In this work, a repeatable assembling and disassembling electrochemical aptamer cytosensor was proposed for the sensitive detection of human liver hepatocellular carcinoma cells (HepG2) based on a dual recognition and signal amplification strategy. A high-affinity thiolated TLS11a aptamer, covalently attached to a gold electrode through Au–thiol interactions, was adopted to recognize and capture the target HepG2 cells. Meanwhile, the G-quadruplex/hemin/aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (G-quadruplex/hemin/aptamer–AuNPs–HRP) nanoprobe was designed. It could be used for electrochemical cytosensing with specific recognition and enzymatic signal amplification of HRP and G-quadruplex/hemin HRP-mimicking DNAzyme. With the nanoprobes as recognizing probes, the HepG2 cancer cells were captured to fabricate an aptamer-cell-nanoprobes sandwich-like superstructure on a gold electrode surface. The proposed electrochemical cytosensor delivered a wide detection range from 1 × 102 to 1 × 107 cells mL−1 and high sensitivity with a low detection limit of 30 cells mL−1. Furthermore, after the electrochemical detection, the activation potential of −0.9 to −1.7 V was performed to break Au–thiol bond and regenerate a bare gold electrode surface, while maintaining the good characteristic of being used repeatedly. The changes of gold electrode behavior after assembling and desorption processes were investigated by electrochemical impedance spectroscopy and cyclic voltammetry techniques. These results indicate that the cytosensor has great potential in disease diagnostic of cancers and opens new insight into the reusable gold electrode with repeatable assembling and disassembling in the electrochemical sensing. 相似文献
A sensitive electrochemical aptasensor for detection of thrombin based on target protein‐induced strand displacement is presented. For this proposed aptasensor, dsDNA which was prepared by the hybridization reaction of the immobilized probe ssDNA (IP) containing thiol group and thrombin aptamer base sequence was initially immobilized on the Au electrode by self‐assembling via Au? S bind, and a single DNA labeled with CdS nanoparticles (DP‐CdS) was used as a detection probe. When the so prepared dsDNA modified Au electrode was immersed into a solution containing target protein and DP‐CdS, the aptamer in the dsDNA preferred to form G‐quarter structure with the present target protein resulting that the dsDNA sequence released one single strand and returned to IP strand which consequently hybridized with DP‐CdS. After dissolving the captured CdS particles from the electrode, a mercury‐film electrode was used for electrochemical detection of these Cd2+ ions which offered sensitive electrochemical signal transduction. The peak current of Cd2+ ions had a good linear relationship with the thrombin concentration in the range of 2.3×10?9–2.3×10?12 mol/L and the detection limit was 4.3×10?13 mol/L of thrombin. The detection was also specific for thrombin without being affected by the coexistence of other proteins, such as BSA and lysozyme. 相似文献
In this work, a sensitive electrochemical aptasensor for the detection of adenosine triphosphate (ATP) has been introduced. A simple and non-enzymatic signal amplification strategy is utilized using silver nanoparticle-decorated graphene oxide (AgNPs–GO) as a redox probe. The modified electrode surface was characterized by scanning electron microscopy, FTIR and UV–Vis spectroscopy, and electrochemical impedance spectroscopy. GO provides an excellent substrate for the presence of the large number of AgNPs, so the monitored oxidation signal of AgNPs has been amplified. ATP-specific DNA aptamer is split into two fragments (F1 & F2) in order to design a sandwich-type assay. For the construction of the sensor, the surface of a graphite screen-printed electrode is modified with electrodeposited gold nanoparticles followed by self-assembling a monolayer of 3-mercaptopropionic acid on the electrode surface. The first amino-labeled fragment, F1, is immobilized on the modified electrode via carbodiimide chemistry. The synthesized AgNPs–GO interacts with F1 via \(\pi{-}\pi\) stacking. In the presence of ATP, the second fragment of the aptamer, F2, forms an associated complex with the immobilized F1 and causes AgNPs–GO to leave the surface. Consequently, a remarkable decrease in the oxidation signal of the AgNPs is observed. The percentage of this decrease has been monitored as an analytical signal, which is proportional to ATP concentration, and delivers a linear response over the range of 10.0 (±0.6) to 850 (±5) nM with a detection limit of 5.0 (±0.2) nM. 相似文献
