It is known that the binding of certain proteins to small molecules in ssDNA/small-molecule chimeras protects the conjugated ssDNA from degradation by exonuclease I (Exo I). This has resulted in numerous methods to specifically detect the interaction between small molecules and proteins. We are presenting here an approach that utilizes the terminal protection strategy in combination with the formation of ssDNA-templated silver nanoclusters (AgNCs), thereby providing a fluorometric tool for the detection of such interactions. A C-rich ssDNA (type 5′-CCCCACCCCT-3′) was labelled with biotin at the 3′ end. In the absence of streptavidin (SA), the biotinylated ssDNA is hydrolyzed in the 3′ to 5′ direction by Exo I to form mononucleotides. The formation of the AgNCs is prevented due to the lack of the DNA scaffold, and this results in weak fluorescence. Conversely, in the presence of SA, the specific binding of SA to the biotinylated ssDNA protects the ssDNA from digestion. As a result, fluorescent AgNCs are being formed. Fluorescence is measured at excitation/emission wavelengths of 625/705 nm. The calibration plot for SA is linear in the 6 to 600 nM concentration range, with a 2.6 nM detection limit. The assay is simple, sensitive and affordable. Conceivably, the method may also be used to detect the binding of other small molecules to proteins.
Graphical abstract A fluorescent sensing platform for small molecule-protein interaction assay has been developed based on terminal protection strategy and ssDNA-templated silver nanoclusters (AgNCs).
Glutathione coated gold and silver nanoclusters (GSH-Au/AgNCs) were synthesized by one-pot reduction methods and are found to be viable fluorescent nanoprobes for cysteine (Cys) and arginine (Arg), with good selectivity over other amino acids. The GSH-Au/AgNCs have two emissions at 616 nm and 412 nm when excited at 360 nm. With the increased concentration of Cys, the ratio of the emission intensities (I616/I412) linearly decreases with Cys in concentration ranging from 0.05 to 10 μM and from 10 to 50 μM, respectively. With increased concentrations of Arg, the ratio of I616/I412 linearly decreases with Arg concentration ranging from 0 to 50 μM and from 50 to 100 μM, respectively. The probe was applied to the determination of Cys and Arg in spiked samples of serum and urine where it gave good recoveries.
Graphical abstract Glutathione-coated gold and silver nanoclusters (GSH-Au/AgNCs) were synthesized by one-pot reduction and are found to be viable fluorescent nanoprobes for cysteine (Cys) and arginine (Arg).
The authors describe a fluorometric assay for microRNA. It is based on two-step amplification involving (a) strand displacement replication and (b) rolling circle amplification. The strand displacement amplification system is making use of template DNA (containing a sequence that is complementary to microRNA-21) and nicking enzyme sites. After hybridization, the microRNA strand becomes extended by DNA polymerase chain reaction and then cleaved by the nicking enzyme. The DNA thus produced acts as a primer in rolling circle amplification. Then, the DNA probe SYBR Green II is added to bind to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA. The method has a wide detection range that covers the10 f. to 0.1 nM microRNA concentration range and has a detection limit as low as 1.0 fM. The method was successfully applied to the determination of microRNA-21 in the serum of healthy and breast cancer patients.
Graphical abstract Schematic of a fluorometric microRNA assay based on two-step amplification involving strand displacement replication and rolling circle amplification. DNA probe SYBR Green II is then bound to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA.
The need for external excitation sources limits the utility of quantum dots (QDs) in multiplexed detection schemes and in in vivo imaging, because it can lead to strong background by surface illumination and tissue autofluorescence. In this work, the authors describe the use of oxidized dextran as a support to conjugate the photoprotein aequorin to QDs in order to obtain self-illuminating QDs and an efficient QD-based bioluminescence (BL) resonance energy system. On addition of Ca2+, BL is generated by immobilized aequorin and transferred to the QDs which thereby become photoexcited. Hence, these QDs will fluoresce without being excited by an external light source and therefore have the typical merits (such as very low background) of bioluminescent systems. The half-life of the BL of aequorin peaking at 460 nm is 1.6 s, and that of the QD-conjugated aequorin (peaking at 528 nm) is 6.4 s. We perceive that by labeling antibodies with these nanocomposites, highly advanced multiplex immunoassays will become possible.
