The authors describe an array for chemiluminescence (CL) based determination of cardiac troponin T (cTnT), an important cardiovascular disease marker. The tracing tag consists of silver nanoparticles (AgNPs) loaded with guanine-rich DNA sequences and detection antibody in a high numerical ratio. The loaded AgNPs were then reacted with hemin to form a hemin/G-quadruplex DNAzyme. A disposable immunosensor array was fabricated by immobilizing capture antibody on corresponding sensing sites on a glass chip. Once a sandwich immunocomplex is formed on the array, the tracing tag catalyzes the CL reaction of the luminol-p-iodophenol and H2O2 system to produce a CL signal, which is collected by a CCD camera. An intuitive CL image is obtained containing all of the spots on the array. Under optimal conditions, the method shows a wide linear range over 4 orders of magnitude (from 0.003 to 270 ng·L?1), a detection limit down to 84 fg·L?1, and a throughput as high as 44 tests·h?1. The results obtained with serum samples are in acceptable agreement with reference values. The AgNP-based tracing tag as well as the immunoassay method shows a promising potential for point-of-care testing for the early clinical diagnosis of cardiovascular disease.
Graphical abstract Schematic presentation of silver nanoparticles (AgNPs) functionalized with hemin/G-quadruplex DNAzyme for highly sensitive chemiluminescence (CL) immunoassay of cardiac troponin T (cTnT) on a glass chip array.
The article describes a colorimetric assay for the determination of thrombin. It is based on the application of a triple enzyme-mimetic activity and a dual aptamer binding strategy. The triple signal amplification relies on oxidation of the chromogenic enzyme substrate 3,3,5,5-tetramethylbenzidine (TMB) that is catalyzed by composites consisting of graphene oxide (GO), gold/platinum nanoparticles (AuPtNP), and aptamer (Apt15), a G-quadruplex/hemin conjugate. The dual-aptamer target binding strategy is based on the fact that thrombin has two active sites to be recognized by its aptamers (Apt15 and Apt29). Magnetic beads (MBs) were modified with Apt29 (Apt29-MB) and then are bound by the GO-AuPtNP-Apt15/G-quadruplex/hemin composites. In the presence of thrombin, Apt29-MB and the GO-AuPtNP-Apt15/G-quadruplex/hemin composites form a sandwich-like superstructure. Thus, the absorbance increases due to the formation of TMB oxide produced by catalysis of the composites. Under optimized conditions, the absorbance at 450 nm increases linearly in the 0.30 to 100 nM thrombin concentration range, and the limit of detection is 0.15 nM. The method is simple, rapid, and does not require complicated instrumentation. Bovine serum albumin, human serum albumin and other proteins were found not to interfere.
Graphical abstract Schematic presentation of the photometric thrombin assay based on a triple enzyme-mimetic activity of combined nanomaterials (consisting of GO, AuPtNPs and the G-quadruplex/hemin DNAzyme) and two aptamers TMB: 3,3,5,5-tetramethylbenzidine, TMBox: 3,3,5,5-tetramethylbenzidine oxide, AuPtNP: gold/platinum nanoparticles).
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.
An aptamer based assay is described for the colorimetric detection of adenosine. The presence of adenosine triggers the deformation of hairpin DNA oligonucleotide (HP1) containing adenosine aptamer and then hybridizes another unlabeled hairpin DNA oligonucleotide (HP2). This leads to the formation of a double strand with a blunt 3′ terminal. After exonuclease III (Exo III)-assisted degradation, the guanine-rich strand (GRS) is released from HP2. Hence, the adenosine-HP1 complex is released to the solution where it can hybridize another HP2 and initiate many cycles of the digestion reaction with the assistance of Exo III. This leads to the generation of a large number of GRS strands after multiple cycles. The GRS stabilize the red AuNPs against aggregation in the presence of potassium ions. If, however, GRS forms a G-quadruplex, it loses its ability to protect gold nanoparticles (AuNPs) from salt-induced AuNP aggregation. Therefore, the color of the solution changes from red to blue which can be visually observed. This colorimetric assay has a 0.13 nM detection limit and a wide linear range that extends from 5 nM to 1 μM.
