Evaluating early apoptosis-related cellular MiRNAs with an ultrasensitive electrochemiluminescence nanoplatform.

It is known that the abnormal expression of specific cellular miRNAs is closely related to cell apoptosis, and so monitoring the level change of these miRNAs can in principle be used to evaluate the process of apoptosis stimulated by drugs. Towards this goal, here we construct an ultrasensitive electrochemiluminescence (ECL) nanoplatform via the target miRNA-triggered immobilization of spherical nucleic acid enzymes (SNAzymes) onto tetrahedral DNA nanostructures on the electrode surface, which catalyzes the luminol-H2O2 reaction to output an ECL signal. This enables the sensitive and specific detection of two apoptosis-related miRNAs, miR-21 and miR-133a, with a detection limit of 33 aM. Furthermore, we employed the developed ECL nanoplatform to monitor the levels of these two miRNAs inside cancer cells stimulated by DOX, showing that the level of miR-21 decreases, while that of miR-133a increases in the early apoptotic cells. This difference highlights the distinct roles of the two target miRNAs, where miR-21 promotes the early apoptosis of cancer cells, whereas miR-133a suppresses it, providing new insight into cell physiological processes.

A simple "signal off-on" fluorescence nanoplatform for the label-free quantification of exosome-derived microRNA-21 in lung cancer plasma.

A simple nanoplatform based on molybdenum disulfide (MoS2) nanosheets, a fluorescence quencher (signal off), and a hybridization chain reaction (HCR) signal amplification (signal on) used for the enzyme-free, label-free, and low-background signal quantification of microRNA-21 in plasma exosome is reported. According to the sequence of microRNA-21, carboxy-fluorescein (FAM)-labeled hybridization probe 1 (FAM-H1) and hybridization probes 2 (FAM-H2) were designed with excitation maxima at 488 nm and emission maxima at 518 nm. MoS2 nanosheets could adsorb FAM-H1 and FAM-H2 and quenched their fluorescence signals to reduce the background signal. However, HCR was triggered when microRNA-21 was present. Consequently, HCR products containing a large number of FAM fluorophores can emit a strong fluorescence at 518 nm and could realize the detection of microRNA-21 as low as 6 pmol/L and had a wide linear relation of 0.01-25 nmol/L. This assay has the ability of single-base mismatch recognition and could identify microRNA-21 with high specificity. Most importantly, this approach was successfully applied to the detection of plasma exosomal microRNA-21 in patients with lung cancer, and it is proposed that other targets can also be detected by changing the FAM-H1 and FAM-H2 corresponding to the target sequence. Thus, a novel, hands-on strategy for liquid biopsy was proposed and has a potential application value in the early diagnosis of lung cancer.

Hexagonal boron nitride nanosheet as an effective nanoquencher for the fluorescence detection of microRNA.

Two-dimensional (2D) hexagonal boron nitride nanosheet (h-BNNS) is proposed as an effective nanoquencher for fluorescence detection of biocompatible microRNA. Compared with bulk hexagonal boron nitride (h-BN), the exfoliated ultrathin nanosheet has a narrow band gap and increased conductivity, thus enabling fast electron transfer with this electron acceptor for more effective fluorescence detection of microRNA. Remarkably, using the nanoprobe consisting of h-BNNS and FAM dye-labeled ssDNA, a low detection limit of 2.39 nM is achieved and a rapid fluorescence response is observed compared with previously reported fluorescence sensing materials. More importantly, this sensing system could also distinguish base-mismatched microRNA, suggesting that the proposed sensing platform held excellent selectivity and great promise for application in the detection of nucleotide polymorphism. This work will benefit microRNA-related fundamental research and disease diagnosis.

A One-Two-Three Multifunctional System for Enhanced Imaging and Detection of Intracellular MicroRNA and Chemogene Therapy.

