Electrochemiluminescence biosensor for miRNA-21 based on toehold-mediated strand displacement amplification with Ru(phen)32+ loaded DNA nanoclews as signal tags.

A novel electrochemiluminescence (ECL) biosensor was developed for high sensitive and selective detection of miRNA-21 based on the efficient and specific toehold-mediated strand displacement (TMSD) amplification with Ru(phen)32+ loaded DNA nanoclews (NCs-Ru(phen)32+) as signal tags. The stable DNA nanoclews, synthesized by a simple rolling circle amplification reaction, were employed to load with Ru(phen)32+ efficiently as ECL signal tags to amplify the signals. As for TMSD, the substrate strand (Sub) was initially hybridized with P1 and P2 to form DNA duplex structures with a toehold 1. miRNA-21 could hybridize with the toehold 1 and trigger the TMSD amplification with the help of assist strand, releasing lots of P1 stands from DNA duplex structures. The TMSD technique realized the converting and amplification of the single miRNA-21 input to lots of output DNA (namely P1) with good selectivity, simultaneously. Output P1 were designed to expand the stem-locked region of HP, which were immobilized on the Au electrodes firstly. Subsequently, the opened HP could hybridize with the Ru(phen)32+, capturing the ECL signal tags closed to the sensing surface. The ECL intensity of the system had a linear relationship with the logarithm of the miRNA-21 concentration in the range of 1.0 fM to 100 pM with a limit of detection of 0.65 fM. The strategy was further applied to detect miRNA-21 in complex samples, and the result was consistent with the qRT-PCR.

Ultrasensitive and Portable Assay for Lead(II) Ions by Electronic Balance as a Readout.

Lead ions (Pb2+) cause harm to human health. Therefore, the development of fast, effective, and convenient sensors for Pb2+ monitoring has received great attention. In this study, a portable method has been proposed for Pb2+ detection using normal electronic balance as a readout. Magnetic bead-catalytic strand is hybridized with platinum nanoparticles (Pt NPs) functioned substrate strand (Pt-Sub) to form double-stranded DNA first. In the presence of Pb2+, the DNAzyme is activated and cleaved at the ribo-adenosine site of the substrate strand and hence causes Pt NPs to be released into the supernatant, which can be easily separated from the Pt-Sub by a magnet. The separated Pt NPs can effectively catalyze the decomposition of H2O2 to produce O2. In a sealed bottle, the pressure inside the bottle is increased by the generation of oxygen so that the water is discharged from the drainage device, and the weight of the water can be easily and precisely measured by a normal electronic balance. The weighting water has a linear relationship with the concentration of Pb2+ in the range of 2.5-100 nM and the detection limit of 0.83 nM (S/N = 3). The proposed method has been applied to detect Pb2+ in water with satisfactory results. Because the electronic balance is one of the most commonly used analytical tools for the laboratory, it is very practical and convenient without the need for expensive instruments and complicated data processing.

Development of an Immunosensor Based on the Exothermic Reaction between H2O and CaO Using a Common Thermometer as Readout.

Thermometers, one of the most commonly used instruments at home, are normally adapted to measure temperature directly with high accuracy but rarely adopted to act as readout in the biosensors. It is necessary to find some ways to establish a relationship between the concentration of the target and the temperature change. In this study, a common thermometer was used as readout to develop a convenient immunosensor. The designed immunosensor comprises three components, including target recognition area, water flow system, and exothermic reaction bottle. The capture antibody for the target [carcinoembryonic antigen (CEA) was selected as a model target] was preloaded on the bottom of the recognition area. In the presence of CEA, a sandwich-type structure was formed between the capture antibody, CEA, and biotinylated detection antibody. Then, the streptavidin-functionalized platinum nanoparticles were labeled on the detection antibody due to biotin-avidin interaction. The captured platinum nanoparticles can effectively catalyze the decomposition of H2O2 into O2. The continuous production of gas resulted in pressure increment inside the reaction bottle and further pushed the water flow into the exothermic reaction bottle. Finally, the water reacted with calcium oxide to generate a large amount of heat in the exothermic reaction bottle; thereby the temperature inside the bottle was enhanced and recorded by a common thermometer easily. The temperature enhancement has a linear relationship with the CEA concentration in the range of 7.81-500 pg/mL with a detection limit of 0.6 pg/mL. Furthermore, by taking advantage of simplicity, compatibility, stability, and high sensitivity, our temperature-based immunoassay has been applied to detect CEA in human serum samples with satisfactory results.

