The authors describe an aptasensor for visual and fluorescent detection of lysozyme via an inner filter effect (IFE). The assay is based on the fact that red gold nanoparticles (AuNPs) act as powerful absorbers of the green fluorescence of CdTe because of spectral overlap. If the lysozyme-binding aptamer is adsorbed onto the surface of the AuNPs, the salt-induced aggregation of AuNPs (that leads to a color change from red to blue) does not occur and the IFE remains efficient. If lysozyme is present, it will bind the aptamer and thereby prevent its adsorption on the AuNPs. As a result, the salt-triggered aggregation of the AuNPs will occur. Consequently, color will change from red to blue, and green fluorescence will pop up because the IFE is suppressed. Under optimum conditions, fluorescence is linearly related to lysozyme concentration in the 1.0 nM to 20 nM concentration range, with a 0.55 nM limit of detection. The method is perceived to be of wider applicability in that it may be used to design other visual and fluorescent assays if appropriate aptamers are available.
Graphical abstract The fluorescence intensity of QDs is quenched by gold nanoparticles (AuNPs) due to an inner filter effect. Aptamers can adsorb on AuNPs to prevent the salt-induced aggregation. AuNPs serve a dual function as fluorescence quencher and colorimetric reporter.
The authors describe a colorimetric method for the determination of DNA based on the deaggregation of gold nanoparticles (AuNPs) induced by exonuclease III (Exo III). DNA amplification is accomplished by Exo III to generate large quantities of the residual DNA. Residual DNA tethers onto the surfaces of AuNPs which prevents their aggregation. Hence, the color of the solution is red. However, in the absence of DNA, salt-induced aggregation is not prevented, and the bluish-purple color of the aggregated AuNPs is observed. The ratio of absorbances at 525 and 625 nm increases up to 150 nM DNA concentrations, and the LOD is as low as 3.0 nM. It is shown that the presence of 300 nM concentrations of random DNA (with a mass up to 10-fold that of target DNA) does not interfere. The method was successfully applied to the analysis of DNA in spiked serum samples. The method is simple, reliable, and does not require complicated amplification steps and expensive instrumentation.
Graphical abstract Schematic of a sensing strategy for DNA detection by exonuclease III-induced deaggregation of gold nanoparticles. DNA concentrations as low as 3 nM can be detected via colorimetric monitoring of the color change from red to purple-blue.
A sensitive visual aptamer-based assay is presented for the determination of ractopamine (RAC) in animal feed beef. In the absence of RAC, the aptamer binds to gold nanoparticles (AuNPs) and this prevents the AuNPs to undergo salt-induced aggregation which usually is accompanied by a color change from red to blue. If however, RAC is present, it will bind to the aptamer while the AuNPs remain uncoated so that aggregation and a color change will occur due to salt-induced aggregation. This can be monitored by spectrophotometer or even with bare eyes. Under optimal conditions, the aptasensor exhibits a linear range that covers the 10 to 400 ng.mL￣1 RAC concentration range. The limit of detection is as low as 10 ng.mL￣1. In order to further improve selectivity, a RAC-selective molecularly imprinted membrane was prepared and used to pre-extract RAC from complex samples. The combined method (molecularly imprinted membrane and aptasensor) was applied to the determination of RAC in spiked animal feed and beef and gave recoveries that ranged from 72.7 % to 87.3 % for complete feed and from 78.2 % to 86.5 % for beef, respectively.
Graphical abstract A sensitive visual aptamer-based assay based on aggregation of gold nanoparticles in combination with a molecularly imprinted polymer was developed for the determination of ractopamine (RAC) in animal feed and beef.
A colorimetric method is presented for the determination of the antibiotic ofloxacin (OFL) in aqueous solution. It is based on the use of an aptamer and gold nanoparticles (AuNPs). In the absence of OFL, the AuNPs are wrapped by the aptamer and maintain dispersed even at the high NaCl concentrations. The solution with colloidally dispersed AuNPs remains red and has an absorption peak at 520 nm. In the presence of OFL, it will bind to the aptamer which is then released from the AuNPs. Hence, AuNPs will aggregate in the salt solution, and color gradually turns to blue, with a new absorption peak at 650 nm. This convenient and specific colorimetric assay for OFL has a linear response in the 20 to 400 nM OFL concentration range and a 3.4 nM detection limit. The method has a large application potential for OFL detection in environmental and biological samples.