Simultaneous imaging, diagnosis, and therapy can offer an effective strategy for cancer treatment. However, the complex probe design, poor drug release efficiency, and multidrug resistance remain tremendous challenges to cancer treatment. Here, a novel one-two-three system is built for enhanced imaging and detection of miRNA-21 (miR-21) overexpressed in cancer cell and chemogene therapy. The system consists of dual-mode DNA robot nanoprobes assembled by two types of hairpin DNAs and three-way branch DNAs modified on gold nanoparticles, with intercalating anticancer drugs (doxorubicin), into DNA duplex GC base pairs. In the system, via intracellular ATP-accelerated cyclic reaction triggered by miR-21, fluorescence and SERS signals were alternated with DNA structure switch, and the precise SERS detection of miRNA and fluorescence imaging oriented "on-demand" release of two types of anticancer drugs (anti-miR-21 and Dox) are achieved. Thus, "one-two-three" means one kind of miR-21-triggered endogenous substance accelerated cyclic reaction, two modes of signal switch, and three functions including enhanced imaging, detection, and comprehensive treatment. The one-two-three system has some notable merits. First, ATP as an endogenous substance promotes DNA structure switching and accelerates the cyclic reaction. Second, the treatment with a dual-mode signal switch is more reliable and accurate and can provide more abundant information than a single-mode treatment platform. Thus, the imaging and detection of intracellular miRNA and effective comprehensive therapy are realized. In vivo results indicate that the system can provide new insights and strategies for diagnosis and therapy.

Electrochemiluminescence from a biocatalysis accelerated N-(aminobutyl)-N-(ethylisoluminol)/dissolved O2 system for microRNA detection.

A kind of biocatalyst, laccase, has been employed as a biocompatible coreactant accelerator to efficiently catalyze coreactant (dissolved O2) for generating high local concentration of superoxide radical (O2•-), acquiring high-intense electrochemiluminescence (ECL) emission of ABEI (N-(aminobutyl)-N-(ethylisoluminol))/dissolved O2 system. Furthermore, a modified strand displacement reaction with excellent amplification efficiency was constructed by replacing traditional single strand DNA to the hairpin DNA as template for triggering the immobilization of more signal probes. As a result, the biosensor for microRNA-21 determination has preeminent selectivity and favorable sensitivity with detection limit down to 80.8 aM. Significantly, the devised strategy has blazed a new path for seeking more coreaction accelerators with splendid biocompatibility thus promoting the application of ternary ECL systems in biological analysis and clinical diagnosis.

Dual-potential electrochemiluminescence of single luminophore for detection of biomarker based on black phosphorus quantum dots as co-reactant.

Simultaneous cathodic and anodic electrochemiluminescence (ECL) emissions of needle-like nanostructures of Ru(bpy)32+ (RuNDs) as the only luminophore are reported based on different co-reactants. Cathodic ECL was attained from RuNDs/K2S2O8 system, while anodic ECL was achieved from RuNDs/black phosphorus quantum dots (BPQDs) system. Ferrocene attached to the hairpin DNA could quench the cathodic and anodic ECL simultaneously. Subsequently, the ECL signals recovered in the presence of tumor marker mucin 1 (MUC1), which made it possible to quantitatively detect MUC1. The variation of ECL signal was related linearly to the concentrations of MUC1 in the range 20 pg mL-1 to 10 ng mL-1, and the detection limits were calculated to 2.5 pg mL-1 (anodic system, 3σ) and 6.2 pg mL-1 (cathodic system, 3σ), respectively. The recoveries were 97.0%, 105%, and 95.2% obtained from three human serum samples, and the relative standard deviation (RSD) is 5.3%. As a proof of concept, this work realized simultaneous ECL emission of  a single luminophore, which initiates a new thought in biomarker ECL detection beyond the traditional ones. Simultaneous cathodic and anodic ECL emissions of RuNDs were reported based on different co-reactants. Ferrocene could quench the ECL emission in the cathode and the anode simultaneously. Thus, an aptasensor was constructed based on the variation of ECL intensity. As a proof of concept, this work realized simultaneous ECL emission of a single luminophore, which initiates a new thought in biomarker ECL detection beyond the traditional ones by avoiding the false positive signals.

