TF cytoplasmic domain. In addition to thrombin-PAR signaling in metastasis and TF-FVIIa-PAR2 signaling in tumor growth, it is likely that additional protease pathways will prove to be crucial activators of PARs in cancer. Transmembrane serine proteases as well as matrix metalloproteinase are prime candidates for accessory pathways to regulate metastasis, tumor expansion, and angiogenesis dependent on specific features of the local tumor microenvironment.
We herein report a novel nanoparticle-based electrochemical DNA detection approach. This DNA sensor is based on a "sandwich" detection strategy, which involves capture probe DNA immobilized on gold electrodes and reporter probe DNA labeled with gold nanoparticles that flank the target DNA sequence. Electrochemical signals are generated by chronocoulometric interrogation of [Ru(NH(3))(6)](3+) that quantitatively binds to surface-confined capture probe DNA via electrostatic interactions. We demonstrated that the incorporation of a gold nanoparticle in this sensor design significantly enhanced the sensitivity and the selectivity. Nanoscale control of the self-assembly process of DNA probes at gold electrodes further increased the sensor performance. As a result of these two combined effects, this DNA sensor could detect as low as femtomolar (zeptomoles) DNA targets and exhibited excellent selectivity against even a single-base mismatch. In addition, this novel DNA sensor showed fairly good reproducibility, stability, and reusability.
Recognition and monitoring proteins in real time and in homogeneous solution has always been a difficult task. Here, we introduce a signal transduction strategy for quick protein recognition and real-time quantitative analysis in homogeneous solutions based on a high-affinity aptamer for protein angiogenin (Ang). The method takes advantage of the sensitive anisotropy signal change of fluorophore-labelled aptamer upon protein/aptamer binding. When the labelled aptamer is bound with its target protein Ang, the increased molecular weight causes the rotational motion of the fluorophore attached to the complex to become much slower. Therefore, increasing the amount of Ang results in a raised anisotropy value of the Ang/aptamer. By monitoring the anisotropy change, we are able to detect the binding events between the aptamer and Ang, and measure Ang concentration quantitatively in homogeneous solutions. This assay is highly selective, with a detection limit of 1 nM of Ang. The dissociation constant of the Ang/aptamer binding is determined in the nanomolar range and changes with increasing salt concentration. One can also use our assay to compare the binding affinities of different ligands for the target molecule. Ang in serum samples of malignant lung cancer was also detected. Efficient protein detection using aptamer-based fluorescence anisotropy measurements is expected to find wide applications in protein monitoring, cancer diagnosis, drug screening and other fields.
The selection of aptamers-nucleic acids that specifically bind low-molecular-weight substrates or proteins-by the SELEX (systematic evolution of ligands by exponential enrichment) procedure has attracted recent efforts directed to the development of new specific recognition units. In particular, extensive activities have been directed to the application of aptamers as versatile materials for the design of biosensors. The Minireview summarizes the recent accomplishments in developing electronic aptamer-based sensors (aptasensors), which include electrochemical, field-effect transistor, and microgravimetric quartz crystal microbalance sensors, and describes methods to develop amplified aptasensor devices and label-free aptasensors.
We report for the first time a simple low-cost electrochemical route to synthesis of diameter-controlled hierarchical flowerlike gold microstructures with "clean" surfaces using gold nanoplates or nanopricks as building blocks without introducing any template or surfactant.
We report a protocol for the amplified detection of target DNA by using a chronocoulometric DNA sensor (CDS). Electrochemistry is known to be rapid, sensitive and cost-effective; it thus offers a promising approach for DNA detection. Our CDS protocol is based on a 'sandwich' detection strategy, involving a capture probe DNA immobilized on a gold electrode and a reporter probe DNA loaded on gold nanoparticles (AuNPs). Each probe flanks one of two fragments of the target sequence. A single DNA hybridization event brings AuNPs, along with hundreds of reporter probes, in the proximity of the electrode. We then employ chronocoulometry to interrogate [Ru(NH3)6]3+ electrostatically bound to the captured DNA strands. This AuNP-amplified DNA sensor can selectively detect as low as femtomolar (zeptomoles) concentrations of DNA targets and conveniently analyze a breast cancer-associated BRCA-1 mutant DNA. The time range for the entire protocol is approximately 3 d, whereas the DNA sensing takes less than 2 h to complete.
A novel label-free immunosensor for the detection of C-reactive protein (CRP) was developed based on a three-dimensional ordered macroporous (3DOM) gold film modified electrode by using the electrochemical impedance spectroscopy (EIS) technique. The electrode was electrochemically fabricated with an inverted opal template, making the surface area of the 3DOM gold film up to 14.4 times higher than that of a classical bare flat one, characterized by the cyclic voltammetric (CV) technique. The 3DOM gold film which was composed of interconnected gold nanoparticles not only has a good biocompatible microenvironment but also promotes the increase of conductivity and stability. The CRP immunosensor was developed by covalently conjugating CRP antibodies with 3-mercaptopropionic acid (MPA) on the 3DOM gold film electrode. The CRP concentration was measured through the increase of impedance values in the corresponding specific binding of CRP antigen and CRP antibody. The increased electron-transfer resistance (R(et)) values were proportional to the logarithmic value of CRP concentrations in the range of 0.1 to 20 ng mL(-1). The detection of CRP levels in three sera obtained from hospital showed acceptable accuracy.
A sensitive method for rapid angiogenin (Ang) detection based on fluorescence resonance energy transfer (FRET) has been described. A dual-labeled probe based on high affinity aptamer for Ang was constructed. As donor and acceptor, 6-carboxyfluorescein (FAM) and 6-carboxy-tetramethylrhodamine (TMR) were labeled at 5'- and 3'-termini of the aptamer probe, respectively. The dual-labeled probe showed obvious fluorescence changes due to the specific binding between aptamer and Ang. By monitoring the fluorescence intensity of donor and acceptor, quantitative Ang detection could be achieved. This assay is highly specific and sensitive, with a detection limit of 2.0 x 10(-10) mol L(-1) and a linear range of 5.0 x 10(-10) to 4.0 x 10(-8) mol L(-1) Ang. Ang in serum samples of health and lung cancer were also detected.