Graphical abstract The photoprotein aequorin was conjugated to CdTe quantum dots coated wit denatured and reduced bovine serum albumin (dBSA) by using oxidized dextran as a cross linker, which leads to the formation of self-illuminating QDs.
CdTe quantum dots (QDs) were integrated with polyethyleneimine-coated carbon dots (PEI-CDs) to form a dually emitting probe for heparin. The red fluorescence of the CdTe QDs is quenched by the PEI-CDs due to electrostatic interactions. In the presence of heparin, the blue fluorescence of PEI-CDs remains unaffected, while its quenching effect on the fluorescence of CdTe QDs is strongly reduced. A ratiometric fluorometric assay was worked out. The ratio of the fluorescences at 595 and 436 nm serves as the analytical signal. Response is linear in the concentration range of 50–600 ng·mL?1 (0.1–1.2 U·mL?1) of heparin. The limit of detection is 20 ng·mL?1 (0.04 U·mL?1). This makes the method a valuable tool for heparin monitoring during postoperative and long-term care. This assay is relatively free from the interference by other analogues which commonly co-exist with heparin in samples, and it is more robust than single-wavelength based assays.
Graphical abstract In the presence of heparin, the fluorescence of polyethyleneimine-coated carbon dots (PEI-CDs) at 436 nm remains unaffected, while its quenching effect on the fluorescence of CdTe at 595 nm is strongly reduced.
The authors describe an aptamer-based fluorescent assay for adenosine (Ade). It is based on the interaction between silver nanoparticles (AgNPs) and CdTe quantum dots (QDs). The beacon comprises a pair of aptamers, one conjugated to Fe3O4 magnetic nanoparticles, the other to AgNPs. In the presence of Ade, structural folding and sandwich association of the two attachments takes place. After magnetic separation, the associated sandwich structures are exposed to the QDs. The AgNPs in sandwich structures act as the signaling label of Ade by quenching the fluorescence of QDs (at excitation/emission wavelengths of 370/565 nm) via inner filter effect, electron transfer and trapping processes. As a result, the fluorescence of QDs drops with increasing Ade concentration. The assay has a linear response in the 0.1 nM to 30 nM Ade concentration range and a 60 pM limit of detection. The assay only takes 40 min which is the shortest among the aptamer-based methods ever reported. The method was successfully applied to the detection of Ade in spiked biological samples and satisfactory recoveries were obtained.
Graphical abstract Schematic of a highly efficient and convenient adenosine (Ade) fluorometric assay. It is based on the interaction between Ag nanoparticles (NPs) and CdTe quantum dots (QDs). Ade aptamers (ABA1 and ABA2) are used as recognition unit and Fe3O4 magnetic nanoparticles act as magnetic separator. The assay exhibits superior sensitivity and speediness.
MicroRNAs are endogenous noncoding RNAs that play critical roles in biological processes and can be considered as molecular markers for early diagnosis and pathogenesis of diseases. The authors describe a highly sensitive electrochemical biosensor for microRNA that is based on the use of tetrahedral DNA nanostructure probes and guanine nanowire amplification. The DNA tetrahedral probe is self-assembled on a gold electrode and enhances reactivity, accessibility, and molecular recognition efficiency. Combined with the tetrahedral probe, the guanine nanowire amplifies the signal and improves the analytical performance of the biosensor. Operated best at a voltage of typically 150 mV (vs. Ag/AgCl), the sensor has a linear response to the logarithmic microRNA concentration in the 500 f. to 10 nM range, with a 176 f. detection limit. It is highly selective and can be applied to real samples. It is concluded that this strategy has a good potential with respect to the determination of microRNA in clinical diagnosis and in biological research.
Graphical abstract Schematic of a tetrahedral DNA nanostructure-based amperometric biosensor coupled to guanine nanowire amplification for analysis of microRNA-21. This strategy is highly selective and also performs well for the detection of microRNA levels of breast cancer patients.
