An electronic channel switching-based aptasensor for ultrasensitive protein detection

Due to the ubiquity and essential of the proteins in all living organisms, the identification and quantification of disease-specific proteins are particularly important. Because the conformational change of aptamer upon its target or probe/target/probe sandwich often is the primary prerequisite for the design of an electrochemical aptameric assay system, it is extremely difficult to construct the electrochemical aptasensor for protein assay because the corresponding aptamers cannot often meet the requirement. To circumvent the obstacles mentioned, an electronic channel switching-based (ECS) aptasensor for ultrasensitive protein detection is developed. The essential achievement made is that an innovative sensing concept is proposed: the hairpin structure of aptamer is designed to pull electroactive species toward electrode surface and makes the surface-immobilized IgE serve as a barrier that separates enzyme from its substrate. It seemingly ensures that the ECS aptasensor exhibits most excellent assay features, such as, a detection limit of 4.44 × 10⁻⁶ μg mL⁻¹ (22.7 fM, 220 zmol in 10-μL sample) (demonstrating a 5 orders of magnitude improvement in detection sensitivity compared with classical electronic aptasensors) and dynamic response range from 4.44 × 10⁻⁶ to 4.44 × 10⁻¹ μg mL⁻¹. We believe that the described sensing concept here might open a new avenue for the detection of proteins and other biomacromolecules.     

Study on the interaction between oxolinic acid aggregates and protein and its analytical application

It was found that oxolinic acid (OA) at high concentration can self-assemble into nano- to micro- meter scale OA aggregates in Tris-HCl (pH 7.48) buffer solution. The nanoparticles of OA were adopted as fluorescence probes in the quantitative analysis of proteins. Under optimum conditions, the fluorescence quenching extent of nanometer scale OA aggregates was in proportion to the concentration of albumins in the range of 3.0 × 10⁻⁸ to 3.0 × 10⁻⁵ g mL⁻¹ for bovine serum albumin (BSA) and 8.0 × 10⁻⁸ to 8.0 × 10⁻⁶ g mL⁻¹ for human serum albumin (HSA). The detection limits (S/N = 3) were 3.4 × 10⁻⁹ g mL⁻¹ for BSA, and 2.6 × 10⁻⁸ g mL⁻¹ for HSA, respectively. Samples were satisfactorily determined. The interaction mechanism of the system was studied using fluorescence, UV-vis, resonance light scattering (RLS) and transmission electron microscope (TEM) technology, etc., indicating that the nonluminescent complex was formed between serum albumin molecular and OA, to disaggregate the self-association of OA, which resulted in the dominated static fluorescence quenching in the system.     

Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork

A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V_H and V_L) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V_H and V_L genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21. The purified scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) protocol for detection of RAC. The average concentration required for 50% inhibition of binding and the limit of detection of the assay were 0.25 ± 0.03 and 0.02 ± 0.004 ng mL⁻¹, respectively, and the linear response range extended from 0.05 to 1.45 ng mL⁻¹. The assay was 10 times as sensitive as the corresponding enzyme-linked immunosorbent assay based on the same fusion protein. Cross-reactivity studies showed that the fusion protein did not cross react with RAC analogs. DcCLEIA was used to analyze RAC spiked pork samples, and the validation was confirmed by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS). The results showed a good correlation between the data of dc-CLEIA and HPLC–MS (R² > 0.99), indicating that the assay was an efficient analytical method for monitoring food safety.     