Recent Applications of Carbon Nanomaterials for microRNA Electrochemical Sensing.

MicroRNA (miRNA) is an important tumor marker in the human body, and its early detection has a great influence on the survival rate of patients. Although there are many detection methods for miRNA at present such as northern blotting, real-time quantitative polymerase chain reaction, microarrays, and others, electrochemical biosensors have the advantages of low detection cost, small instrument size, simple operation, non-invasive detection and low consumption of reagents and solvents, and thus they play an important role in the early detection of cancer. In addition, with the development of nanotechnology, nano-biosensors show great potential. The application of various nanomaterials in the development of electrochemical biosensor has greatly improved the detection sensitivity of electrochemical biosensor. Among them, carbon nanomaterials which have unique electrical, optical, physical and chemical properties have attracted increasing attention. In particular, they have a large surface area, good biocompatibility and conductivity. Therefore, carbon nanomaterials combined with electrochemical methods can be used to detect miRNA quickly, easily and sensitively. In this review, we systematically review recent applications of different carbon nanomaterials (carbon nanotubes, graphene and its derivatives, graphitic carbon nitride, carbon dots, graphene quantum dots and other carbon nanomaterials) for miRNA electrochemical detection. In addition, we demonstrate the future prospects of electrochemical biosensors modified by carbon nanomaterials for the detection of miRNAs, and some suggestions for their development in the near future.

Electrochemical biosensor for detection of MON89788 gene fragments with spiny trisoctahedron gold nanocrystal and target DNA recycling amplification.

The shape-controlled synthesis of gold nanocrystals via shape induction of hexadecyltrimethylammonium chloride, potassium bromide, and potassium iodide and enantioselective direction of L-cysteine is reported. The resulting gold nanocrystals (STO-Au) offer spiny trisoctahedron nanostructures with good monodispersity and enhanced exposed high-index facets and high catalytic activity. Construction of the electrochemical sensing platform for MON89788 gene involves the modification of STO-Au, thionine (Thi), and labeled bipedal DNA probe 1 or 2 (P1 or P2) for target DNA-induced recycling amplification. In the detection, two surface DNA probes were immobilized on gold electrode via the Au-S bond. Then, hairpin DNA 1 (H1), Thi-STO-Au-P1, and Thi-STO-Au-P2 self-assemble into two-dimensional DNA nanopores (DNPs) on the electrode surface. Target DNA hybridizes with hairpin DNA 2 (H2) to open hairpin structure of H2. The opened H2 binds with H1 in the DNPs to release Thi-STO-Au-P1, Thi-STO-Au-P2, and target DNA by toehold-mediated strand-displacement. The utilization of target DNA-induced recycling allows one target DNA to release 2N STO-Au-labeled DNA strands, promoting significant signal amplification. The detection signal is further enhanced by the catalyzed redox reaction of Thi with STO-Au. The differential pulse voltammetric signal, best measured at - 0.18 V vs. Ag/AgCl, decreases linearly with increasing concentration of MON89788 in the range 0.02-8 × 104 fM, and the detection limit is 0.0048 fM (S/N = 3). The proposed method was successfully applied for electrochemical detection of MON89788 gene fragments in the PCR products from genetically modified soybean. Graphical Abstract We develop l-cysteine controlled synthesis of spiny trisoctahedron gold nanocrystals with good monodispersity and highly exposed high-index facets. The architecture achieves to ultrahigh catalytic activity. The electrochemical biosensor based on gold nanocrystals and target DNA recycling amplification provides advantage of sensitivity, repeatability, and regeneration-free.

Experimental and theoretical study for miR-155 detection through resveratrol interaction with nucleic acids using magnetic core-shell nanoparticles.

