Electrochemiluminescent functional nucleic acids-based sensors for food analysis.

Foodborne contaminants widely exist in foods, which can lead to various foodborne diseases and food safety issues. The development of quick, sensitive and universal analytical approaches for foodborne contaminants is imperative. Electrochemiluminescent functional nucleic acids (ECL FNAs)-based sensors are a series of sensing devices using FNAs as the recognition elements and ECL as the transducer. Contributing to the specific recognition ability of FNA and the high sensitivity of ECL, ECL FNA-based sensors are considered to be of great application potential for foodborne contaminants monitoring. This review mainly presents the applications of ECL FNA-based sensors for foodborne contaminants (including microorganisms, mycotoxins, allergens, antibiotics, heavy metal ions, pesticides and some illegal additives). In general, the application of ECL FNA-based sensors in the field of food analysis is just in its infancy. Although there are several limitations and challenges, it is envisaged that ECL FNA-based sensors will have broad prospects for food analysis in the future.

Fluorometric determination of the activity of inorganic pyrophosphatase and its inhibitors by exploiting the peroxidase mimicking properties of a two-dimensional metal organic framework.

A copper(II)-based two-dimensional metal-organic framework with nanosheet structure (CuBDC NS) that possesses peroxidase (POx) mimicking activity was prepared. In the presence of hydrogen peroxide, the system catalyses the oxidation of terephthalic acid to a blue-fluorescent product (excitation = 315 nm; emission = 425 nm). Pyrophosphate has a very strong affinity for Cu2+ ion and blocks the POx-mimicking activity of the CuBDC NS. If, however, inorganic pyrophosphatase is present, the POx mimicking activity is gradually restored because pyrophosphate is hydrolyzed. The findings were used to design a method for the determination of the activity of inorganic pyrophosphatase by fluorometry. Fluorescence increases linearly in the 1-50 mU·mL-1 inorganic pyrophosphatase activity range. The limit of detection is 0.6 mU·mL-1 (S/N = 3). Graphical abstract A copper(II)-based two-dimensional metal-organic framework (CuBDC NS) is described that possesses POx-mimicking activity. Inorganic pyrophosphate (PPi) was hydrolyzed to phosphate in the presence of inorganic pyrophosphatase (PPase). Hence, it cannot coordinate with Cu2+ in CuBDC NS, its structure was well-conserved to catalyses the oxidation of terephthalic acid (H2BDC) to produce a blue fluorescent product (oxBDC) in the presence of hydrogen peroxide (H2O2).

A calcium alginate sponge with embedded gold nanoparticles as a flexible SERS substrate for direct analysis of pollutant dyes.

A surface-enhanced Raman scattering (SERS) substrate with good flexibility and high water absorbing capacity is reported. It consists of a calcium alginate sponge incorporating gold nanoparticles. These are in close contact with the sponge without the need for amino or sulfhydryl modification. The substrate is capable of detecting the dyes crystal violet (CV) and malachite green (MG) in water directly and rapidly by immersing it into the liquid sample. Preconcentration and separation are not required. The dyes absorbed on the sponge can be detected without drying and thus the whole analytical process can be completed within 3 min. The results show that the lowest detectable concentrations are 0.1 and 0.25 µg⋅L-1 for CV and MG, respectively. This is lower than the minimum required performance limits set by the European Commission and the US EPA. Moreover, MG and CV can be simultaneously detected in liquid samples due to their different SERS bands (at 1216 and 1534 cm-1, respectively). It should be noted that the molecular structures of MG and CV are very similar. Therefore, the method has a large potential for determination of several analytes simultaneously even in complex sample metrics. Graphical abstract Schematic presentation of the fabrication of a sodium alginate sponge loaded with gold nanoparticles. Gold nanoparticles together with gel-like alginate were freeze-dried to form the sponge. The sponge was cross-linked by CaCl2 solution and then it was freeze-dried again.

A Simple and Convenient Aptasensor for Protein Using an Electronic Balance as a Readout.

