The authors describe an aptamer-based fluorometric assay for the insecticide acetamiprid. It is based on target-induced release of the fluorescein-labeled complementary strand of the aptamer (CS) from the aptamer/CS conjugate (dsDNA). Three kinds of nanoparticles with opposite effects on the fluorophore (FAM) were used. These include gold nanoparticles (AuNPs), single-walled carbon nanotubes (SWNTs) and silica nanoparticles (SiNPs) coated with streptavidin. In the presence of acetamiprid, FAM-labeled CS is released from the dsDNA-modified SNP-streptavidin complex and accumulates in the supernatant (phase I) after centrifugation. Fluorescence intensity decreases on addition of the supernatant to the SWNTs and AuNPs because they act as quenchers (phase II). In the absence of acetamiprid, the dsDNA-modified SiNP-streptavidin complex remains intact and no labeled CS is present in the supernatant containing the AuNPs and SWNTs. So, the relative fluorescence intensity is quite low. The assay is highly selective for acetamiprid and has a limit of detection (LOD) as low as 127 pM. The method was successfully applied to the determination of acetamiprid in spiked serum and water where it gave LODs of 198 and 130 pM, respectively.
Graphical abstract In the absence of acetamiprid, the dsDNA-modified silica nanoparticle (SiNP)-streptavidin conjugate remains intact, leading to a very weak relative fluorescence intensity. In the presence of target, the dsDNA-modified SiNP-streptavidin complex is disassembled and FAM-labeled CS is released from the aptamer (Apt), resulting in a very strong relative fluorescence intensity.
The authors describe a competitive aptamer based assay for detection of the platelet-derived growth factor BB (PDGF-BB; used as a model protein). The assay is making use of thrombin (a serine protease) as an enzyme label for reporting signals. It is taking advantage of a highly selective aptamer and of the fairly specific enzymatic activity of thrombin in terms of cleaving artificial fluorogenic peptide substrates. In a first step, the surface of wells of microplates is coated with PDGF-BB. On addition of a sample containing PDGF-BB, free and bound PDGF-BB compete with each other for binding to a DNA probe that consists of an aptamer sequence for PDGF-BB and a 29-mer aptamer sequence for thrombin. After washing, thrombin is added and will attach to the DNA probe that bound to the PDGF-BB on the microplates. Following addition of a fluorogenic peptide substrate, the bound thrombin will catalyze the cleavage of the substrate to generate a fluorescent product whose fluorescence intensity is measured at excitation/emission wavelengths of 370/440 nm. Fluorescence intensity decreases with increasing PDGF-BB concentration in the sample because less thrombin will bind to the PDGF-BB coated surface of the microplate. Under optimal conditions, PDGF-BB can be quantified in the 0.125 to 3 nM concentration range. This assay was successfully applied to the determination of PDGF-BB in spiked 100-fold diluted human serum.
Graphical abstract In a competitive thrombin-linked aptamer assay, free platelet-derived growth factor BB (PDGF-BB) sample competes with the PDGF-BB coated on microplates for binding to a DNA probe containing PDGF-BB-binding aptamer and thrombin-binding aptamer. The labeled thrombin cleaves substrate into product, achieving signal generation.
In order to develop an aptamer based fluorescence resonance energy transfer (FRET) assay for 19-nortestosterone, a 76-mer 17β-estradiol aptamer was split into two pieces (referred to as P1 and P2, respectively). P1 was labeled with a quencher (BHQ), and P2 with a fluorophore (6FAM). The two aptamer pieces were employed to detect NT via FRET quenching in a homogeneous solution. This method has a low detection limit (5 μM) within a wide dynamic range (5 to 1000 μM). The approach was used to analyze spiked urine samples, and the results showed that the average recovery of three samples containing different NT concentrations ranged from 58 to 118 % with a relative standard deviation (RSD) of less than 1 %. In our perception, the method has a wide scope for future applications to other analytes by using dually labeled split aptamers.