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.

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.

An electrochemical sensor based on poly(procaterol hydrochloride)/carboxyl multi-walled carbon nanotube for the determination of bromhexine hydrochloride.

Poly(procaterol hydrochloride) (p-ProH) polymeric film was successfully deposited onto the carboxyl multi-walled carbon nanotube (CMWCNT) modified glass carbon electrode (GCE) to construct a p-ProH/CMWCNT composite modified GCE. Due to the synergistic effect of p-ProH and CMWCNT in the composite, the developed sensor can enormously enhance the oxidation peak current of bromhexine hydrochloride (BrH) at ca. + 0.90 V. Based on this appearance, an electrochemical method was established for the sensitive and selective determination of BrH with differential pulse voltammetry (DPV). Various conditions affecting the peak current response of BrH were studied and optimized. Under the best conditions, the oxidation peak current of BrH is linear to its concentration in two linear dynamic ranges of 0.2-1.0 µmol L-1 (R = 0.9948) and 1.0-8.0 µmol L-1 (R = 0.9956), with a detection limit of 0.1 µmol L-1 (S/N = 3). Interference experiment indicated that the as-prepared electrochemical sensor showed wonderful selectivity to the recognition of BrH and was free from disturbance of many other electro-active substances such as dopamine, ascorbic and uric acid. Finally, the practicability of the BrH sensor was verified by the satisfactory results acquired from the BrH determination in pharmaceutical preparation and human serum.

Label-free sensitive luminescence biosensor for immunoglobulin G based on Ag6Au6 ethisterone cluster-estrogen receptor α aggregation and graphene.

A specific and label-free "on-off-on" luminescence biosensor based on a novel heterometallic cluster [Ag6Au6(ethisterone)12]-estrogen receptor α (Ag6Au6Eth-ERα) aggregation utilizing graphene oxide (GO) as a quencher to lead a small background signal was firstly constructed to detect immunoglobulin G (IgG) with a simple process and high selectivity. The efficient photoluminescent (PL) Ag6Au6Eth-ERα aggregation is strongly quenched by GO. In the presence of IgG, the PL of this system will be restored, and perceivable by human eyes under UV lamp excitation (365 nm). The quenching mechanism of GO on Ag6Au6Eth-ERα and enhancement mechanism of IgG on Ag6Au6Eth-ERα-GO were investigated in detail. Under the optimum conditions, the biosensor for high sensitive IgG detection expressed a wider linear range of 0.0078-10 ng/mL and a lower detection limit of 0.65 pg/mL with good stability and repeatability, which provided a new approach for label-free IgG detection.

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.

Highly sensitive colorimetric aptasensor for ochratoxin A detection based on enzyme-encapsulated liposome.

A simple, low-cost, and sensitive liposome-based colorimetric aptasensor has been developed to detect ochratoxin A (OTA). Specifically, a dumbbell-shaped probe was designed, including magnetic beads (MBs), double-stranded DNA (dsDNA), and enzyme-encapsulated liposome. The dsDNA formed by the hybridization between OTA aptamer and its complementary probe. And the dsDNA was used to contact the MBs and the enzyme-encapsulated liposome. In the presence of OTA, the aptamer preferred to combine with OTA to form G-quadruplex, resulting in the release of the detection probe and the enzyme-encapsulated liposome. Each liposome contained a large amount of HRP. Thus, when the liposome was lysed by adding the mixed solution of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2, a large number of HRP were released. HRP could catalyze the H2O2-mediated oxidation of TMB and hence resulted in the color change from colorless to blue with the OTA concentration varying, and this variation can be observed by naked eyes easily. The result showed that the absorption intensity at 652 nm enhanced with the increase of OTA concentration ranging from 0.05 to 2.0 ng mL-1, and the limit of detection was calculated to be 0.023 ng mL-1 (S/N = 3). The developed colorimetric aptasensor has been applied to detect OTA concentration in corn samples with satisfied results.