The electronic balance, one of the most common pieces of equipment in the laboratory, is normally used to directly measure the weight of a target with high accuracy. However, little attention has been paid to the extension of its applications. In this study, an electronic balance was used as a readout to develop a novel aptasensor for protein quantification for the first time. Thrombin was selected as a model target, and its two aptamers recognizing different sites of the protein were used (one aptamer was immobilized on the surface of magnetic microparticles and the other aptamer was functionalized with platinum nanoparticles). The two aptamers were specifically bound with the thrombin to form a sandwich structure; thus, the platinum nanoparticles were linked to the magnetic microparticles, and they were separated by a magnet easily. The captured platinum nanoparticles effectively catalyzed the decomposition of H2O2, generating a large volume of O2 to discharge a certain amount of water in a drainage device, because the pressure in the vial is higher than that outside of the vial. The weight of water was accurately measured by an electronic balance. The weight of water increased with the increasing of the thrombin concentration in the range of 0 to 100 nM with a detection limit of 2.8 nM. This is the first time the use of an electronic balance as a signal readout for biomolecule quantitation in bioassay has been reported.

Target-Induced Horseradish Peroxidase Deactivation for Multicolor Colorimetric Assay of Hydrogen Sulfide in Rat Brain Microdialysis.

Hydrogen sulfide (H2S) is important for normal neural functions, which involves protecting neurons from oxidative stress and neuronal transmission modulation in brain. The detection of H2S is significant for revealing its role in the diagnosis of various disease. In this study, a novel multicolor colorimetric assay based on the etching of gold nanorods (Au NRs) is proposed to evaluate H2S level with the naked eye. This measurement relies on the catalytic oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) via horseradish peroxidase (HRP) to produce TMB2+, which could etch the Au NRs quickly and accompany with a distinct color change. The vivid colors can be easily distinguished with the naked eye without any sophisticated instruments. The presence of H2S can cause the deactivation of HRP, which affects the amount of TMB2+ produced and consequently affects the color changing of the system. Based on this mechanism, a simple but sensitive multicolor colorimetric assay is developed for H2S detection with a linear range of 0.05-50 µM. The proposed method is demonstrated for monitoring extracellular H2S in rat brain coupled with microdialysate.

Cationic Carbon Dots for Modification-Free Detection of Hyaluronidase via an Electrostatic-Controlled Ratiometric Fluorescence Assay.

Carbon dots (CDs) emerge as excellent fluorescent nanomaterials, but the full exploitation and application of their exceptional properties in the development of fluorescence assay are still rare. In this work, cationic carbon dots (C-CDs) covered with plenty of positive charges on the surface were synthesized through a facile ultrasonic method. Negatively charged hyaluronic acid (HA) caused the aggregation of positively charged C-CDs and neutral red (NR) along its linear chain via electrostatic adsorption, leading to a remarkable Förster resonance energy transfer (FRET) from C-CDs to NR. However, the presence of hyaluronidase (HAase) resulted in the enzymolysis of HA, as well as the liberation of C-CDs and NR. The corresponding change of fluorescence color from red to green-yellow afforded a reliable ratiometric assay for HAase. Also the ratio of fluorescence intensity for C-CDs (I525) to that for NR (I630) was used for quantitative detection of HAase. The proposed sensing system was easily operated in aqueous media with a detection limit of 0.05 U/mL. This strategy provides a new approach for the wider application of some special CDs in detecting biomolecules.

Highly Uniform Gold Nanobipyramids for Ultrasensitive Colorimetric Detection of Influenza Virus.

Gold nanoparticles (AuNPs) have been frequently utilized for the construction of diverse colorimetric biosensors. Normally, AuNPs with sharp edges could have better sensitivity. However, the poor monodipersity of AuNPs with sharp edges seriously confines their utility for colorimetric biosensing. Herein, we demonstrate the utility of highly uniform gold nanobipyramids (Au NBPs) for ultrasensitive colorimetric detection of H5N1 virus. The proposed method is based on the fact that alkaline phosphatase (ALP) could catalyze the decomposition of 4-aminophenyl phosphate (4-APP) to generate 4-aminophenol (4-AP), which would then reduce silver nitrate to metal silver and then deposited on Au NBPs. The metal silver shell coated on the Au NBPs changed the refractive index of gold and thus resulted in a blue shift of longitudinal localized surface plasmon resonance (LSPR) and accompanied a vivid color change. This method exhibited a higher sensitivity than that of other Au NPs such as gold nanorods due to the high-index-faceted on the tips of the Au NBPs. This method was used to detect the activity of ALP. It exhibited a linear range of 0.1-5 mU/mL with a limit of detection (LOD) of 0.086 mU/mL. Finally, the proposed method was used in immunoassay to detect H5N1 virus. The results showed that the corresponding linear range for the detection of H5N1 virus antigen was 0.001-2.5 ng/mL, and the LOD was determined to be 1 pg/mL, which is more sensitive than those in most of the colorimetric biosensors reported previously.