Graphical abstract A split aptamer-based fluorescence resonance energy transfer assay for 19-nortestosterone was developed with a wide dynamic range of 5 to 1000 μM and low detection limit (5 μM). The average recovery from spiked urine samples ranged from 58 to 118 %, with a relative standard deviation (RSD) of less than 1 %.
The authors report that the peroxidase-like activity of Au@Pt core-shell nanohybrids (Au@PtNHs) is selectively inhibited by cysteine. This finding has led to a highly sensitive colorimetric assay for cysteine that is based on the nanohybrid-catalyzed oxidation of TMB by H2O2 to form a blue product. The method has a detection limit of 5.0 nM and a linear range from 10 nM to 20 μM. The assay is highly selective over other amino acids. It was successfully applied to the determination of cysteine in an injection containing a mixture of amino acids.
Graphical abstract The peroxidase-like activity of Au@Pt core-shell nanohybrids (Au@PtNHs) is selectively inhibited by cysteine, enabling the determination of cysteine.
A fluorometric ATP assay is described that makes use of carbon dots and graphene oxide along with toehold-mediated strand displacement reaction. In the absence of target, the fluorescence of carbon dots (with excitation/emission maxima at 360/447 nm) is strong and in the “on” state, because the signal probe hybridizes with the aptamer strand and cannot combine with graphene oxide. In the presence of ATP, it will bind to the aptamer and induce a strand displacement reaction. Consequently, the signal probe is released, the sensing strategy will change into the “off” state with the addition of graphene oxide. This aptasensor exhibits selective and sensitive response to ATP and has a 3.3 nM detection limit.
Graphical abstract Schematic of signal amplification by strand displacement in a carbon dot based fluorometric assay for ATP. This strategy exhibits high sensitivity and selectivity with a detection limit as low as 3.3 nM.
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 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 an electrochemical method for the determination of the blood clotting factor IX (FIX). A nanogapped dielectrode (with a < 100 nm junction) was modified with antibody against FIX, and the resulting system was characterized by both impedance spectroscopy and voltammetry. In order to attain the improved sensitivity, gold nanoparticles were electrostatically attached to FIX. The current to voltage (I-V) measurement was carried out from 1 V to 5 V, where the entire calibration plot with 5 V was taken. This results in a limit of detection as low of 1 pM, which is much lower compared to the real concentration of FIX (87 nM) in human blood serum. The analytical range extends from 1 pM to 0.1 μM. The electrode is highly specific over other serum proteins.
Graphical abstract A nanogapped dielectrode with a <100 nm junction was modified with antibody against blood clotting factor IX and characterized by voltammetry. Gold nanoparticles were electrostatically attached to the analyte (blood clotting factor IX), and current/voltage (I-V) measurements were performed. Factor IX can be quantified with a limit of detection as low as 10 pM.
The authors describe a surface plasmon resonance (SPR) based aptasensor for the carcinogenic mycotoxin aflatoxin B1 (AFB1) in a direct assay format. The aptamer is immobilized on the surface of a commercial sensor chip, and the SPR signal increases on binding of AFB1. The sensor chip can be fully regenerated by passing a flow of buffer over it upon which bound AFB1 dissociates from the aptamer. The biosensor works in the 0.4 nM to 200 nM AFB1 concentration range and has a 0.4 nM detection limit. It allows AFB1 to be determined in complex samples such as diluted red wine and beer. The assay is sensitive, and the chip is easily regenerated and stable. The method therefore overcomes certain limitations of antibody-based SPR assays and of competitive SPR assays for AFB1.
Graphical abstract Schematic presentation of the assay: Aptamer is coated on the chip of SPR, and the binding between aflatoxin B1 (AFB1) and the aptamer on chip causes SPR responses, allowing sensitive detection of AFB1.