A colormetric and fluorescent chemosensor for adenosine-5′-triphosphate based on rhodamine derivative

A rhodamine spirolactam derivative (1) was developed as a colormetric and fluorescent chemosensor for adenosine-5′-triphosphate (ATP) via hydrogen bonds interaction. As far as we know, this is the first case to explore ATP-induced ring-opening of spirolactam in rhodamine derivatives. It exhibited a highly sensitive “turn-on” fluorescent response toward ATP with a 47-fold fluorescence intensity enhancement under 20 equiv. of ATP added. The chemosensor can be applied to the quantification of ATP with a linear range covering from 1.0 × 10⁻⁷ to 2.0 × 10⁻⁴ M and a detection limit of 2.5 × 10⁻⁸ M. The experiment results show that the response behavior of 1 toward ATP is pH independent in medium condition (pH 6.0–8.0). Most importantly, the novel chemosensor has well solved the problem of serious interferences from other nucleoside polyphosphates such as ADP and AMP generally met by previously reported typical fluorescent chemosensors for ATP. Moreover, the response of the chemosensor toward ATP is fast (response time less than 3 min). In addition, the chemosensor can be used for the fluorescence assay for protein kinase activity with satisfactory results. The chemosensor for ATP based on hydrogen bonds interaction provided a novel strategy for the design of colormetric and ratiometric fluorescent probes for other target anions with high sensitivity and selectivity.     

Ultrasensitive cysteine sensing using citrate-capped CdS quantum dots

The importance of cysteine (Cys) in biological systems has stimulated a great deal of efforts in the development of analytical methods for the determination of this amino acid. In this work, a novel fluorescent probe for Cys based on citrate (Cit)-capped CdS quantum dots (QDs) is reported. The Cit-capped CdS QDs fluorescent probe offers good sensitivity and selectivity for detecting Cys. A good linear relationship was obtained from 1.0 × 10⁻⁸ mol L⁻¹ to 5.0 × 10⁻⁵ mol L⁻¹ for Cys. The detection limit was calculated as 5.4 × 10⁻⁹ mol L⁻¹. The proposed method was applied to detect Cys in human urine samples, which showed satisfactory results. This assay is based on both the lability of Cit and the strong affinity of thiols to the surface of CdS QDs. The addition of Cys improved the passivation of the surface traps of CdS QDs and enhanced the fluorescence intensity.     

A functionalized gold nanoparticles and Rhodamine 6G based fluorescent sensor for high sensitive and selective detection of mercury(II) in environmental water samples

A gold-nanoparticles (Au NPs)-Rhodamine 6G (Rh6G) based fluorescent sensor for detecting Hg (II) in aqueous solution has been developed. Water-soluble and monodisperse gold nanoparticles (Au NPs) has been prepared facilely and further modified with thioglycolic acid (TGA). Free Rh6G dye was strongly fluorescent in bulk solution. The sensor system composing of Rh6G and Au NPs fluoresce weakly as result of fluorescence resonance energy transfer (FRET) and collision. The fluorescence of Rh6G and Au NPs based sensor was gradually recovered due to Rh6G units departed from the surface of functionalized Au NPs in the presence of Hg(II). Based on the modulation of fluorescence quenching efficiency of Rh6G-Au NPs by Hg(II) at pH 9.0 of teraborate buffer solution, a simple, rapid, reliable and specific turn-on fluorescent assay for Hg(II) was proposed. Under the optimum conditions, the fluorescence intensity of sensor is proportional to the concentration of Hg(II). The calibration graphs are linear over the range of 5.0 × 10⁻¹⁰ to 3.55 × 10⁻⁸ mol L⁻¹, and the corresponding limit of detection (LOD) is low as 6.0 × 10⁻¹¹ mol L⁻¹. The relative standard deviation of 10 replicate measurements is 1.5% for 2.0 × 10⁻⁹ mol L⁻¹ Hg(II). In comparison with conventional fluorimetric methods for detection of mercury ion, the present nanosensor endowed with higher sensitivity and selectivity for Hg(II) in aqueous solution. Mercury(II) of real environmental water samples was determined by our proposed method with satisfactory results that were obtained by atomic absorption spectroscopy (AAS).     