Creating BHb-imprinted magnetic nanoparticles with multiple binding sites.

A kind of protein imprinted over magnetic Fe3O4@Au multifunctional nanoparticles (NPs) with multiple binding sites was synthesized and investigated. Magnetic Fe3O4@Au NPs as carrier materials were modified with 4-mercaptophenylboronic acid (MPBA) and mercaptopropionic acid (MPA) to introduce boronic acids and carboxyl groups. Using Bovine Hemoglobin (BHb) as a template, a polydopamine(PDA)-based molecular imprinted film was fabricated to produce a kind of magnetic molecularly imprinted nanoparticle (MMIP), possessing multiple binding sites with benzene-diol, amino groups, boronic acids and carboxyl groups. The MMIPs exhibited an excellent imprinting effect and adsorption capacity (89.65± 0.38 mg g-1) toward the template protein. The results show that the MMIPs reached saturated adsorption at 0.5 mg mL-1 within 90 min. The synthesized MMIPs are suitable for the removal and enrichment of the template protein in proteomics. The strategy of multiple binding sites paves the way for the preparation of functional nanomaterials in molecular imprinting techniques.

Fluorescence biosensor for inorganic pyrophosphatase activity.

A highly sensitive and selective fluorescence biosensor for inorganic pyrophosphatase (PPase) activity has been developed based on special click ligation trigger hyperbranched rolling circle amplification (CLT-HRCA). Pyrophosphate ion (PPi) can coordinate with Cu2+ to form stable PPi/Cu2+ complex and Cu2+ in the complex cannot be reduced to Cu+. The addition of PPase causes the hydrolysis of PPi into orthophosphate (Pi) and therefore induces the releasing of Cu2+ from the stable PPi/Cu2+ complex, and the free Cu2+ is easily reduced to Cu+ by sodium ascorbate. Then Cu+ catalyzes the cyclization reaction between the specially designed 5'-azide and 3'-alkyne tagged padlock probes through Cu+ catalyzed azide-alkyne cycloaddition (CuAAC), which in turn initiates the hyperbranched rolling circle amplification (HRCA). Given that the CLT-HRCA products contain large amounts of double-stranded DNAs (dsDNAs), the addition of SYBR Green I resulted in the enhanced fluorescence signal. There was a linear relationship between the enhanced fluorescence intensity and the logarithm PPase activity ranging from 0.05 to 25 mU with a detection limit of 0.02 mU. Such proposed biosensor has been successfully applied to screen the potential PPase inhibitors and has accessed the related inhibit ability with high efficiency.

Highly sensitive aptamer based on electrochemiluminescence biosensor for label-free detection of bisphenol A.

Bisphenol A (BPA), a typical endocrine disruptor, is widely used as a key monomer in the packaging industry. Residual monomer can transfer from the package material to the food and thereby pose a risk to the health of the consumer, so determination of BPA migration is highly important for food safety control. In this study, a simple but sensitive electrochemiluminescence (ECL) biosensor, which combines the characteristics of high selectivity of an aptamer and high sensitivity of ECL, has been developed to detect BPA from package materials. The aptamer was immobilized on a gold electrode surface through Au-S interaction. The aptamer was then hybridized with complementary DNA (CDNA) to form double-stranded DNA (dsDNA). Ru(phen)32+ can intercalate into the grooves of dsDNA and acts as an ECL indicator; high ECL intensity can therefore be detected from the electrode surface. In the presence of BPA, which can competitively bind with the aptamer owing to their high affinity, Ru(phen)32+ is released from the electrode surface and the ECL of the system is decreased. The decreasing ECL signal has a linear relationship with BPA in the range of 0.1-100 pM with a detection limit of 0.076 pM. The developed biosensor has been applied to detect migration of BPA from different categories of canned drink with satisfactory results.

Exploring the electrochemiluminescent behavior of procaterol hydrochloride in the presence of Ru(bpy)32+ and its analytical application in pharmaceutical preparation.