This article reports on a novel aptamer-based platform for the quantitation of urea by using an aptamer with high affinity and selectivity for urea. The surface of a glassy carbon electrode (GCE) was modified by drop casting a cocktail consisting of carbon nanotubes and reduced graphene oxide (rGO) decorated with platinum-gold nanoparticles. The urea aptamer was then immobilized on the nanocomposite via covalent conjugation. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to trace the modification of the GCE. Binding of urea caused the aptamer to be folded, and this result in an inhibition of the interfacial charge transfer rate when using hexacyanoferrate as an electrochemical redox probe. The change in redox current was quantified by differential pulse voltammetry, typically at a working voltage of 0.22 V vs. Ag/AgCl. The assay has a 1.9 pM detection limit, and the response is linear up to 150 nM concentration of urea. The superior selectivity and affinity of aptamer-modified GCE makes it a most useful tool for analysis of urea present in very low concentrations.
The authors describe a method for the fabrication of a nanohybrid composed of carbon dots (C-dots) and gold nanoparticles (AuNPs) by in-situ reduction of C-dots and hydroauric acid under alkaline conditions. The process does not require the presence of surfactant, stabilizing agent, or reducing agent. The hybrid material was deposited in a glassy carbon electrode (GCE), and the modified GCE exhibited good electrocatalytic activity toward the oxidation of nitrite due to the synergistic effects between carbon dots and AuNPs. The findings were used to develop an amperometric sensor for nitrite. The sensor shows a linear response in the concentration range from 0.1 μmol?L-1 to 2 mmol?L-1 and a low detection limit of 0.06 μmol?L-1 at the signal-to-noise ratio of 3.
Graphical abstract Fabrication, characterization and electrochemical behavior of a glassy carbon electrode modifid with carbon dots and gold nanoparticles for sensing nitrite in lake water.
The authors introduce a method for spatially arranged DNA immobilization on 10-nm gold nanoparticles (GNP) deposited on a silicon substrate carrying nanogapped interdigitated electrodes. The GNPs are covalently bound to the surface via silane chemistry, and the single steps of fabrication are monitored by FTIR spectroscopy and atomic force microscopy. This GNP deposition technique is shown to reduce the size of the nanogaps to 130 nm. FTIR also was used to monitor the immobilization of DNA on the surface of the interdigitated electrodes. This method allows DNA to be immobilized in a uniform and homogenous way. The utility of the method is demonstrated by immobilizing probe DNA on the surface and detecting target DNA specific for the human papilloma virus via fluorescence with a detection limit as low as 1 pM. In our perception, this method for GNP-mediated DNA immobilization enables high-performance sensing of a wide range of target (analyte) DNA.
Graphical abstract Schematic presentation of gold nanoparticle-mediated and spatially resolved deposition of DNA on nano-gapped interdigitated electrodes. The method was applied to the chemiluminescent determination of the human papillomavirus
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.
A simple method is described for the determination of copper(II) ions based on the cathodic electrochemiluminescence (ECL) of lucigenin which is quenched by Cu(II). The blue ECL is best induced at ?0.45 V (vs. Ag/AgCl) at a scan rate of 50 mV·s?1. Under optimum conditions, the calibration plot is linear in the 3.0 to 1000 nM Cu(II) concentration range. The limit of detection is 2.1 nM at a signal-to-noise ratio of 3. Compared to other analytical methods, the one presented here is simple, fast, selective and cost-effective. It has been successfully applied in the analysis of copper ions in spiked tap water samples with recoveries ranging from 93.0% (at 50 nM concentration) to 105.7% (at 150 nM).
Graphical abstract The inhibitory effect of Cu(II) on the cathodic electrochemiluminescence of lucigenin enables determination of Cu(II) with a 2.1 nM detection limit.