Graphical abstract Schematic of a sensitive and simple colorimetric aptasensor for ofloxacin (OFL) detection in tap water and synthesic urine. The assay is based on the salt-induced aggregation of gold nanoparticles which results in a color change from red to purple.
A colorimetric method is described for the determination of Pt(II). It is based on the use of gold nanoparticles (AuNPs) which are known to aggregate in the presence of a cationic polymer such as poly(diallyldimethylammonium chloride) (PDDA). If, however, a mismatched aptamer (AA) electrostatically binds to PDDA, aggregation is prevented. Upon the addition of Pt(II), it will bind to the aptamer and induce the formation of a hairpin structure. Hence, interaction between aptamer and PDDA is suppressed and PDDA will induce the aggregation of the AuNPs. This is accompanied by a color change from red to blue. The effect can be observed with bare eyes and quantified by colorimetry via measurement of the ratio of absorbances at 610 nm and 520 nm. Response is linear in the 0.24–2 μM Pt(II) concentration range, and the detection limit is 58 nM. The assay is completed within 15 min and selective for Pt(II) even in the presence of other metal ions. It was successfully applied to the rapid determination of Pt(II) in spiked soil samples.
Graphical abstract Schematic representation of the method for detection of Pt(II) based on the use of a cationic polymer and gold nanoparticles. In the presence of Pt(II), aptamer interacts with the Pt(II) and prevents the interaction between aptamer and cationic polymer. Hence, cationic polymer induce the aggregation of the AuNPs and lead to the color change from red to blue.
The article reports an aptamer based assay for cocaine by employing graphene oxide and exonuclease III-assisted signal amplification. It is based on the following scheme and experimental steps: (1) Exo III can digest dsDNA with blunt or recessed 3-terminus, but it has limited activity to ssDNA or dsDNA with protruding 3-terminus; (2) GO can absorb the FAM-labeled ssDNA probe and quench the fluorescence of probe, while the affinity between FAM-labeled mononucleotide and GO is negligible; (3) Cocaine aptamer can be split into two flexible ssDNA pieces (Probe 1 and Probe 2) without significant perturbation of cocaine-binding abilities; (4) The triple complex consisting of Probe 1, Probe 2 and cocaine can be digested by Exo III with the similar efficiency as normal dsDNA. Cocaine aptamer is split into two flexible ssDNA pieces (Probe 2 and 3′-FAM-labeled Probe 1). Cocaine can mediate the cocaine aptamer fragments forming a triplex. The triple complex has unique characteristic with 3′-FAM-labeled blunt end at the Probe 1 and 3′-overhang end at Probe 2. If exonuclease III is added, it will catalyze the stepwise removal of fluorescein (FAM) labeled mononucleotides from the 3-hydroxy termini of the special triplex complex, resulting in liberation of cocaine. The cocaine released in this step can produce a new cleavage cycle, thereby leading to target recycling. Through such a cyclic bound-hydrolysis process, small amounts of cocaine can induce the cleavage of a large number of FAM-labeled probe 1. The cleaved FAM-labeled mononucleotides are not adsorbed on the surface of graphene oxide (GO), so a strong fluorescence signal enhancement is observed as the cocaine triggers enzymatic digestion. Under optimized conditions, the assay allows cocaine to be detected in the 1 to 500 nM concentration range with a detection limit of 0.1 nM. The method was applied to the determination of cocaine in spiked human plasma, with recoveries ranging from 92.0 to 111.8 % and RSD of <12.8 %.
The authors describe a new kind of adenosine triphosphate (ATP) assay. It is based on the use of gold nanoparticles (AuNPs) coated with 4-mercaptophenylboronic acid (MPBA) as the recognition element for ATP. MPBA has a specific affinity for AuNPs through Au-S interaction, and three boronic acid groups undergo condensation to form a boroxine ring. This induces the aggregation of AuNPs and a visible color change from red to blue. However, in the presence of ATP, the boronic acid group of MPBA preferentially binds to the 2’, 3’-hydroxy group of ATP to form a stable boronate ester. Hence, the aggregation of AuNPs is progressively decreased as the concentration of ATP increases, and the color change is increasingly reversed. The ratio of absorbance at 520 and 683 nm increases linearly in the 8 to 100 μM ATP concentration range, with a 0.12 μM limit of detection (at an S/N ratio of 3 σ). The colorimetric assay was successfully applied to the determination of ATP in T47D breast cancer cells and in cultured cells with added anticarcinogen.