We report on a highly sensitive competitive immunoassay for the mycotoxin Ochratoxin (OTA) using magnetic silica nanoparticles (NPs) fluorescently labeled with rhodamine 123 (Rho123) as signal intensifier. The method is based on the measurement of fluorescence resonance energy transfer (FRET) that occurs from CdTe quantum dots covered with anti-OTA antibody to the dye Rho123 on the surface of the NPs. The immunoreaction between anti-OTA antibody and OTA brings the fluorophore (acting as the acceptor) in close proximity of the QDs (acting as the donor), and this causes FRET to occur upon photo-excitation of the QDs. The size and polydispersity of the silica coated magnetic NPs was studied via TEM. The method has a detection limit of 0.8 pg of OTA per mL. It was applied to the determination of OTA in spiked human serum. A linear relationship is found between the increase in the fluorescence intensity of Rho 123 at 580 nm and the concentration of OTA in spiked samples over the 8 to 48 pg?mL?1 concentration range. This highly sensitive homogeneous competitive detection scheme is simple, rapid and efficient. It does not require multiple separation steps and excessive washing.
Graphical abstract Following photoexcitation of immobilized quantum dots (QDs), FRET occurs between the QDs and Rhodamine 123. The close proximity of Rho 123 and the magnetic silica core/shell particles leads to strongly intensified emission to result in an assay for Ochratoxin A that has a detection limit as low as 0.8 pg?mL-1
The article describes a method for determination of tannic acid in extracts of medicinal plants. Tannic acid (TA) is an antioxidant and has anticancer and antimicrobial properties. Titanium dioxide nanoparticles (TiO2) were co-sensitized with 5-methylphenazinium methosulfate (PMS) and carboxy-functionalized cadmium telluride quantum dots (CdTe QDs), and immobilized on a fluorine-doped tin oxide electrode. The surface morphology and electrochemical properties of the modified electrode were investigated by scanning electron microscopy and amperometry, respectively. A composite consisting of TiO2, PMS and CdTe QDs in a nafion film has a response to TA under LED light higher than that observed for each separate component. Under optimized experimental conditions and at an applied voltage of +0.4 V vs Ag/AgCl, the photoelectrochemical sensor has a linear response in the 0.2 to 200 μmol L?1 TA concentration range and a detection limit of 60 nmol L?1. The sensor was successfully applied to the determination of TA in spiked extracts from three medicinal plants, with recovery values between 98.3 and 103.9 %.
Graphical abstract Schematic diagram for photoelectrochemical detection of tannic acid based on a fluorine doped tin oxide electrode modified with titanium oxide, 5-methylphenazinium methosulfate and carboxy-functionalized cadmium telluride quantum dots
The authors report on a new approach for the determination of the breast cancer biomarker microRNA-155 (miRNA-155). It is based on the measurement of the fluorescence shift of oligonucleotide-templated copper nanoclusters (DNA-CuNC). A probe DNA was designed that acts as a template for the preparation of CuNC which, under 400 nm excitation, exhibit strong fluorescence enhancement at 490 nm and a 90 nm Stokes shift after binding to target miRNA-155 and formation of a DNA-RNA heteroduplex. Under the optimal conditions, the fluorescence of the DNA-CuNC increases with increasing concentration of miRNA-155 in the range from 50 pM to 10 nM, with a 11 pM detection limit. The assay has excellent selectivity over noncomplementary RNA. The method was applied to the determination of miRNA-155 in the presence of human plasma and saliva.
Graphical abstract Schematic of the detection strategy that relies on the fluorescence shift of DNA-CuNCs resulting from the specific binding of DNA-CuNCs with target miRNA-155. Fluorescence intensities are linearly proportional to the concentrations of target RNA from 50 pM to 10 nM.
The authors describe a method for the determination of cobalt(II) ions based on the use of luminescent and water-soluble ZnO quantum dots capped with β-cyclodextrin (β-CD@ZnO QDs). The modified QDs display strong yellow-green fluorescence with a peak at 537 nm under 360 nm excitation. High-resolution transmission electron microscopy, Fourier transform infrared spectroscopy, luminescence, and UV-visible absorption spectroscopy were used to characterize the β-CD@ZnO QDs. The fluorescence of the QDs is quenched by Co(II) ions. This finding was exploited to design a quenchometric assay designed for the detection of Co(II) in water solution. The detection limit is 0.34 μM (based on the 3σ/slope criterion), and the linear range extends from 1.0 to 10 μM. The method was applied to quantify Co(II) in spiked real samples. The quenching mechanism was studied, and this showed that aggregation-induced quenching causes the main effect.