Graphical abstract Schematic presentation of a colorimetric aptamer biosensor for adenosine detection based on DNA cycling amplification and salt-induced aggregation of gold nanoparticles.
The authors describe an SPR sensor chip coated with gold nanoparticles (AuNPs) that enables highly sensitive determination of genetically modified (GM) crops. Detection is based on localized surface plasmon resonance (LSPR) with its known sensitivity to even minute changes in refractive index. The device consists of a halogen light source, a light detector, and a cuvette cell that contains a sensor chip coated with AuNPs. It is operated in the transmission mode of the optical path to enhance the plasmonic signal. The sample solution containing target DNA (e.g. from the GM crop) is introduced into the cuvette with the sensor chip whose surface was functionalized with a capture DNA. Following a 30-min hybridization, the changes of the signal are recorded at 540 nm. The chip responds to target DNA in the 1 to 100 nM concentration range and has a 1 nM detection limit. Features of this sensor chip include a short reaction time, ease of handling, and portability, and this enables on-site detection and in-situ testing.
Graphical abstract A localized surface plasmon resonance (LSPR)-based nanoplasmonic spectroscopic device enabling a highly sensitive biosensor is developed for the detection of genetically modified (GM) DNA founded in Roundup Ready (RR) soybean.
A method is described for the determination of the polarity of mixed organic solvents by using the fluorescent probe Hostasol Red (HR) desposited on the outer surface of nanosized zeolite L. Organic solvents and their mixtures can be roughly classified according to their polarity with bare eyes and fluorometrically. Emission peaks range from 520 to 640 nm. Some solvents act as quenchers. The method is studied with series of protic and nonprotic solvents, and with selected mixtures of organic solvents.
Graphical abstract The dye Hostalene Red adsorbed on nanosized zeolite shows strong fluorescence solvatochromism. This can be exploited to quickly assess the polarity of solvents and solvent mixtures.
A toehold-aided DNA recycling amplification technology was developed based on the combination of toehold-aided DNA recycling and the hemin/G-quadruplex label. The dsDNA formed between aptamer and DNA1 was first immobilized on magnetic beads. On addition of target analyte (exemplified here for riboflavin), the aptamer-riboflavin complex is formed and DNA1 is released by the beads. After magnetic separation, the supernatant containing the released DNA1 is added to a solution containing the hairpin capture DNA on magnetic beads. DNA1 will hybridize with the hairpin capture DNA via toehold binding and branch migration. This process will open the hairpin structure, and an external toehold is formed in the newly formed dsDNA. On addition of reporter DNA containing the G-quadruplex, it will interact with the formed dsDNA via toehold binding and branch migration, resulting in the releasing of DNA1 and capturing of reporter DNA on the magnetic beads. The released DNA1 will bind to another hairpin capture DNA which can start another round of DNA1 recycling. Chemiluminescence (CL) is generated by the G-quadruplex-hemin-luminol CL reaction system. Under optimal conditions, the calibration plot is linear in the 0.1 to 700 nM riboflavin concentration range, with a 30 pM detection limit (at a signal-to-noise ratio of 3). The method was successfully applied to the quantitation of riboflavin in spiked urine samples.
The fucosylated Golgi protein 73 (fuc-GP73) has been used as a criterion to distinguish hepatocellular carcinoma (HCC) from other chronic liver diseases. We describe an amperometric aptasensor for ultrasensitive detection of fuc-GP73 that uses a thiolated aptamer against GP73 as the capture probe, and gold nanoparticles (AuNPs) modified with Avidinlens culinaris agglutinin (A-LCA) as the detection probe. The AuNPs on the surface of a gold electrode provide a large surface for immobilization of A-LCA, so that they can be heavily loaded with biotinylated horse radish peroxidase (B-HRP) via avidin-biotin interactions. This results in enhanced analytical sensitivity. Under optimized conditions and a typical working potential as low as 48 mV (vs. SCE), the dynamic response of the electrode covers the 10 pg·mL?1 to 25 ng·mL?1 fuc-GP73 concentation range, with a 7 pg·mL?1 detection limit (for an S/N ratio of 3). The assay is precise, selective and reproducible. It was applied to the determination of fuc-GP73 in serum.