Graphical abstract Schematic of a colorimetric assay for the visualization and sensitive and selective detection of adenosine triphosphate (ATP) based on the use of gold nanoparticles (AuNPs) coated with 4-mercaptophenylboronic acid (MPBA). The assay was applied to the determination of ATP in T47D breast cancer cells and in cultured cells with added anticarcinogen.
The authors describe a method for the colorimetric determination of unamplified microRNA. It is based on the use of citrate-capped gold nanoparticles (AuNPs) and, alternatively, a microRNA-probe hybrid or a magnetically extracted microRNA that serve as stabilizers against the salt-induced aggregation of AuNPs. The absorbance ratios A525/A625 of the reacted AuNP solutions were used to quantify the amount of microRNA. The assay works in the range of 5–25 pmol microRNA. The lower limit of detection (LOD) is 10 pmol. The performance of the method was tested by detection of microRNA-210-3p in totally extracted urinary microRNA from normal, benign, and bladder cancer subjects. The sensitivity and specificity for qualitative detection of urinary microRNA-210-3p using the assay are 74% and 88% respectively, which is consistent with real time PCR based assays. The assay was applied to the determination of specific microRNA by using its specific oligo targeter or following magnetic isolation of the desired microRNA. The method is simple, cost-efficient, has a short turn-around time and requires minimal equipment and personnel.
Graphical abstract Schematic of the two detection schemes: In the first approach, matched microRNA hybridizes with its specific probe to stabilize gold nanoparticles (AuNPs) against salt induced aggregation and to leave the red color of the AuNPs unchanged. In the second one, microRNA extracted via magnetic nanoparticles (MNP) stabilizes AuNPs against aggregation.
An efficient approach is demonstrated for preparing particles consisting of a silver core and a shell of molecularly imprinted polymer (Ag@MIP). The MIP is prepared by using bisphenol A (BPA) as the template and 4-vinylpyridine as the functional monomer. The Ag@MIP fulfills a dual function in that the silver core acts as a SERS substrate, while the MIP allows for selective recognition of BPA. The Ag@MIP is characterized by scanning electron microscopy, transmission electron microscopy, X-ray powder diffraction, thermogravimetric analysis and Raman spectroscopy. The Raman intensity of Ag@MIP is higher than that of bare silver microspheres. The detection limit for BPA is as low as 10?9 mol·L?1.
Graphical abstract Schematic illustration of the preparation of silver microspheres coated with a molecularly imprinted polymer (Ag@MIPs) for detecting bisphenol A (BPA) by surface enhanced Raman scattering (SERS).
The authors describe an aptamer based assay for determination of ractopamine (RAC) by using PicoGreen (PG) as a fluorescent probe specific for dsDNA. In the absence of RAC, the aptamer forms a duplex structure with a complementary sequence that results in enhanced PG fluorescence. Upon binding to RAC, the aptamer undergoes a structural switch. This reduces the number of DNA duplexes formed and causes a reduction of fluorescence intensity of PG as measured at excitation/emission wavelengths of 480/520 nm. Under optimized conditions, the dynamic calibration plot covers the 50 pM to 50 μM concentration range, with a 50 pM detection limit. This meets the safety supervision regulations of the European Commission in terms of residue limits of RAC in food. The method displays high selectivity over other β-adrenergic agonists including clenbuterol, dopamine and salbutamol. The assay was successfully applied to samples of swine urine at spiking levels of 7.4 nM, 22.2 nM and 37 nM. Average recoveries ranged from 95 to 110%, with an RSD of <1.5%. The method is expected to represent a promising tool for simple, rapid and sensitive on-site detection of RAC in animal products.
Graphical abstract An aptamer based fluorescent assay for determination of ractopamine was developed with a dynamic range of 50 pM to 50 μM. The average recovery from spiked urine samples ranged from 95 to 110%, with an RSD of <1.5%.