Fluorescence enhancement effect for the determination of proteins with morin-Al3+ -cetyltrimethylammonium bromide

It is found that the fluorescence intensity of morin-Al³⁺ complex can be greatly enhanced by proteins in the presence of cetyltrimethylammonium bromide (CTAB). It is considered that protein and CTAB provide a hydrophobic environment with low polarity and large viscosity, resulting in the fluorescence enhancement of morin-Al³⁺ complex. The experiments indicate that under optimum conditions, the enhanced intensity of fluorescence is in proportion to the concentration of proteins (such as bovine serum albumin (BSA), human serum albumin (HSA) and egg albumin (EA)) in the wide range, and their detection limits (S/N = 3) are 2.6 × 10⁻⁹, 1.4 × 10⁻⁸ and 1.6 × 10⁻⁸ g ml⁻¹, respectively. This method has satisfactorily been used for the determination of protein in actual sample. In comparison with most of fluorimetric methods reported, this method is quick and simple, and has high sensitivity, wide linear range and good stability.     

A novel Lu fluorescent nano-chemosensor using new functionalized mesoporous structures

A new Lu³⁺ sensitive fluorescent chemosensor is designed using 8-hydroxyquinoline functionalized mesoporous silica with highly ordered structure (LUS-SPS-Q). The characterization of LUS-SPS-Q showed that the organized structure has been preserved after the post grafting procedure. The synthesized material showed a selective interaction with Lu³⁺ ion, most probably due to the presence of the fluorophore moiety at its surface. The emission intensity of the Lu³⁺-bound mesoporous material increases with an increase in concentrations of Lu³⁺ ion. Addition of other mono-, di-, trivalent ions resulted in insignificant change in the fluorescent intensity. The enhancement of fluorescence is attributed to the strong covalent binding of Lu³⁺ ion. The linear response range of Lu³⁺ chemo-sensor was from 1.6 × 10⁻⁷ to 1.0 × 10⁻⁵ mol L⁻¹. The Limit of detection obtained was 8.2 × 10⁻⁸ mol L⁻¹ and the pH range which the proposed chemo-sensor can be applied was 3.3–8.3.     

One-step synthesis and applications of fluorescent Cu nanoclusters stabilized by l-cysteine in aqueous solution

Herein, an innovative and simple strategy for synthesizing high fluorescent Cu nanoclusters was successfully established while l-cysteine played a role as the stabilizer. Meaningfully, the current Cu nanoclusters together with a quantum yield of 14.3% were prepared in aqueous solution, indicating their extensive applications. Subsequently, the possible fluorescence mechanism was elucidated by fluorescence, UV–vis, HR-TEM, FTIR, XPS, and MS. Additionally, the CuNCs were employed for assaying Hg²⁺ on the basis of the interactions between Hg²⁺ and l-cysteine; thus facilitating the quenching of their fluorescence. The proposed analytical strategy permitted detections of Hg²⁺ in a linear range of 1.0 × 10⁻⁷ mol L⁻¹ × 10⁻³ mol L⁻¹, with a detection limit of 2.4 × 10⁻⁸ mol L⁻¹ at a signal-to-noise ratio of 3. Significantly, this CuNCs described here were further applied for coding and fluorescent staining, suggesting may broaden avenues toward diverse applications.     

Multiplex sensor for detection of different metal ions based on on–off of fluorescent gold nanoclusters

In this study, a multiplex fluorescence sensor for successive detection of Fe³⁺, Cu²⁺ and Hg²⁺ ions based on “on–off” of fluorescence of a single type of gold nanoclusters (Au NCs) is described. Any of the Fe³⁺, Cu²⁺ and Hg²⁺ ions can cause quenching fluorescence of Au NCs, which established a sensitive sensor for detection of these ions respectively. With the introduction of ethylene diamine tetraacetic acid (EDTA) to the system of Au NCs and metal ions, a restoration of fluorescence may be found with the exception of Hg²⁺. A highly selective detection of Hg²⁺ ion is, thus, achieved by masking Fe³⁺ and Cu²⁺. On the other hand, the masking of Fe³⁺ and Cu²⁺ leads to the enhancement of fluorescence of Au NCs, which in turn provides an approach for successive determination of Fe³⁺ and Cu²⁺ based on “on–off” of fluorescence of Au NCs. Moreover, this assay was applied to the successful detection of Fe³⁺, Cu²⁺ and Hg²⁺ in fish, a good linear relationship was found between these metal ions and the degree of quenched fluorescent intensity. The dynamic ranges of Hg²⁺, Fe³⁺ and Cu²⁺ were 1.96 × 10⁻¹⁰–1.01 × 10⁻⁹, 1.28 × 10⁻⁷–1.27 × 10⁻⁶ and 1.2 × 10⁻⁷–1.2 × 10⁻⁶ M with high sensitivity (the limit of detection of Fe³⁺ 2.0 × 10⁻⁸ M, Cu²⁺ 1.9 × 10⁻⁸ M and Hg²⁺ 2 × 10⁻¹⁰ M). These results indicate that the assay is suitable for sensitive detection of these metal ions even under the coexistence, which can not only determine all three kinds of metal ions successively but also of detecting any or several kinds of metal ions.     