Graphical abstract The fluorescence of β-cyclodextrin-capped ZnO quantum dots (β-CD@ZnO QDs) is quenched by cobalt ions, and this finding is exploited in a fluorescence assay for cobalt ions in aqueous solutions.
A method is described for the determination of the activity of alkaline phosphatase (ALP). It is based on the reversible modulation of the fluorescence of WS2 quantum dots (QDs). The fluorescence of the QDs is quenched by Cr(VI) but restored by free ascorbic acid (AA). The detection scheme relies on the fact that ALP hydrolyzes the substrate ascorbic acid 2-phosphate to produce AA, and that enzymatically generated AA can restore the fluorescence of the QDs. The signal (best measured at excitation/emission peak wavelengths of 365/440 nm) increases linearly in the 0.5 to 10 U·L?1 ALP activity range, with a detection limit of 0.2 U·L?1. The method was applied to the determination of ALP activity in human serum samples and demonstrated satisfactory results.
Graphical abstract The fluorescence of chromate-loaded tungsten disulfide quantum dots (QDs) is quenched but restored after reaction with ascorbic acid that is formed by the catalytic action of alkaline phosphatase (ALP) on ascorbic acid 2-phosphate (AAP). The increase in fluorescence can be related to the activity of ALP.
Stable copper nanoclusters (CuNCs) were prepared by utilizing D-penicillamine as both the stabilizer and reductant. The emission of the CuNCs (with excitation/emission peaks at 390/645 nm) is largely stabilized by coating with poly(sodium-p-styrenesulfonate) (PSS). Cytochrome c (Cyt c) quenches the fluorescence of the PSS-coated CuNCs, and this effect was exploited to design a quenchometric fluorometric assay for Cyt c. If trypsin is added to the loaded CuNCs, it will hydrolyze Cyt c to form peptide fragments, and fluorescence is gradually restored. A highly sensitive and fluorometric turn-off-on assay was constructed for sequential detection of Cyt c and trypsin. The linear ranges for Cyt c and trypsin are from 8.0 nM to 680 nM, and from 0.1 to 6.0 μg mL?1, and the lower detection limits are 0.83 nM and 20 ng mL?1 for Cyt c and trypsin, respectively.
Graphical abstract Schematic illustration of the fluorometric assay for trypsin based on the electron transfer between poly(p-styrenesulfonate)-protected copper nanoclusters (PSS-CuNCs) and cytochrome c (Cyt c).
The authors describe a method for highly sensitive and selective determination of the activity of protein kinase (PKA). It is based on the finding that silver nanoclusters (AgNCs) can act as a nucleus to catalyze further deposition of silver nanoparticles. This causes the color of a solution to change from pale yellow to black. In the detection scheme presented here, the substrate peptide is phosphorylated by PKA in the presence of ATP. The resulting phosphopeptides bind to oligonucleotide-stabilized AgNCs in the presence of Zr(IV) ions due to electrostatic interactions between Zr(IV) and the phosphate groups, thereby capping the AgNCs. The silver enhancement process (leading to a color change to black) does not work if the AgNCs are capped. The degree of inhibition is proportional to the activity of the kinase. The color change can be detected visually or photographically in a microplate format by exploiting the changes in the grey values of the digital photos. In addition, the DNA-AgNCs display fluorescence emission at 635 nm when excited at 565 nm. Electrochemical assays were performed (at a working voltage as low as 38 mV vs. Ag/AgCl) by using a glassy carbon electrode modified with a solution containing AgNCs, Zr(IV) ions and the peptide, and immersing it into the silver enhancement solution. The assay is highly sensitive and selective. It was applied to the determination of PKA in lysates of HeLa cells. The detection limits typically are between 32 and 37 U? L-1 based on a signal-to-noise ratio of 3.
Graphical Abstract A method for colorimetric and electrochemical determination of the activity of protein kinase activity is described that is based on silver nanocluster (AgNC) based signal amplification. AgNCs act as nucleus for further deposition of silver nanoparticles, but protein kinase can inhibit this process.