The authors describe a gold nanoparticle (AuNP) based aggregation assay for colorimetric determination of silver ions. The detection scheme is based on the release of aptamers from the surface of AuNPs that is triggered by the formation of C-Ag(I)-C links. In the absence of Ag(I) ions, the aptamers are readily adsorbed on the surface of the AuNPs. This prevents the aggregation of AuNPs and warrants the stability of the red colloidal solution at high salt concentration. In the presence of Ag(I) ions, the aptamers are released from the surface of AuNPs due to binding to Ag(I). Hence, salt-induced aggregation of AuNPs will occur which is accompanied by a gradual color change from red to blue. The color change occurs in the 1 to 500 nM Ag(I) concentration range, and the detection limit is 0.77 nM. The method was successfully applied to the determination of Ag(I) in spiked tap water samples.
Graphical abstract Schematic of a gold nanoparticle-based aggregation assay for colorimetric determination of silver ions. Visual quantitation also is posssible due to a gradual color change from red to blue.
An electrochemiluminescent (ECL) aptamer based method is described for the determination of thrombin. Three-dimensional nitrogen-doped graphene oxide (3D-NGO) was placed on a glassy carbon electrode (GCE) to provide an electrode surface that displays excellent electrical conductivity and acts as a strong emitter of ECL. The modified electrode was further coated with chitosan via electrodeposition. Finally, the amino-modified aptamer was immobilized on the modified GCE. The interaction between thrombin and aptamer results in a decrease in ECL. The assay has a linear response in the 1 fM to 1 nM thrombin concentration range and a 0.25 fM lower detection limit (at an S/N ratio of 3). The method was applied to the determination of thrombin in spiked human plasma samples, and recoveries ranged between 94 and 105% (with RSDs of <3.6%). The calibration plot was recorded at potential and wavelength of fluorescence emission (wavelength:?445 nm; potential:?0 to -2 V).
Graphical abstract A bare glassy carbon electrode (GCE) does not display electrochemiluminescence (ECL). If, however, nitrogen-doped graphene quantum dots, chitosan, and three-dimensional nitrogen-doped graphene oxide (NGQD-chitosan/3D-NGO) are electrodeposited on the GCE, strong ECL can be observed. The ECL intensity decreased after aptamer and bovine serum albumin (BSA) were dropped onto the electrode (curve a). However, the ECL further decreases after addition of thrombin (TB; curve b).
A fluorometric patulin (PAT) assay is presented that is based on the use of magnetic reduced graphene oxide (rGO) and DNase I. The fluorescence of the PAT aptamer labelled with 6-carboxyfluorescein (FAM) is quenched by magnetized reduced graphene oxide (rGO-Fe3O4) due to fluorescence resonance energy transfer (FRET). However, in the presence of PAT, the labelled aptamer is stripped off from rGO-Fe3O4. The rGO-Fe3O4 is then magnetically separated so that the fluorescence of free labelled PAT aptamer is restored. DNase I cannot hydrolyze the aptamer on rGO-Fe3O4, but it can cleave the free aptamer-PAT complex. This will release FAM and PAT which can undergo a number of additional cycles to trigger the cleavage of abundant aptamer. Recycling of DNase I-assisted target therefore leads to a strong amplification of fluorescence and consequently to an assay with low limit of detection. The detection limit for PAT is as low as 0.28 μg L?1 which is about 13 times lower than that without using DNase I. The method offers a new approach towards rapid, sensitive and selective detection based on an aptamer. Conceivably, it has a wide scope in that it may be applied to numerous other analytes if appropriate aptamers are available.
Abstract Schematic of a fluorometric assay based on the use of magnetic graphene oxide and DNase I. It was applied to the determination of patulin. DNase I was introduced for recycling amplification. The detection limit is about 13 times lower than that without using DNase I. Figure a contains poor quality of text in image. Otherwise, please provide replacement figure file.Thank you. I will provide the figure file.