In this paper, we report a novel and more general signal-on strategy for the fabrication of electrochemical aptamer-based (E-AB) biosensor. The principle is that the interaction between the target and the aptamer strand may induce the formation and subsequent dissociation of target–aptamer complex from an electrode surface, and consequently, the remaining DNA strand on the electrode surface can hybridize again with a ssDNA containing an electrochemical probe. Differential pulse voltammetric studies have revealed that this target induced disassociation (TID) strategy is an effective signal-on method for the detection of ATP molecules with good selectivity. The TID strategy may also have several advantages, such as independence on the specific structure of either the aptamers or their complementary sequences and promotion of the generalization of E-AB sensors, the more convincible results due to the signal-on model, and the unnecessity to label the aptamers, which provides the optimized status for the reaction with the targets, etc. 相似文献
A highly sensitive and selective label-free electrochemical sensor was developed for the determination of kanamycin. To improve the sensitivity of the electrochemical sensor, graphene-gold nanoparticles were prepared by a one-step electrochemical coreduction process and were modified on the surface of a glassy carbon electrode. The double-stranded DNA(ds-DNA) duplex probe was immobilized onto the graphene-gold nanoparticle-modified electrode. The introduction of target kanamycin induced the displacement of aptamer from the ds-DNA duplex into the solution. Methylene blue(MB) as a redox indicator monitored the current change using differential pulse voltammetry. Under optimal conditions, the designed electrochemical aptasensor exhibited a wide linear range from 0.1 pmol/L to 10 pmol/L with a detection limit of 0.03 pmol/L for kanamycin. The experimental strategy enabled the direct analysis of milk samples, and the results showed high sensitivity and good selectivity. 相似文献
A novel aptamer-based label-free electrochemical impedance spectroscopy biosensor for 17β-estradiol has been fabricated. The aptamers were firstly immobilized on the gold electrode through Au-S interaction; the aptamer probe was then bound with the addition of 17β-estradiol to form the estradiol/aptamer complex on the electrode surface. This leads to a significantly larger interfacial electron transfer resistance than that without the addition of 17β-estradiol. The change in the resistance had a linear relationship with 17β-estradiol concentration in the range of 1.0 × 10(-8) to 1.0 × 10(-11) mol L(-1), with a detection limit of 2.0 × 10(-12) mol L(-1). The biosensor showed high selectivity to 17β-estradiol and good stability. The designed biosensor has been applied to detect 17β-estradiol in human urine with satisfactory results. 相似文献
Previous aptamers for porphyrins and metalloporphyrins were all guanine-rich sequences that can fold in G-quadruplex structures. Due to stacking-based binding, these aptamers can hardly tell different porphyrins apart, and they can also bind other planar molecules, hindering their practical applications. In this work, we used the capture selection method to obtain aptamers for hemin and protoporphyrin IX (PPIX). The hemin aptamer (Hem1) features two highly conserved repeating binding loops, and it cannot form a G-quadruplex, which was supported by its Mg2+-dependent but K+-independent hemin binding and CD spectroscopy. Isothermal titration calorimetry revealed much higher enthalpy change for the new aptamer, and the best aptamer showed a Kd of 43 nM hemin. Hem1 can also enhance the peroxidase-like activity of hemin. This work demonstrates that aptamers have alternative ways to bind porphyrins allowing selective recognition of different porphyrins. 相似文献
An electrochemical sensing system for oxytetracycline (OTC) detection was developed using ssDNA aptamer immobilized on gold interdigitated array (IDA) electrode chip. A highly specific ssDNA aptamer that bind to OTC with high affinity was employed to discriminate other tetracyclines (TCs), such as doxycycline (DOX) and tetracycline (TET). The immobilized thiol-modified aptamer on gold electrode chip served as a biorecognition element for the target molecules and the electrochemical signals generated from interactions between the aptamers and the target molecules was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV). The current decrease due to the interference of bound OTC, DOX or TET was analyzed with the electron flow produced by a redox reaction between ferro- and ferricyanide. The specificity of developed EC-biosensor for OTC was highly distinguishable from the structurally similar antibiotics (DOX and TET). The dynamic range was determined to be 1-100 nM of OTC concentration in semi-logarithmic coordinates. 相似文献