p-Dimethylaminobenzaldehyde thiosemicarbazone: a simple novel selective and sensitive fluorescent sensor for mercury(II) in aqueous solution

A novel and simple fluorophore, p-dimethylaminobenzaldehyde thiosemicarbazone (DMABTS), was prepared in order to find available fluorescent chemosensor for mercuric ion in aquesous solution. DMABTS emitted fluorescence at 448 nm in aqueous solution and its fluorescence intensity was completely quenched upon interaction with Hg²⁺ ions, which should be attributed to the 1:1 complex formation between DMABTS and Hg²⁺. The binding constant of the complex was determined as 7.48 × 10⁶ mol l⁻¹. The linear range of quantitative detection of 0 to 5.77 × 10⁻⁶ mol l⁻¹ and the detection limit of 7.7 × 10⁻⁷ mol l⁻¹ for Hg²⁺ in the 6.3 × 10⁻⁶ mol l⁻¹ DMABTS aqueous solution were obtained from a calibration curve. The coexistence of several transition metal ions and anions did interfere the fluorometric titration of Hg²⁺ ion by less than 4% in the emission change.     

High sensitive determination of zinc with novel water-soluble small molecular fluorescent sensor

A high sensitive method of quantitative analysis for the determination of zinc in the nutrition supplements has been developed by using a novel water-soluble fluorescent sensor HQ3: (8-pyridylmethyloxy-2-methyl-quinoline). Under the optimized condition of 67 mM phosphate buffer, pH 7.4, and 5% (v/v) DMSO, the zinc concentration showed good linear relationship with fluorescence intensity in the range of 7.5 × 10⁻⁸ to 2.5 × 10⁻⁵ M with the detection limit of 1.5 × 10⁻⁸ M. HQ3 exhibited high selectivity to zinc comparing with other metal ions except for cadmium. The developed analytical method was successfully used for determining the content of zinc in a real sample of zinc gluconate solution of Sanchine.     

Fluorescence single-molecule counting assays for protein quantification using epi-fluorescence microscopy with quantum dots labeling

A single-molecule counting approach for quantifying the antibody affixed to a surface using quantum dots and epi-fluorescence microscopy is presented. Modifying the glass substrates with carboxyl groups provides a hydrophilic surface that reacts with amine groups of an antibody to allow covalent immobilization of the antibody. Nonspecific adsorption of single molecules on the modified surfaces was first investigated. Then, quantum dots were employed to form complexes with surface-immobilized antibody molecules and used as fluorescent probes for single-molecule imaging. Epi-fluorescence microscopy was chosen as the tool for single-molecule fluorescence detection here. The generated fluorescence signals were taken by an electron multiplying charge-coupled device and were found to be proportional to the sample concentrations. Under optimal conditions, a linear response range of 5.0 × 10⁻¹⁴-3.0 × 10⁻¹² mol L⁻¹ was obtained between the number of single molecules and sample concentration via a single-molecule counting approach.     