The authors have developed a straightforward colorimetric method for the rapid determination of lysozyme by using citrate-capped gold nanoparticles (AuNPs) with different particle sizes but without any further surface modification. It is found that AuNPs (15 nm i.d.) undergo aggregation in the presence of lysozyme owing to the high abundance of amino groups in lysozyme. Aggregation leads to a color change of the solution from red over purple to bluish-purple that can be detected visually or by photometry. The limit of detection is 20 nM. We further show that the use of AuNPs with 5 and 15 nm i.d. can improve the sensitivity of the assay compared to using bare AuNPs by adding HAuCl4 and NH2OH to the solution which induces the growth of AuNPs and leads to a smaller interparticle space between AuNPs. This gives rise to differently colored solutions, with color transitions from red to bluish-purple to colorless. The LODs are 0.1 nM for both the 5-nm and 15-nm AuNPs. Compared to the LOD when using a solution of 15-nm AuNPs and without chloroauric acid and hydroxylamine, the LOD (0.1 nM) is lower by a factor of 200. The method is sensitive, specific, and does not require sophisticated equipment. Its feasibility was demonstrated by analyzing lysozyme in samples of egg white.
Graphical abstract We utilized 4 kinds of gold AuNPs with different particle sizes (5, 15, 30, and 50 nm) as colorimetric probes for lysozyme analysis.
An ultrasensitive conformation-dependent colorimetric assay has been developed for the detection of mercury(II) ions. It is based on the use of exonuclease III (Exo III)-assisted target recycling and gold nanoparticles (AuNPs). In the absence of Hg(II), the hairpin-shaped DNA probe (H-DNA) binds to AuNPs and stabilizes them in solutions of high ionic strength. In the presence of Hg(II), on the other hand, the sticky termini of the H-DNA form a rigid DNA duplex stem with a blunt 3′-terminus. Thus, Exo III is activated as a biocatalyst for selective and stepwise removal of mononucleotides from the 3′-terminus of the H-DNA. As a result, Hg(II) is released from the T?Hg(II)?T complexes. The guanine-rich sequences released from the H-DNA are then self-assembled with potassium ion to form a stable G-quadruplex conformation. In solutions of high ionic strength, this results in aggregation of AuNPs and a color change from red to blue which can be seen with bare eyes. The method is highly sensitive and selective. It has a linear response in the 10 pM to 100 nM Hg(II) concentration range, and the detection limit is as low as 3.2 pM (at an S/N ratio of 3). The relative standard deviation at a level of 0.5 nM of Hg(II) is 4.9% (for n?=?10). The method was applied to the detection of Hg(II) in spiked environment water samples, with recoveries ranging from 92% to 106%.
Graphical abstract A conformation-dependent colorimetric system was fabricated for label-free detection of mercury(II) by utilizing exonuclease III(Exo III)-assisted target recycling and gold nanoparticles (AuNPs).
An isothermal colorimetric method is described for amplified detection of the CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on (a) target DNA-triggered unlabeled molecular beacon (UMB) termini binding, and (b) exonuclease III (Exo III)-assisted target recycling, and (c) hemin/G-quadruplex (DNAzyme) based signal amplification. The specific binding of target to the G-quadruplex sequence-locked UMB triggers the digestion of Exo III. This, in turn, releases an active G-quadruplex segment and target DNA for successive hybridization and cleavage. The Exo III impellent recycling of targets produces numerous G-quadruplex sequences. These further associate with hemin to form DNAzymes and hence will catalyze H2O2-mediated oxidation of the chromogenic enzyme substrate ABTS2? causing the formation of a green colored product. This finding enables a sensitive colorimetric determination of GMO DNA (at an analytical wavelength of 420 nm) at concentrations as low as 0.23 nM. By taking advantage of isothermal incubation, this method does not require sophisticated equipment or complicated syntheses. Analyses can be performed within 90 min. The method also discriminates single base mismatches. In our perception, it has a wide scope in that it may be applied to the detection of many other GMOs.
Graphical abstract An isothermal and sensitive colorimetric method is described for amplified detection of CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on target DNA-triggered molecular beacon (UMB) termini-binding and exonuclease III assisted target recycling, and on hemin/G-quadruplex (DNAzyme) signal amplification.