Detection of aflatoxin B1 in food samples based on target-responsive aptamer-cross-linked hydrogel using a handheld pH meter as readout.

Aflatoxin B1 (AFB1) can cause great threat to human health, so the development of convenient and portable device for sensitive detection of AFB1 is highly desired. The portable pH meter has the characters of facile operation, low cost, and easy availability. Therefore, in this study, we investigate the applicability of utilizing a pH meter as the readout to develop a portable sensor for AFB1. The specific detection of AFB1 is realized via the combination of AFB1-responsive aptamer-cross-linked hydrogel. Upon the addition of AFB1, AFB1 binds to its aptamer with high affinity in lieu of aptamer/DNA complex, causing the collapse of hydrogel network and results in the releasing of urease into the solution. The released urease can catalyse the hydrolysis of urea and result in the rise of pH value. The change of pH value has a direct relationship to the concentration of AFB1 in the range of 0.2-20µM with a detection limit of 0.1µM (S/N = 3). The proposed portable device is successfully applied to assay AFB1 in the food samples with satisfied results.

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.

Preparation of an Efficient Ratiometric Fluorescent Nanoprobe (m-CDs@[Ru(bpy)3]2+) for Visual and Specific Detection of Hypochlorite on Site and in Living Cells.

Hypochlorite (ClO-) is one of the most important reactive oxygen species (ROS), which plays an important role in sustaining human innate immunity during microbial invasion. Moreover, ClO- is a powerful oxidizer for water treatment. The safety of drinking water is closely related to its content. Herein, m-phenylenediamine (mPD) is used as a precursor to prepare carbon dots (named m-CDs) with highly fluorescent quantum yield (31.58% in water), and our investigation shows that the strong fluorescent emission of m-CDs can be effectively quenched by ClO-. Based on these findings, we developed a novel fluorescent nanoprobe (m-CDs) for highly selective detection of ClO-. The linear range was from 0.05 to 7 µM (R2 = 0.998), and the limit of detection (S/N = 3) was as low as 0.012 µM. Moreover, a portable agarose hydrogel solid matrix-based ratiometric fluorescent nanoprobe (m-CDs@[Ru(bpy)3]2+) sensor was subsequently developed for visual on-site detection of ClO- with the naked eyes under a UV lamp, suggesting its potential in practical application with low cost and excellent performance in water quality monitoring. Additionally, intracellular detection of exogenous ClO- was demonstrated via ratiometric imaging microscopy.

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.

Highly sensitive antibody-aptamer sensor for vascular endothelial growth factor based on hybridization chain reaction and pH meter/indicator.

Vascular endothelial growth factor (VEGF) is a crucial signaling protein for the tumor growth and metastasis, which is also acted as the biomarkers for various diseases. In this research, we fabricate an aptamer-antibody sensor for point-of-care test of VEGF. Firstly, target VEGF is captured by antibody immobilized on the microplate, and then binds with aptamer to form the sandwich structure. Next, with the assist of glucose oxidase (GOx)-functionalized ssDNAs, hybridization chain reaction occurs using the aptamer as the primer. Thus, GOx are greatly gathered on the microplate, which catalyzes the oxidization of glucose, leading to the pH change. As a result, the detect limit at a signal-to-noise was estimated to be 0.5pg/mL of target by pH meter, and 1.6pg/mL of VEGF was able to be distinguished by naked eyes. Meanwhile, this method has been used assay VEGF in the serum with the satisfactory results.

Boron nitride nanosheets as a platform for fluorescence sensing.

Hexagonal boron nitride nanosheets (BNNS), one kind of inorganic graphene analogue, exhibit high fluorescence quenching ability via the photo-induced electron transfer (PET) and different affinity toward single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). As a proof of concept, the BNNS was first used as a sensing platform for the rapid detection of DNA and small molecules with high sensitivity and selectivity in this study. This strategy is versatile and quick fluorescence sensing of DNA and extensive DNA related analytes such as metal cations and small molecules. Moreover, this strategy might be available for the practical application in future. This work provides an avenue for understanding the interaction between two-dimensional nanomaterials and biomolecules and designing novel sensing strategies for extending the applications of nanomaterials in bioanalysis.