A novel electrochemical nanobiosensor for the detection of miR-155 (as breast cancer biomarker) is introduced . Fe3O4NPs@Ag core-shell nanoparticles were synthesized and their shape and characteristics were confirmed by scanning electron microscope (SEM) imaging, Fourier-transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) methods. Synthesized nanoparticles were applied onto the magnetic bar carbon paste electrode and then the amine-modified anti-miR-155 (single-stranded probes) was applied on the modified electrode surface and upon hybridization with target miR-155, resveratrol (RSV) was eventually applied as an electrochemical label on the double-strand oligonucleotide. Differential pulse voltammetry (DPV) of the oxidation peak of RSV was assumed as the final signal by sweeping potential from 0 to 0.6 V (vs. Ag/AgCl). The fabrication process was optimized through a series of experiments and the optimized process was confirmed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The linear range of the fabricated nanobiosensor was 0.5 fM to 1.0 nM and the detection limit was 0.15 fM. The nanobiosensor was able to pass reproducibility and specificity tests using different types of mismatched target sequences.Spiked real samples of human serum were used to confirm that the nanobiosensor enables detection of miR-155 without any significant interferences from other moieties and molecules. Finally, the molecular dynamics simulation of the RSV interaction with single- and double-stranded oligonucleotide was performed and confirmed the preferential binding of RSV to double-stranded DNA; therefore, it can be used as the electrochemical label of DNA and/or miRNA hybridization-based biosensors. Graphical abstract.

Determination of miRNAs in serum of cancer patients with a label- and enzyme-free voltammetric biosensor in a single 30-min step.

The preparation of an integrated biosensor for the easy, fast, and sensitive determination of miRNAs is described based on a direct hybridization format and a label-free voltammetric detection. The biosensor involves a disposable carbon electrode substrate doubly nanostructured with reduced graphene oxide (rGO) and AuNPs modified with pyrene carboxylic acid (PCA) and 6-ferrocenylhexanethiol (Fc-SH), respectively. A synthetic amino terminated DNA capture probe was covalently immobilized on the CO2H moieties of PCA/rGO, while Fc-SH was used as a signaling molecule. Differential pulse voltammetry was employed to record the decrease in the oxidation peak current of Fc after the hybridization due to the hindering of the electron transfer upon the formation of the DNA-RNA duplex on the electrode surface. The stepwise biosensor preparation was characterized by surface and electrochemical techniques showing the role played by each biosensor component as well as the reliability of the target miRNA determination. The determination of the oncogene miRNA-21 synthetic target allowed quantification in the low femtomolar range (LOD of 5 fM) with a high discrimination of single-base mismatched sequences in a single 30-min incubation step. The bioplatform allowed the determination of the target miRNA in a small amount of total RNA extracted from breast cancer (BC) cells or directly in serum samples collected from BC patients without the need for prior extraction, purification, amplification, or reverse transcription of the genetic material and with no matrix effect. Graphical abstract.

Au@Ag core-shell nanoparticles for microRNA-21 determination based on duplex-specific nuclease signal amplification and surface-enhanced Raman scattering.

A novel surface-enhanced Raman scattering (SERS) analysis strategy has been designed combining Au@DTNB@Ag core-shell nanoparticles (DTNB attachment on gold nanoparticles, then encapsulated in Ag shell nanoparticles named as ADANPs) and duplex-specific nuclease signal amplification (DSNSA) platform. Firstly, ADANPs and magnetic substrate of Fe3O4 nanoparticles were covalently attached to the 3'- and 5'- end of capture probe (CP) targeting miRNA-21. Upon the addition of target miRNA-21, these heteroduplexes were specifically cleaved by DSN and resulted in ADANPs that were released from the surface of Fe3O4 nanoparticles (Fe3O4 NPs). At the same time, miRNA-21 remained intact and can rehybridize another DNA probe to trigger the signal-amplifying reaction. Based on this principle, the developed SERS method exhibited good linearity in the range 0 to 1 nM for miRNA-21 with a limit of detection (LOD) of 0.084 fM and has an ability to differentiate even a single-base mismatched sequence on the target sequence or other miRNA sequence. The results provide a novel SERS method which can successfully been applied to the miRNA-21 detection in human serum. Graphical abstract a shows the synthesis of Fe3O4 NPs and the conjugation of Au@DTNB@Ag NPs (ADANPs) for the detection of miRNA-21, b shows the operating principle of DSN-assisted signal amplification strategy for miRNA detection based on Fe3O4@CP@ADA NPs.