This study describes a new kind of aptamer-based electrochemical sensor that is not based on the target binding-induced conformational change of the aptamers by using a 15-mer thrombin-binding aptamer (5'-GGTTGGTGTGGTTGG-3') as the model oligonucleotide. The sensors are developed by first self-assembling the aptamer (i.e. a thrombin-binding aptamer) onto an Au electrode and then hybridizing the assembled aptamer with a ferrocene (Fc)-labeled short aptamer-complementary DNA oligonucleotide to form an electroactive double-stranded DNA (ds-DNA) oligonucleotide onto the Au electrode. The binding of the target (i.e. thrombin) towards the aptamer essentially destroys the Watson-Crick helix structure of the ds-DNA oligonucleotide assembled onto the electrode and leads to the dissociation of the Fc-labeled short complementary DNA oligonucleotide from the electrode surface to the solution, resulting in a decrease in the current signal obtained at the electrode, which can be used for the determination of the target. With the thrombin-binding aptamer as the model oligonucleotide, the current decrease obtained with the aptamer-based electrochemical sensors is linear with the concentration of thrombin within the concentration range from 0 to 10 nM (DeltaI/nA = 6.7C(thrombin)/nM + 2.8, gamma = 0.975). Unlike most kinds of existing aptamer-based electrochemical sensor, the electrochemical aptasensors demonstrated here are not based on the conformational change of the aptamers induced by the specific target binding. Moreover, the aptasensors are essentially label-free and are very responsive toward the targets. This study may pave a facile and general way to the development of aptamer-based electrochemical sensors. 相似文献
The aim of this study is to develop a selective adenosine aptamer sensor using a rational approach. Unlike traditional RNA
aptamers developed from SELEX, duplex DNA containing an abasic site can function as a general scaffold to rationally design
aptamers for small aromatic molecules. We discovered that abasic site-containing triplex DNA can also function as an aptamer
and provide better affinity than duplex DNA aptamers. A novel adenosine aptamer sensor was designed using such a triplex.
The aptamer is modified with furano-dU in the binding site to sense the binding. The sensor bound adenosine has a dissociation
constant of 400 nM, more than tenfold stronger than the adenosine aptamer developed from SELEX. The binding quenched furano-dU
fluorescence by 40%. It was also demonstrated in this study that this sensor is selective for adenosine over uridine, cytidine,
guanosine, ATP, and AMP. The detection limit of this sensor is about 50 nM. The sensor can be used to quantify adenosine concentrations
between 50 nM and 2 μM. 相似文献
Oxidative damage is an important factor in causing various human disease and injury. As an oxidative DNA damage product, 8-hydroxy-2′-deoxyguanosine (8-OHdG) is a key marker, which is widely used to study oxidative damage mechanism in diseases. Most reported electrochemical methods were based on oxidation current of 8-OHdG. In this work, a simple electrochemical biosensor for ultrasensitive detection of 8-OHdG was proposed based on it triggered polyaniline (PANI) deposition on tetrahedral DNA nanostructure (TDN). TDN was immobilized onto a gold electrode surface based on self-assembly between three thiolated nucleotide sequences. 8-OHdG-aptamer on the top of TDN formed a hemin/G-quadruplex structure in the presence of 8-OHdG and hemin, which have high catalytic activity to trigger PANI deposition. Numerous negative charges on the duplex DNAs contained in hemin/G-quadruplex and TDN supplied exquisite environment for PANI deposition, which improved the detection sensitivity greatly by increasing the DPV current to10-fold (∼3 μA) compared to our previously reported method without TDN. The response signals correlated linearly with the concentration of 8-OHdG ranging from 10 pM to 2 nM, with a detection limit of 1 pM (S/N = 3). The sensitivity was improved to almost 300-fold when compared with most of previously reported electrochemical methods. The method was also simple and reliable, avoiding complex, expensive label procedures and nanomaterial synthesized procedures. The method had been successfully applied to quantify 8-OHdG in urine and human serum samples with satisfactory results. 相似文献