Electrochemical investigation and determination of procaterol hydrochloride on poly(glutamic acid)/carboxyl functionalized multiwalled carbon nanotubes/polyvinyl alcohol modified glassy carbon electrode.

Poly(glutamic acid) (P-GLU)/carboxyl functionalized multiwalled carbon nanotubes (MWCNTs-COOH)/polyvinyl alcohol (PVA) modified glassy carbon electrode (GCE) has been successfully prepared and the electrochemical behavior of procaterol hydrochloride (ProH) was studied. The results show that the as-prepared modified electrode exhibits a good electrocatalytic property towards the oxidation of ProH in 0.2M phosphate buffer solution (PBS) (pH 6.0) due to the enhanced oxidation peak current at ~+0.59V. Under optimal reaction conditions, the oxidation peak current of ProH is proportional to its concentration in the linear dynamic ranges of 0.060 - 8.0µM (R = 0.9974), with a detection limit of 8.0 × 10-9M. Finally, this method was efficiently used for the determination of ProH in tablets and human urine with recoveries of 88.5~98.7% and 89.2 ~ 108.0%, respectively.

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.

A Portable Immunosensor with Differential Pressure Gauges Readout for Alpha Fetoprotein Detection.

A portable, affordable and simple detector is requested in a "Point-of-Care-Testing" (POCT) system. In this study, we exploited the potentialities of Differential Pressure Gauge (DPG) to the orientation of POCT technology. Alpha fetoprotein (AFP) was chosen as a model analyte that could specifically recognized by its antigen, and a tiny outfits equipped with a DPG was employed as the signal readout. Pt/SiO2 nanospheres were synthesized and modified with the detection antibody. In the presence of target, a sandwich of immunocomplex specifically formed and the Pt/SiO2 had been modified on the capture antibody. Which then can be dissolved to release plenty of Pt and the suspensions were transferred into a closed vial filled with appropriated amount of hydrogen peroxide. Subsequently, hydrogen peroxide was decomposed to produce oxygen, resulting in the enhancement of pressure in the closed vial and which can be detected by DPG easily. Under the optimized conditions, the read out signal from DPG had a direct relationship with AFP concentrations in the range of 10~200 ng/mL, and the detection limit was as low as 3.4 ng/mL. The proposed portable sensor had been successfully applied to detect AFP in serum samples with satisfactory results. This strategy holds a great promising in biological analysis as its convenient operations, reliable results and flexible apparatus.

Highly sensitive colorimetric immunosensor for influenza virus H5N1 based on enzyme-encapsulated liposome.

Development of simple but sensitive biosensor for influenza detection is highly important in immediate and effective clinical treatment. In this study, a sensitive colorimetric immunosensor which combines the advantages of high selectivity of immunoassay and simplicity of colorimetric detection has been developed to detect influenza virus H5N1 based on enzyme-encapsulated liposome. Biotin-tagged liposome encapsulated with large amount of horseradish peroxidase (HRP) was firstly synthesized. In the presence of H5N1, H5N1 co-bound with the capture antibody and the biotinylated detection antibody to form sandwich immunocomplex. Subsequently, the HRP-encapsulated liposome was introduced to conjugate with the detection antibody through biotin-avidin-biotin linkage. Upon the addition of substrate (mixture of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2), the liposome was directly lysed to release large amount of HRP by TMB. The released HRP catalyzed the H2O2-mediated oxidation of TMB, resulting in color change of the system, which was observed by naked eyes or UV-vis spectra. The result showed that the absorption intensity enhanced with the increase of H5N1 concentration ranging from 0.1 to 4.0 ng/mL, and the detection limit was calculated to be 0.04 ng/mL. The sensitivity of the proposed biosensor is much higher than that of conventional enzyme-linked immunosorbent assay method. The proposed immunosensor is relatively simple, low-cost, sensitive, and selective without using any sophisticated instruments, therefore it may have a promising prospect for detecting targets in clinical medicine, food safety analysis, and environmental monitoring.