Graphical abstract Schematic of an electrochemical aptasensor for the determination of fucosylated golgi protein 73 (fuc-gp73) based on the avidin-Lens culinaris agglutinin (A-LCA) and biotinylated horse radish peroxidase (B-HRP). It was applied to serum analysis with good sensitivity, selectivity and reproducibility.
The authors describe a dye-sensitized photoelectrochemical immunoassay for the tumor marker carcinoembryonic antigen (CEA). The method employs the rhodamine dye Rh123 with red color and absorption maximum at 500 nm for spectral sensitization, and a 3D nanocomposite prepared from graphene oxide and MoS2 acting as the photoelectric conversion layer. The nanocomposite with flower-like 3D architectures was characterized by transmission electron microscopy, scanning electron microscopy, X-ray powder diffraction, and UV-vis diffuse reflectometry. A photoelectrochemical sandwich immunoassay was developed that is based on the use of the nanocomposite and based on the specific binding of antibody and antigen, and by using a secondary antibody labeled with Rh123 and CdS (Ab2-Rh123@CdS). Under optimal conditions and at a typical working voltage of 0 V (vs. Hg/HgCl2), the photocurrent increases linearly 10 pg mL?1 to 80 ng mL?1 CEA concentration range, with a 3.2 pg mL?1 detection limit.
Graphical abstract Flower-like GO-MoS2 complex with high efficiency of electron transport was synthesized to construct photoelectrochemical platform. The sandwich-type immunoassay was built on this platform based on specific binding of antigen and antibody. Carcinoembryonic antigen in sample was detected sensitively by using sensitization of rhodamine dye Rh123 as signal amplification strategy.
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).
Hetero-dimeric magnetic nanoparticles of the type Au-Fe3O4 have been synthesised from separately prepared, differently shaped (spheres and cubes), monodisperse nanoparticles. This synthesis was achieved by the following steps: (a) Mono-functionalising each type of nanoparticles with aldehyde functional groups through a solid support approach, where nanoparticle decorated silica nanoparticles were fabricated as an intermediate step; (b) Derivatising the functional faces with complementary functionalities (e.g. amines and carboxylic acids); (c) Dimerising the two types of particles via amide bond formation. The resulting hetero-dimers were characterised by high-resolution TEM, Fourier transform IR spectroscopy and other appropriate methods.
Graphical Abstract Nano-LEGO: Assembling two types of separately prepared nanoparticles into a hetero-dimer is the first step towards complex nano-architectures. This study shows a solid support approach to combine a gold and a magnetite nanocrystal.
The authors have prepared a super-hydrophilic polymer consisting of a poly-polyhedral oligomeric silsesquioxane (POSS)-formaldehyde (PPF) composite. The polymerization process does not require a catalyst and results in a material with excellent hydrophilic properties and abundant functional groups. The PFF composite, even if not chemically modified, can selectively bind glycoproteins due to strong hydrophilic interactions. It is shown that glycoproteins can be selectively captured by the composite that has a binding capacity as large as 542 mg g?1 for the model protein ovalbumin. The PPF was applied to the selective capture and isolation of ovalbumin from complex biological samples.
Graphical abstract Super-hydrophilic poly-polyhedral oligomeric silsesquioxane formaldehyde (PPF) is prepared via a catalyst-free polymerization route. PPF exhibits high capturing and adsorption selectivity towards glycoproteins due to its strong hydrophilic interaction with glycan groups. Favorable capturing capacity is also achieved.
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.
The authors describe an oligonucleotide-based lateral flow test for visual detection of Ag(I). The assay is based on cytosine-Ag(I)-cytosine [C-Ag(I)-C] coordination chemistry to capture gold nanoparticle (AuNP) tags in the test zone. A thiolated C-rich oligonucleotide probe was immobilized on the AuNPs via gold-thiol chemistry, and a biotinylated C-rich oligonucleotide probe was immobilized on the test zone. The AuNPs labelled with C-rich oligonucleotides are captured by Ag(I) ions in the test zone through the C-Ag(I)-C coordination. The resulting accumulation of AuNPs produces a readily visible red band in the test zone. Under optimized conditions, the test is capable of visually detecting 1.0 ppb of Ag(I) which is 50 times lower than the maximum allowable concentration as defined by the US Environmental Protection Agency for drinking water. Hence, the test is inexpensive and highly sensitive. It was applied to the detection of Ag(I) in spiked samples of tap water and river water. In our perception, the test is a particularly valuable tool in limited resource settings.