Simple and sensitive fluorometric sensing of malachite green with native double-stranded calf thymus DNA as sensing material

A novel fluorometric sensing of malachite green is proposed in this paper. The native double-stranded calf thymus DNA was used as sensing material. In the presence of native double-stranded calf thymus DNA, malachite green could interact with the DNA, which resulted in a strong fluorescence emission. The fluorescent intensity was linear with malachite green concentration in the range of 4.0 × 10⁻¹⁰ − 1.8 × 10⁻⁷ g ml⁻¹ and the limit of detection was 2.0 × 10⁻¹⁰ g ml⁻¹. Before fluorescence measurement, the only required operation is the mixing of two solutions. So, this method is rather simple and rapid. The method is very safe for the analyst. Furthermore, the mechanism for fluorescence enhancing of native double-stranded calf thymus DNA on MG was proposed based on a series of experiments. The results suggest that the interaction between MG and calf thymus DNA is intercalation in nature.     

A reversible chemosensor for nitrite based on the fluorescence quenching of a carbazole derivative

The carbazole derivative, with an amino group in 9-position (9-methylacryloylamino carbazole (MAC), has been utilized to prepare a fluorescent sensor and used for the determination of NO₂⁻ based on the reaction between nitrite (NO₂⁻) and excess I⁻ to form I₃⁻, which can quench the fluorescence of carbazole derivative. MAC, as a fluorescent carrier, has a terminal double bond and is covalently immobilized on a quartz glass plate surface by photo-polymerization to prevent the leakage of the dye. The sensor shows sufficient repeatability, selectivity, operational lifetime of 8 weeks, and a fast response of less then 30 s. NO₂⁻ can be determined in the range between 1.0×10⁻⁶ and 1.0×10⁻⁴ mol l⁻¹ with a detection limit of 8.0×10⁻⁷ mol l⁻¹ at pH of 2.0. The quenching mechanism is discussed. Most commonly coexisting ions do not interfer with the NO₂⁻ assay.     

Colorimetric and fluorescent chemosensor for citrate based on a rhodamine and Pb complex in aqueous solution

In this paper we unveil a novel rhodamine compound based fluorescent chemosensor (1-Pb²⁺) for colormetric and fluorescent detection of citrate in aqueous solution. This is the first fluorescent chemosensor for citrate based on rhodamine compound. The comparison of this method with some other fluorescence methods for citrate indicates that the method can detect citrate in aqueous solution by both color changes and fluorescent changes with long emission wavelength. In the new developed sensing system, 1-Pb²⁺ is fluorescent due to Pb²⁺-induced fluorescence enhancement of 1. However, the addition of citrate may release 1 into the solution with quenching of fluorescence. The chemosensor can be applied to the quantification of citrate with a linear range covering from 1.0 × 10⁻⁷ to 5.0 × 10⁻⁵ M and a detection limit of 2.5 × 10⁻⁸ M. The experiment results show that the response behavior of 1-Pb²⁺ towards citrate is pH independent in medium condition (pH 6.0–8.0). Most importantly, the fluorescence changes of the chemosensor are remarkably specific for citrate in the presence of other anions (even those that exist in high concentration), which meet the selective requirements for practical application. Moreover, the response of the chemosensor toward citrate is fast (response time less than 1 min). In addition, the chemosensor has been used for determination of citrate in urine samples with satisfactory results.     

Fluorometric method for the determination of hydrogen peroxide and glucose with Fe3O4 as catalyst

In this paper, we utilized the instinct peroxidase-like property of Fe₃O₄ magnetic nanoparticles (MNPs) to establish a new fluorometric method for determination of hydrogen peroxide and glucose. In the presence of Fe₃O₄ MNPs as peroxidase mimetic catalyst, H₂O₂ was decomposed into radical that could quench the fluorescence of CdTe QDs more efficiently and rapidly. Then the oxidization of glucose by glucose oxidase was coupled with the fluorescence quenching of CdTe QDs by H₂O₂ producer with Fe₃O₄ MNPs catalyst, which can be used to detect glucose. Under the optimal reaction conditions, a linear correlation was established between fluorescence intensity ratio I₀/I and concentration of H₂O₂ from 1.8 × 10⁻⁷ to 9 × 10⁻⁴ mol/L with a detection limit of 1.8 × 10⁻⁸ mol/L. And a linear correlation was established between fluorescence intensity ratio I₀/I and concentration of glucose from 1.6 × 10⁻⁶ to 1.6 × 10⁻⁴ mol/L with a detection limit of 1.0 × 10⁻⁶ mol/L. The proposed method was applied to the determination of glucose in human serum samples with satisfactory results.     