Based on the strong enhancement effect of procaterol hydrochloride on the electrochemiluminescence (ECL) of Ru(bpy)32+ (bpy = 2,2'-bipyridine) in an alkaline H3 PO4 -NaOH buffer solution on a bare Pt electrode, a simple, rapid and sensitive method was developed for the determination of procaterol hydrochloride. The optimum conditions for the enhanced ECL have been developed in detail in this work. Under optimum conditions, the logarithmic ECL enhancement vs. the logarithmic concentration of procaterol hydrochloride is linear over a wide concentration range of 2.0 × 10-7 to 2.0 × 10-4  M (r = 0.9976), with a limit of detection of 1.1 × 10-8  M (S/N = 3), and a relative standard deviation of 2.1% (n = 7, c = 5.0 × 10-6  M). The proposed method was applied to the determination of this drug in tablets with recoveries of 89.7%-98.5%. In addition, a possible mechanism for the enhanced ECL of Ru(bpy)32+ , which is caused by ProH, has also been proposed.

An ultrasensitive conformation-dependent colorimetric probe for the detection of mercury(II) using exonuclease III-assisted target recycling and gold nanoparticles.

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).

Multicolor ELISA based on alkaline phosphatase-triggered growth of Au nanorods.

Seed-mediated synthesis of gold nanorods (AuNRs) has been widely used for diverse applications in the past decade. In this work, this synthetic process is demonstrated for multicolor biosensing for the first time. Our investigation reveals that ascorbic acid acts as a key factor to mediate the growth of AuNRs. This phenomenon is incorporated into the alkaline phosphatase (ALP)-enzyme-linked immunosorbent assay (ELISA) system based on the fact that ALP can catalyze the conversion of ascorbic acid-phosphate into ascorbic acid with high efficiency. This allows us to develop a multicolor ELISA approach for sensitive detection of disease biomarkers with the naked eye. We show the proof-of-concept multicolor ELISA for the detection of prostate-specific antigen (PSA) in human serum. The results show that different colors are presented in response to different concentrations of PSA, and a detection limit of 3 × 10(-15) g mL(-1) in human serum was achieved. The proposed multicolor ELISA could be a good supplement to conventional ELISA for POC diagnostics.

An immobilization free DNAzyme based electrochemical biosensor for lead determination.

DNAzyme based electrochemical biosensors have the characteristics of high sensitivity and selectivity, but traditional DNAzyme based electrochemical biosensors need the immobilization of DNAzyme on the electrode surface first, and the procedures are time consuming and tedious, which limit their real application. In this study, a simple but sensitive immobilization free DNAzyme based electrochemical biosensor has been proposed and lead has been chosen as a model target because of the severe effects of lead toxicity. The different diffusivity and electrostatic repulsion between long and short DNA on the negatively charged ITO electrode can be used to discriminate the short and long DNA. Lead dependent DNAzyme was hybridized with its substrate (which was modified with methylene blue at the 3' terminal) beforehand. Since the DNAzyme/substrate complex contains a large negative charge, it cannot diffuse easily to the negatively charged ITO electrode surface and little electrochemical signal has been detected. The presence of lead would trigger the cleavage of the DNAzyme/substrate complex and cause the release of a methylene blue-labeled short-oligonucleotide into the solution. The methylene blue-labeled short-oligonucleotide can diffuse easily to the surface of the negatively charged ITO electrode and results in the enhanced electrochemical response being detected. Each lead can cleave a lot of DNAzyme/substrate complex to realize signal amplification. The enhanced electrochemical signal has a linear relationship with the Pb(2+) concentration in the range of 0.05-1 µM with a detection limit of 0.018 µM (S/N = 3). The proposed biosensor has been applied to detect Pb(2+) in water samples with satisfactory results.

Label-free electrochemiluminescence biosensor for ultrasensitive detection of telomerase activity in HeLa cells based on extension reaction and intercalation of Ru(phen)3 (2.).

Telomerase is one of the most common markers of human malignant tumors, such as uterine, stomach, esophageal, breast, colorectal, laryngeal squamous cell, thyroid, bladder, and so on. It is necessary to develop some sensitive but convenient detection methods for telomerase activity determination. In this study, a label-free and ultrasensitive electrochemiluminescence (ECL) biosensor has been fabricated to detect the activity of telomerase extracted from HeLa cells. Thiolated telomerase substrate (TS) primer was immobilized on the gold electrode surface through gold-sulfur (Au-S) interaction and then elongated by telomerase specifically. Then, it was hybridized with complementary DNA to form double-stranded DNA (dsDNA) fragments on the electrode surface, and Ru(phen)3 (2+) has been intercalated into the dsDNA grooves to act as the ECL probe. The enhanced ECL intensity has a linear relationship with the number of HeLa cells in the range of 5∼5000 and with a detection limit of 2 HeLa cells. The proposed ECL biosensor has high specificity to telomerase in the presence of common interferents. The relative standard deviations (RSDs) were <5 % at 100 HeLa cells. The proposed method provides a convenient approach for telomerase-related cancer screening or diagnosis.