Plasmonic nanobiosensors for detection of microRNA cancer biomarkers in clinical samples.

MicroRNAs (miRNAs) play an important role in the regulation of biological processes and have demonstrated great potential as biomarkers for the early detection of various diseases, including esophageal adenocarcinoma (EAC) and Barrett's esophagus (BE), the premalignant metaplasia associated with EAC. Herein, we demonstrate the direct detection of the esophageal cancer biomarker, miR-21, in RNA extracted from 17 endoscopic tissue biopsies using the nanophotonics technology our group has developed, termed the inverse molecular sentinel (iMS) nanobiosensor, with surface-enhanced Raman scattering (SERS) detection. The potential of this label-free, homogeneous biosensor for cancer diagnosis without the need for target amplification was demonstrated by discriminating esophageal cancer and Barrett's esophagus from normal tissue with notable diagnostic accuracy. This work establishes the potential of the iMS nanobiosensor for cancer diagnostics via miRNA detection in clinical samples without the need for target amplification, validating the potential of this assay as part of a new diagnostic strategy. Combining miRNA diagnostics with the nanophotonics technology will result in a paradigm shift in achieving a general molecular analysis tool that has widespread applicability for cancer research as well as detection of cancer. We anticipate further development of this technique for future use in point-of-care testing as an alternative to histopathological diagnosis as our method provides a quick result following RNA isolation, allowing for timely treatment.

Cationic liposome-triggered luminol chemiluminescence reaction and its applications.

Liposomes are spherical phospholipid bilayer vesicles. In the present study, we found that cationic liposomes made by (2,3-dioleoyloxy-propyl)-trimethylammonium (DOTAP) could enhance the luminol-H2O2 chemiluminescence (CL) reaction. Mechanism studies showed that the positive charge on the surface of liposomes plays an important role in the CL process. We speculated that the cationic liposomes with quaternary ammonium groups on the surface may be capable of catalyzing the decomposition of H2O2 leading to the formation of oxygen-related free radicals including ˙OH, 1O2, and O2˙-. The luminol anions tend to move close to the surface of the cationic liposomes and then to be oxidized by the oxidizing radical species which may be around the surface of cationic liposomes forming excited-state 3-aminophthalate* (3-APA*). When the 3-APA* returns to the ground state, an enhanced CL is observed. In addition, the single-strand DNA (ssDNA) showed a significant inhibition effect on the proposed CL reaction. The CL intensity decreased linearly with an increasing amount of DNA from 0.05 to 2 pmol. We assumed that the binding of ssDNA with cationic liposomes would neutralize the positive charge on the surface of liposomes and inhibit the catalytic activity of DOTAP cationic liposomes. Based on the ssDNA-inhibited luminol-H2O2-cationic liposome CL reaction, simple label-free CL sensing platforms were developed for the detection of sequence-specific DNA related to the hepatitis B virus (HBV) gene and for the detection of ATP (as a model analyte) using an anti-ATP aptamer as the recognition element.

Mass spectrometric quantification of microRNAs in biological samples based on multistage signal amplification.

This work describes a novel method for quantification of miRNAs based on multistage signal amplification (MSA) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The multistage signal amplification involves hybridization enrichment of miRNA targets with a DNA probe-magnetic bead conjugate, target recycling amplification with a duplex-specific nuclease, and acid hydrolysis of the reporter molecules producing free nucleobases. Nucleobases thus generated are quantified by LC-ESI-MS/MS with specificity and repeatability. Taking miR-21 as the model target, biological samples such as serum and cell cultures were analyzed by using the present protocol. The analytical results indicate that facile and cost-effective quantifications of miRNA targets can be achieved by using the popular LC-ESI-MS/MS technique, and very importantly, without an isolation of total RNAs from the sample prior to the quantitative assay. The assay for miR-21 detection had a linear calibration curve in the range from 0.2 pM to 0.25 nM with a limit of detection of 60 fM. Analysis of MCF-7 cells treated with toremifene (a potent inhibitor of breast cancer cell growth) revealed that the content of miRNA-21 decreased by ca. 50%, and the decrease was dose-dependent.