The authors describe a fluorescence based aptasensor for adenosine (AD), a conceivable biomarker for cancer. The assay is based on the immobilization of capture DNA on newly synthesized quaternary CuInZnS quantum dots (QDs) and the conjugation of probe DNA on gold nanoparticles (AuNPs). The capture DNA is an adenosine-specific aptamer that is partly complementary to the probe DNA. Once the capture aptamer hybridizes probe DNA, the fluorescence of the QDs (measured at excitation/emission wavelengths of 522/650 nm) is quenched by the AuNPs. However, when AD is added, it will bind to the aptamer and restrain the hybridization between capture DNA and probe DNA. Therefore, the fluorescence of the QDs will increase with increasing AD concentration. Under optimal conditions, fluorescence is linearly related to the AD concentration in the range from 50 to 400 μM, the detection limit being 1.1 μM. This assay is sensitive, selective, reproducible and acceptably stable. It was applied to the determination of AD in spiked human serum samples where it gave satisfactory results.
An aptamer based method is described for the electrochemical determination of ampicillin. It is based on the use of DNA aptamer, DNA functionalized gold nanoparticles (DNA-AuNPs), and single-stranded DNA binding protein (ssDNA-BP). When the aptamer hybridizes with the target DNA on the AuNPs, the ssDNA-BP is captured on the electrode surface via its specific interaction with ss-DNA. This results in a decreased electrochemical signal of the redox probe Fe(CN)63? which is measured best at a voltage of 0.188 mV (vs. reference electrode). In the presence of ampicillin, the formation of aptamer-ampicillin conjugate blocks the further immobilization of DNA-AuNPs and ssDNA-BP, and this leads to an increased response. The method has a linear reposne that convers the 1 pM to 5 nM ampicillin concentration range, with a 0.38 pM detection limit (at an S/N ratio of 3). The assay is selective, stable and reproducible. It was applied to the determination of ampicillin in spiked milk samples where it gave recoveries ranging from 95.5 to 105.5%.
Graphical abstract Schematic of a simple and sensitive electrochemical apta-biosensor for ampicillin detection. It is based on the use of gold nanoparticles (AuNPs), DNA aptamer, DNA functionalized AuNPs (DNA-AuNPs), and single-strand DNA binding protein (SSBP).
An electrochemical nanoaptasensor is described that is based on the use of a glassy carbon electrode (GCE) modified with electrodeposited silver nanoparticles (AgNPs). An aptamer (Apt) against trinitrotoluene (TNT) was then immobilized on the AgNPs. The addition of TNT to the modified GCE leads to decrease in peak current (typically measured at a potential of ?0.45 V vs. Ag/AgCl) of riboflavin which acts as an electrochemical probe. Even small changes in the surface (as induced by binding of Apt to TNT) alter the interfacial properties. As a result, the LOD is lowered to 33 aM, and the dynamic range extends from 0.1 fM to 10 μM without sacrificing specificity.
Graphical abstract Schematic presentation of a nanoaptasensor which is based on a glassy carbon electrode (GCE) modified with electrodeposited silver nanoparticles (AgNPs) and aptamer (Apt). It was applied to the detection of 2,4,6-trinitrotoluene (TNT) with the help of riboflavin (RF) as a redox probe.
The authors describe a colorimetric method for the determination of the staphylococcal enterotoxin B (SEB) that also allows for visual readout. The assay is based on the growth of gold nanoparticles (AuNPs) mediated by a hemin/G-quadruplex DNAzyme which generates a color change from red to blue in the presence of SEB. The method is enzyme-free and does not require a label. The kinetics of the formation of the AuNPs is controlled by the hemin/G-quadruplex DNAzyme and this is key to the signal generation mechanism. In the presence of SEB, the reactions between aptamer and target modulated the amount of single probe G strands that form DNAzyme capable of consuming hydrogen peroxide. The growth process of AuNPs is influenced by the resulting concentration of H2O2 and leads to the color change. Under optimal conditions, a linear relationship exists between absorbance and SEB concentration in the range from 0.1 to 500 pg·mL￣1 which covers the clinically relevant range. In case of visual detection, the lower limit of detection is 1 pg·mL?1. The assay described here is sensitive, comparably inexpensive and can detect SEB rapidly without the need for sophisticated equipment. In our perception, the method has a wide scope in that it may be adapted to various nucleic acids, proteins and other biomolecules if respective aptamers are available.