Fluorometric method for inorganic pyrophosphatase activity detection and inhibitor screening based on click chemistry.

A fluorometric method for pyrophosphatase (PPase) activity detection was developed based on click chemistry. Cu(II) can coordinate with pyrophosphate (PPi), the addition of pyrophosphatase (PPase) into the above system can destroy the coordinate compound because PPase catalyzes the hydrolysis of PPi into inorganic phosphate and produces free Cu(II), and free Cu(II) can be reduced by sodium ascorbate (SA) to form Cu(I), which in turn initiates the ligating reaction between nonfluorescent 3-azidocoumarins and terminal alkynes to produce a highly fluorescent triazole complex, based on which, a simple and sensitive turn on fluorometric method for PPase can be developed. The fluorescence intensity of the system has a linear relationship with the logarithm of the PPase concentration in the range of 0.5 and 10 mU with a detection limit down to 0.2 mU (S/N = 3). This method is cost-effective and convenient without any labels or complicated operations. The proposed system was applied to screen the potential PPase inhibitor with high efficiency. The proposed method can be applied to diagnosis of PPase-related diseases.

Luminescent Ag6Au6 heterometallic ethisterone cluster and probe for estrogen receptor α.

A heterometallic cluster [Ag6Au6(ethisterone)12] of an unprecedented topology was synthesized and characterized. A sensitive and specific probe for estrogen receptor α (ERα) has been developed for the first time based on the enhancement of the Ag6Au6 luminescence.

A novel fluorescent reagent for recognition of triplex DNA with high specificity and selectivity.

A fluorescent agent (DMT) was screened for recognizing triplex DNA with a specific and selective characteristic, which was embedded into the triplex DNA structure. The triplex DNA was firstly formed by a complementary target sequence through two distinct and sequential events. The conditions including pH and hybridization time, fluorescent agent concentration and embedding time were optimized in the experiment. Under the optimum conditions, the fluorescence signal was enhanced up to 9-fold in comparison with the DMT embedding into the ssDNA, dsDNA and G-quadruplexes. Under the same fluorescence conditions, the changes of the fluorescence signal were also investigated by several kinds of base mismatched DNAs in the experiment. The results showed that our biosensor provided excellent discrimination efficiency toward the perfectly mismatched target DNA with no formation of triplex DNA. We preliminarily deduced the mechanism of the fluorescent reagent for recognizing triplex DNA with high specificity and selectivity.

An electrochemical impedimetric sensor based on biomimetic electrospun nanofibers for formaldehyde.

Herein, simple molecular recognition sites for formaldehyde were designed on electrospun polymer nanofibers. In order to improve the conductivity of the electrospun polymer nanofibers, carbon nanotubes were introduced into the resulting nanofibers. By employing these functionalized nanocomposite fibers to fabricate a biomimetic sensor platform, an obvious change caused by recognition between recognition sites and formaldehyde molecules was monitored through electrochemical impedance spectroscopy (EIS). The experimental conditions were optimized and then a quantitative method for formaldehyde sensing in low concentration was established. The relative results demonstrated that the sensor based on biomimetic recognition nanofibers displays an excellent recognition capacity toward formaldehyde. The linear response range of the sensor was between 1 × 10(-6) mol L(-1) and 1 × 10(-2) mol L(-1), with the detection limit of 8 × 10(-7) mol L(-1). The presented research provided a fast, feasible and sensitive method for formaldehyde with good anti-interference capabilities and good stability, which could meet the practical requirement for formaldehyde assay.

Highly selective colorimetric bacteria sensing based on protein-capped nanoparticles.

A rapid and cost-effective colorimetric sensor has been developed for the detection of bacteria (Bacillus subtilis was selected as an example). The sensor was designed to rely on lysozyme-capped AuNPs with the advantages of effective amplification and high specificity. In the sensing system, lysozyme was able to bind strongly to Bacillus subtilis, which effectively induced a color change of the solution from light purple to purplish red. The lowest concentration of Bacillus subtilis detectable by the naked eye was 4.5 × 10(3) colony-forming units (CFU) mL(-1). Similar results were discernable from UV-Vis absorption measurements. A good specificity was observed through a statistical analysis method using the SPSS software (version 17.0). This simple colorimetric sensor may therefore be a rapid and specific method for a bacterial detection assay in complex samples.