Various kinds of nanomaterials have been described in recent years that represent stable and low-cost alternatives to biomolecules (such as enzymes) for use in (bio)analytical methods. The materials typically include, metal/metal oxides, metal complexes, nanocomposites, porphyrins, phthalocyanines, smart polymers, and carbonaceous nanomaterials. Due to their biomimetic and other properties, such nano-materials may replace natural enzymes in chemical sensors, biosensors, and in various kinds of bioassays. This overview (with 252 references) highlights the analytical potential of such nanomaterials. It is divided into sections on (a) the types of nanomaterials according to their intrinsic nature, (b) non-enzymatic sensor designs (including electrochemical, colorimetric, fluorescent and chemiluminescent methods), and (c), applications of non-enzymatic sensors in the biomedical, environmental and food analysis fields. We finally address current challenges and future directions.
Graphical abstract This review discusses different types of nanomaterials, which are explored as a potential biomimetic material to replace the natural enzyme in the field of biosensors, and have found widespread applications in biomedical, food and environmental analysis.
A composite consisting of chitosan containing azidomethylferrocene covalently immobilized on sheets of reduced graphene oxide was drop-casted on a polyester support to form a screen-printed working electrode that is shown to enable the determination of nitrite by cyclic voltammetry and chronoamperometry. Both reduction and oxidation of nitrite can be accomplished due to the high electron-transfer rate of this electrode. Under optimal experimental conditions (i.e. an applied potential of 0.7 V vs. Ag/AgCl in pH 7.0 solution), the calibration plot is linear in the 2.5 to 1450 μM concentration range, with an ~0.35 μM limit of detection (at a signal-to-noise ratio of 3). The sensor was successfully applied to the determination of nitrite in spiked mineral water samples, with recoveries ranging between 95 and 101 %.
Graphical abstract We describe the design of ferrocene-functionalized reduced graphene oxide electrode and its electrocatalytic properties towards the determination of nitrite. Compared to a reduced graphene oxide electrode, the sensor exhibits enhanced electrocatalytic activity towards both oxidation and reduction of nitrite.
We have prepared graphene quantum dot-europium(III) complex composites by noncovalently connecting chelating ligands dibenzoylmethane (DBM) and 1,10-phenanthroline (Phen) with graphene quantum dots (GQDs) first, followed by coordination to Eu(III). The resulting composites are well water-soluble and display red fluorescence of high color purity. The composites were characterized by transmission electron microscopy, X-ray photoelectron spectroscopy and X-ray diffraction. Aqueous solutions of the composites under 365 nm excitation display fluorescence with a peak at 613 nm and a quantum yield as high as 15.5 %. The good water solubility and stable photoluminescence make the composites very different from other Eu(III)-based coordination complexes. The composites are cell viable and can be used to label both the cell membrane and the cytoplasm of MCF-7 cells. They are also shown to act as bioprobes for in-vivo localization of tumorous tissue. In our perception, such composites are expected to possess wide scope because of the many functionalizations that are possible with GQDs.
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.
The authors describe a method for amperometric determination of thiodiglycol (TDG), the main hydrolysis product of sulfur mustard. The electrode consists of a mixture of graphene nanosheets, silver nanoparticles and the ionic liquid octylpyridinium hexafluorophosphate. Electrochemical oxidation of TDG was performed by cyclic voltammetry at pH 4 and revealed a pair of well-defined redox peaks at potentials of 0.43 and 0.19 V (vs. Ag/AgCl). Amperometric detection was accomplished over a dynamic range that is linear in the 10–3700 μM concentration range. The detection limit (at an S/N of 3) is 6 μM. The electrode was applied to the determination of TDG in (spiked) waste water and gave recoveries that ranged from 98.2 to 103.3 %.
Graphical abstract The article describes an amperometric sensor for the determination of thiodiglycol, the main hydrolysis product of sulfur mustard. The electrode was constructed by using graphene nanosheets, silver nanoparticles and an ionic liquid electrode, and it was successfully applied to the determination of thiodiglycol in (spiked) waste water samples.
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.