Gold-silver nanoclusters (Au-AgNCs) were synthesized by simultaneous chemical reduction of Au(III) and Ag(I) ions in one pot, using bovine serum albumin as both a template and a reductant. The Au-AgNCs have an average size of 2.4 nm and display strong red fluorescence (with an emission peak at 610 nm on excitation at 360 nm). The fluorescence quantum yield can reach 18.6%. Fluorescence is strongly quenched by hypochlorite, while other common anions have minor (or no) effects on fluorescence. Based on these findings, a fluorometric method was developed for the determination of hypochlorite. The method has a linear response in the 0.7 to 15 μM concentration range, with a limit of detection as low as 80 nM. It was successfully applied to the determination of hypochlorite in (spiked) tap water.
Graphical abstract Gold-silver nanoclusters with strong red fluorescence were synthesized by simultaneous chemical reduction of Au(III) and Ag(I) ions in one pot, and a sensitive and selective method for the detection of hypochlorite was developed based on the quenching of the fluorescence of the nanoclusters.
Under visible-light irradiation, a cathodic photoelectrochemical (PEC) sensor is presented for highly sensitive determination of Cr(VI) at a potential of ?0.25 V (vs SCE). PbS quantum dots (QDs) were capped with mercaptoacetic acid and assembled on the surface of an indium tin oxide (ITO) electrode via the linker poly(diallyl dimethyl ammonium chloride) providing a photoactive sensor. Cr(VI) accepts the photoelectrons generated by the PbS QDs. This promotes the separation of electron holes and enhances the cathodic photocurrent generated by a 470-nm LED. The sensor has 10 pM detection limit and a linear working range from 0.02 nM to 2 μM of chromate. The method was successfully applied to the determination of Cr(VI) and total chromium in spiked environmental water samples.
Graphical abstract Schematic illustration of the photocurrent enhancement response of ITO/PbS toward chromium(VI). In the presence of Cr(VI) (red line), Cr(VI) accepts the photoelectrons generated by the PbS QDs under 470-nm LED irradiation, resulting in improved photocurrent of ITO/PbS.
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.
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.
The authors describe a method for the differentiation of penicillamine (PA) enantiomers by using CdSe/ZnS quantum dots (QDs) modified with β-cylodextrin (β-CD-CdSe/ZnS QDs). Selective enantiorecognition of L-PA and D-PA was accomplished by virtue of selective host-guest interaction between the PAs and the β-CD pockets on the QDs. The fluorescence intensity of the modified QDs decreases in the presence of L-PA. On the contrary, it increases in the presence of D-PA. These findings form the basis for a new method for recognition of PA enantiomers. Under optimized conditions, a linear relationship exists between fluorescence intensity and D-PA concentration in the 0.1 to 5.0 mg L?1 range, and between 0.8 and 5.0 mg L?1 for L-PA. Detection limits are 0.06 mg L?1 for D-PA, and 0.2 mg L?1 for L-PA. The potential of this method has been demonstrated by the determination of D-PA in pharmaceutical formulations and L-PA in (spiked) environmental samples.
Graphical abstract Selective and specific enantiorecognition of penicillamine (PA) enantiomers using β-cylodextrin modified CdSe/ZnS quantum dots is described. Fluorescence intensity increases in the presence of D-PA, but it decreases in the presence of L-PA. Results were the basis for analytical applications.
A novel electrochemiluminescent (ECL) method for highly sensitive detection of gene mutations was designed based on the amplification strategy of dual-functional aluminum(III). A film composed of nafion and polyaniline (Nafion-PANI) was placed onto glassy carbon electrode (GCE) in order to improve conductivity and stability, and then cadmium sulfide quantum dots (CdS QDs) were attached as an ECL label. Al(III) was introduced in order to enhance the ECL signal intensity of the CdS QDs by filling the surface electronic defects of CdS QDs. The Al(III) ions also assist by improving sensitivity by promoting the electron transfer at the GCE and by retaining plenty of single-stranded DNA (ssDNA). The ECL is generated at typically ?1.5 V in the presence of containing K2S2O8. Compared to conventional ECL based DNA biosensors, the one described here – based on the use of dually functional Al(III) ions – enables ssDNA to be detected in the 1 f. to 10 nM concentration range, with a 6 f. detection limit. This method was applied to the quantitation of target ssDNA with different mismatching status in human serum. In our perception, it represents a highly attractive tool for the detection of ssDNA and has a particular potential in the diagnosis of hereditary diseases.
Graphical abstract Preparation and schematic illustration of dual-functional aluminum(III)-based electrochemiluminescent for detection of target ssDNA.