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.
The authors describe a novel assay for the detection of methylated DNA site. Rolling circle amplification and CdSe/ZnS quantum dots with high fluorescence efficiency are applied in this method. The CdSe/ZnS quantum dots act as electron donors, and hemin and oxygen (derived from hydrogen peroxide act as acceptors in photoinduced electron transfer. The assay, best performed at excitation/emission peaks of 450/620 nm, is sensitive and specific. Fluorometric response is linear in the 1 pM to 100 nM DNA concentration range, and the lowest detectable concentration of methylated DNA is 142 fM (S/N =?3). The method is capable of recognizing 0.01% methylated DNA in a mixture of methylated/unmethylated DNA.
Graphical abstract A novel method for methylated sites detection in DNA is established. Rolling circle amplification and photoinduced electron transfer. CdSe/ZnS quantum dots with high fluorescence efficiency act as the electron donor, while G-quadruplex/hemin and hydrogen peroxide derived oxygen act as electron acceptor. It presents a linear response towards 1 pM to 100 nM methylated DNA with a correlation coefficient of 0.9968, and the lowest detectable concentration of methylated DNA was 142 fM, with selectivity significantly superior to other methods.
The family of zearalenones (ZENs) represents a major group of mycotoxins with estrogenic activity. They are produced by Fusarium fungi and cause adverse effects on human health and animal production. The authors describe here a label-free amperometric immunosensor for the direct determination of ZENs. A glassy carbon electrode (GCE) was first modified with polyethyleneimine-functionalized multi-walled carbon nanotubes. Next, gold and platinum nanoparticles (AuPt-NPs) were electro-deposited. This process strongly increased the surface area for capturing a large amount of antibodies and enhanced the electrochemical performance. In a final step, monoclonal antibody against zearalenone was orientedly immobilized on the electrode, this followed by surface blocking with BSA. The resulting biosensor was applied to the voltammetry determination of ZENs, best at a working voltage of 0.18 V (vs SCE). Under optimized conditions, the method displays a wide linear range that extends from 0.005 to 50 ng mL?1, with a limit of detection of 1.5 pg mL?1 (at an S/N ratio of 3). The assay is highly reproducible and selective, and therefore provides a sensitive and convenient tool for determination of such mycotoxins.
Graphical abstract An amperometric immunosensor for the direct determination of ZENs has been developed by immobilizing anti-ZEN monoclonal antibody on multi-walled carbon nanotubest hat were deposited, along with gold and platinum nanoparticles, on a glassy carbon electrode modified with Staphylococcus protein A.
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.
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).
The negatively charged ruthenate(II) complex [Ru(bpy)(PPh3)(CN)3]? and gold nanoparticles (AuNPs) were used for detecting lysozyme (LYS). The luminescence of the ruthenate(II) complex is quenched by AuNPs, and this induces the aggregation of AuNPs and a color change from red to blue. After addition of lysozyme, the positively charged lysozyme and the negatively charged ruthenate(II) complex bind each other by electrostatic interaction firstly. This prevents AuNPs from aggregation and quenches the emission of the ruthenate(II) complex. Its luminescence and the degree of aggregation of the AuNPs can be used to quantify LYS. The fluorometric calibration plot is linear in the 0.01 to 0.20 μM LYS concentration range, and the calibration plot is linear between 0.02 and 0.20 μM of LYS. The color of the solution can be easily distinguished by bare eyes at 0.08 μM or higher concentration of LYS. The applicability of the method was verified by the correct analysis of LYS in chicken egg white.
Graphical abstract Schematic of a luminometric and colorimetric probe based on the induced aggregation of gold nanoparticles by an anionic luminescent ruthenate(II) complex or sensitive lysozyme detection.