A sensitive enzymatic method for paraoxon detection based on enzyme inhibition and fluorescence quenching

A sensitive and selective method for the paraoxon detection based on enzyme inhibition and fluorescence quenching was presented in this study. Under the catalytic effect of acetylcholinesterase (AChE), acetylthiocholine (ATCh) hydrolysis released thiocholine (TCh) which could react with N-(7-dimethylamino-4-methylcoumarin-3-yl) maleimide (DACM) to produce a blue fluorescence compound. Subsequently, AChE catalytic activity was inhibited with the addition of paraoxon, which caused TCh decreased, leading to a significant decrease of the blue fluorescent compound. Meanwhile, p-nitrophenol, the hydrolysis product of paraoxon, would lead to a quenching of the fluorescence. Therefore, fluorescence intensity of the system would decrease dramatically by a combined effect of enzyme inhibition and fluorescence quenching. Under optimal experimental conditions, an excellent linear relationship between the decrease of fluorescence intensity and paraoxon concentration over the range from 5.5 × 10⁻¹² to 1.8 × 10⁻¹⁰ mol L⁻¹ was obtained. Fluorescence background caused by nonenzymatic hydrolysis of ATCh or other matters was relatively low, the proposed approach offered adequate sensitivity for the detection of paraoxon at 3.5 × 10⁻¹² mol L⁻¹.     

Protein determination using methylene blue in a synchronous fluorescence technique

A new method for detecting protein by synchronous fluorescence enhancement was developed, based on the combination of near infrared (NIR) fluorescence and the dedimerization phenomenon of methylene blue (MB). Under analytical conditions, there are linear relationships between the enhancing extent of synchronous fluorescence of MB-sodium dodecyl benzene sulfonate (SDBS)-protein at 667 nm and the concentration of protein in the range of 8.0 × 10⁻⁸-4.0 × 10⁻⁵ g mL⁻¹ for bovine serum albumin (BSA), 1.0 × 10⁻⁷-3.5 × 10⁻⁵ g mL⁻¹ for egg albumin (EA). The detection limits (S/N = 3) of BSA and EA are 8.9 ng mL⁻¹ and 10.0 ng mL⁻¹, respectively. The fluorescence enhancement mechanism is discussed in detail. Results from multiple techniques indicate that the fluorescence enhancement of the system originates from the hydrophobic microenvironment provided by BSA and SDBS, and the formation of an MB-SDBS-BSA complex, as well as the deaggregation of some MB dimer.     

Fluorescence enhancement of CdTe/CdS quantum dots by coupling of glyphosate and its application for sensitive detection of copper ion

A novel fluorescent probe for Cu²⁺ determination based on the fluorescence quenching of glyphosate (Glyp)-functionalized quantum dots (QDs) was firstly reported. Glyp had been used to modify the surface of QDs to form Glyp-functionalized QDs following the capping of thioglycolic acid on the core–shell CdTe/CdS QDs. Under the optimal conditions, the response was linearly proportional to the concentration of Cu²⁺ between 2.4 × 10⁻² μg mL⁻¹ and 28 μg mL⁻¹, with a detection limit of 1.3 × 10⁻³ μg mL⁻¹ (3δ). The Glyp-functionalized QDs fluorescent probe offers good sensitivity and selectivity for detecting Cu²⁺. The fluorescent probe was successfully used for the determination of Cu²⁺ in environmental samples. The mechanism of reaction was also discussed.