LMP1 gene detection using a capped gold nanowire array surface plasmon resonance sensor in a microfluidic chip.

Surface plasmon resonance (SPR) nanowire array chips with a microfluidic system are an effective detection method for a rapid test device. This study investigated a capped gold nanowire array and a microfluidic test platform to provide a fundamental understanding of the kinetic binding of SPR nanowires and the surface gold refractive index. The device sensitivity of the SPR nanowire array was 485 nm RIU-1 and the detection limit was 4.1 × 10-5 RIU. Moreover, a kinetic binding analysis also indicated that a peak shift resulted from a specific hybridization of the target molecule with the immobilized probe on the gold nanostructures. The peak shift (red-shift) value of latent membrane protein 1 (LMP1) DNA was 2.21 nm. The results demonstrated that this new method had high sensitivity to detect amplified DNA products without labeling or complex sample treatment. The SPR nanowire chip can detect the PCR products at lower cycle numbers compared to gel electrophoresis due to probe and DNA specificity. Furthermore, the mechanisms of SPR nanowire array fabrication and the detection of the LMP1 gene were studied. The findings can assist in improving the biosensing of DNA-amplified products and in developing rapid detection devices with a small-footprint nanostructured SPR chip.

Double signal amplification strategy for ultrasensitive electrochemical biosensor based on nuclease and quantum dot-DNA nanocomposites in the detection of breast cancer 1 gene mutation.

Rapid and efficient detection of microRNA (miRNA) of breast cancer 1 gene mutation (BRCA1) at their earliest stages is one of the crucial challenges in cancer diagnostics. In this study, a highly-sensitive electrochemical DNA biosensor was fabricated by double signal amplification (DSA) strategy for the detection of ultra-trace miRNA of BRCA1. In the presence of target miRNA of BRCA1, the well-matched RNA-DNA duplexes were specifically recognized by double-strand specific nuclease (DSN), and the DNA part of the duplexes were then cleaved and miRNAs were released to trigger another following cycle, which produced a primarily amplified signal by such a cyclic enzymatic signal amplification (CESA). Then triple-CdTe quantum dot labelled DNA nanocomposites (3-QD@DNA NC) was selectively hybridized with the cleaved DNA probe on the electrode and produced multiply amplified signals. The biosensor exhibited a high sensitivity for the detection of miRNA of BRCA1 in concentrations ranging from 5 aM to 5 fM, and its detection limit of 1.2 aM was obtained, which is two or three orders of magnitude lower than those by single signal amplification strategy such as CESA or QD-labeled DNA probes. The as-prepared biosensor was successfully used to detect the miRNA of BRCA1 in human serum samples with acceptable stability, good reproducibility, and good recovery. The proposed DNA biosensor based on double signal amplification strategy provided a feasible, rapid, and sensitive platform for early clinical diagnosis and practical applications.

Dark-Field Microwells toward High-Throughput Direct miRNA Sensing with Gold Nanoparticles.

MicroRNA (miRNA) is a class of short RNA that is emerging as an ideal biomarker, as its expression level has been found to correlate with different types of diseases including diabetes and cancer. The detection of miRNA is highly beneficial for early diagnostics and disease monitoring. However, miRNA sensing remains difficult because of its small size and low expression levels. Common techniques such as quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridization and Northern blotting have been developed to quantify miRNA in a given sample. Nevertheless, these methods face common challenges in point-of-care practice as they either require complicated sample handling and expensive equipment, or suffer from low sensitivity. Here we present a new tool based on dark-field microwells to overcome these challenges in miRNA sensing. This miniaturized device enables the readout of a gold nanoparticle assay without the need of a dark-field microscope. We demonstrate the feasibility of the dark-field microwells to detect miRNA in both buffer solution and cell lysate. The dark-field microwells allow affordable miRNA sensing at a high throughput which make them a promising tool for point-of-care diagnostics.