The authors describe a method for signal amplification in electrochemical aptasensors. It is based on the induction of an increased electrochemical current by the aptamer captured on a glassy carbon electrode (GCE). The phosphate groups on the aptamer backbone are brought to reaction with added molybdate to form a redox-active molybdophosphate precipitate on the surface of the GCE that generates a strong electrochemical current. To further enhance sensitivity, gold nanorods (GNRs) were selected as a support for the immobilization of aptamers. The aptasensor was applied to the determination of the cancer biomarker carcinoembryonic antigen (CEA) in a sandwich format. Antibody against CEA, CEA (antigen) and GNRs modified with CEA aptamer were sequentially captured on the GCE. The resulting aptasensor, best operated at a voltage as low as 0.18 V vs. Ag/AgCl, is highly sensitive and has a wide linear range that extends from 0.1 pg·mL?1 to 10 ng·mL?1 of CEA. This amplification strategy uses an aptamer as both the recognition probe and signal probe and therefore simplifies signal transduction. Conceivably, this detection scheme may be adapted to numerous other electrochemical bioassays if respective antibodies and aptamers are available.
Graphical abstract Schematic presentation of an electrochemical aptasensor based on aptamer induced electrochemical current for the detection of cancer biomarker carcinoembryonic antigen (CEA). Gold nanorods (GNR) are chosen for the immobilization of aptamers to increase the loading of aptamers.
In this work, by incorporating a specific DNAzyme sequence into a hairpin aptamer probe, we describe a label-free and sensitive method for electrochemical detection of cytokines using recombinant human IFN-γ as the model analyte. The hairpin aptamer probes are immobilized on a gold electrode through self-assembly. The presence of IFN-γ opens the hairpin structure and forms the hemin/G-quadruplex peroxidase-mimicking DNAzyme with subsequent addition of hemin. The peroxidase-mimicking DNAzyme catalyzes the electro-reduction of H(2)O(2) and amplifies the current response for IFN-γ detection, which enables the monitoring of IFN-γ at the sub-nanomolar level. The proposed sensor also shows high selectivity towards the target analyte. Our strategy thus opens new opportunities for label-free and amplified detection of different types of cytokines. 相似文献
A novel electrochemiluminescence (ECL) aptasensor was proposed for sensitive and cost-effective detection of the target thrombin adopted an aptamer-based sandwich format. To detect thrombin, capture aptamers labeled with gold nanoparticles (AuNPs) were first immobilized onto the thio-silanized ITO electrode surface through strong Au-S bonds. After catching the target thrombin, signal aptamers tagged with ECL labels were attached to the assembled electrode surface. As a result, an AuNPs-capture-aptamer/thrombin/ECL-tagged-signal-aptamer sandwich type was formed. Treating the resulting electrode surface with tri-n-propylamine (TPA) and applying a swept potential to the electrode, ECL response was generated which realized the detection of target protein. Spectroscopy and electrochemical impedance techniques were used to characterize and confirm the fabrication of the ECL aptasensor. AuNPs amplification and smart sensor fabrication art were implemented for the sensitive and cost-effective detection purpose. Signal-to-dose curve excellently followed a sandwich format equation and could be used to quantify the protein, and the detection limit was estimated to be 10 nM. Other forms of thrombin such as β- and γ-thrombins had negligible response, which indicated a high specificity of α-thrombin detection. The aptasensor opened up new fields of aptamer applications in ECL domain, a highly sensitive technique, and had a promising perspective to be applied in microarray analysis. 相似文献
The binding affinity of 17β-estradiol with an immobilized DNA aptamer was measured using capillary electrophoresis. Estradiol captured by the immobilized DNA was injected into the separation capillary using pH-mediated sample stacking. Stacked 17β-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Standard addition was used to quantify the concentration of estradiol bound to the aptamer. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 ± 10 μM. The method was also used to screen binding affinity of the aptamer for estrone and testosterone. This study demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers. 相似文献