Electrochemical nucleic acid hybridization biosensor based on poly(L-Aspartic acid)-modified electrode for the detection of short oligonucleotide sequences related to hepatitis C virus 1a.

This work describes, for the first time, the fabrication of poly(L-aspartic acid) (PAA) film modified pencil graphite electrode (PGE) for the detection of hepatitis C Virus 1a (HCV1a). The presence of PAA on the electrode surface can provide free carboxyl groups for covalent binding of biomolecules. The PGE surface was first coated with PAA via electropolymerization of the L-aspartic acid, and avidin was subsequently attached to the PAA modified electrode by covalent attachment. Biotinylated HCV1a probes were immobilized on avidin/PAA/PGE via avidin-biotin interaction. The morphology of PAA/PGE was examined using a scanning electron microscope. The hybridization events were monitored with square wave voltammetry using Meldola's blue (MDB). Compared to non-complementary oligonucleotide sequences, when hybridization was carried out between the probe and its synthetic targets or the synthetic polymerase chain reaction analog of HCV1a, the highest MDB signal was observed. The linear range of the biosensor was 12.5 to 100 nM and limit of detection was calculated as 8.7 nM. The biosensor exhibited favorable stability over relatively long-term storage. All these results suggest that PAA-modified electrode can be used to nucleic acid biosensor application and electropolymerization of L-aspartic acid can be considered as a good candidate for the immobilization of biomolecules.

Preparation of DNA-functionalized surfaces for simultaneous homeotropic orientation of liquid crystals and optical recognition of analytes: application to the determination of progesterone.

The work describes a simplified method for the preparation of liquid crystal (LC) bioassay using DNA-based capture molecules and having lower detection limits. The capture DNA probes of the stem-loop structure were immobilized on the surface of a glass slide. A homeotropic orientation of LC molecules can be obtained with the proper surface coverage of capture DNA probes. In the presence of analytes (specifically shown here for the progesterone as a model analyte), the molecular binding between capture DNA probes and progesterone opens the loop of the capture DNA probes. The opened sequence is then amenable to hybridization with a reporter DNA probe that is immobilized on gold nanoparticles. This changes the surface microstructure, disrupts the orientation of LC molecules, and results in an enhanced optical response, expressed as the average grey value of the images. This new kind of surface treatment for simultaneous recognition of target molecules and homeotropic anchoring of LCs reduces the number of preparation steps and makes the process of LC bioassay easier. This method has a detection limit as low as 0.1 pmol·L-1 of progesterone. Graphical abstract Schematic presentation of the liquid crystal-based DNA assay. DMOAP: Dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride; TEA: Triethoxsilylbutyraldehyde; 5CB: 4-cyano-4'-pentylbiphenyl; P4: progesterone.

A highly sensitive and low-background fluorescence assay for pesticides residues based on hybridization chain reaction amplification assisted by magnetic separation.

Due to the concern over food safety, it is important to detect the pesticides residues in agricultural products. Here, a highly sensitive and low background fluorescent strategy for the detection of pesticides residues has been developed. The fluorescence intensity of N-methyl mesoporphyrin IX (NMM) binding G-quadruplex could be turn off because of inhibiting effect of the pesticides on the acetylcholinesterase (AChE) activity. For that, four single-stranded DNAs (named linker, trigger, H1 and H2, respectively) are rational designed and T-Hg-T mismatches duplex DNAs as a recognizer combined with the separation of magnetic beads. The design of hybridization chain reaction (HCR) amplification strategy assisted by magnetic separation has been adopted to improve the detection sensitivity. In the presence of pesticides, the amount of the thiol group generated by hydrolysis reaction of acetylcholine (ACh) is reduced, lead to release of less trigger DNA. Therefor subsequent HCR process is retarded with decreased fluorescence intensity. The reduced fluorescence intensity has a quantitative relationship with the pesticide concentration. The limit of detection of chlorpyrifos was estimated to be 2.0 ng ml-1. It has been applied to detect the pesticides residues in real samples.