Simultaneous detection of cancer biomarkers holds great promise for the early diagnosis of cancer. In the present work, an ultrasensitive and reliable surface-enhanced Raman scattering (SERS) sensor has been developed for simultaneous detection of multiple liver cancer related microRNA (miRNA) biomarkers. We first proposed a novel strategy for the synthesis of nanogap-based SERS nanotags by modifying gold nanoparticles (AuNPs) with thiolated DNA and nonfluorescent small encoding molecules. We also explored a simple approach to a green synthesis of hollow silver microspheres (Ag-HMSs) with bacteria as templates. On the basis of the sandwich hybridization assay, probe DNA-conjugated SERS nanotags used as SERS nanoprobes and capture DNA-conjugated Ag-HMSs used as capture substrates were developed for the detection of target miRNA with a detection limit of 10 fM. Multiplexing capability for simultaneous detection of the three liver cancer related miRNAs with the high sensitivity and specificity was demonstrated using the proposed SERS sensor. Furthermore, the practicability of the SERS sensor was supported by the successful determination of target miRNA in cancer cells. The experimental results indicated that the proposed strategy holds significant potential for multiplex detection of cancer biomarkers and offers the opportunity for future applications in clinical diagnosis.

We have developed a novel method for detecting nicotinamide adenine dinucleotide (NAD+) based on fluorescent silver nanoclusters (AgNCs) stabilized by a dumbbell-shaped DNA template containing two cytosine-loops joined in a dsDNA stem. The design involves two types of components: a dumbbell-shaped DNA template and three enzymes. In the presence of NAD+ as a cofactor, Escherichia coli DNA ligase (E.coli DNA ligase) catalyzes template ligation to generate a sealed (no terminal nucleotides) dumbbell-shaped structure, preventing digestion by exonuclease III (Exo III) and exonuclease I (Exo I). The loop regions of the intact template serve as sites for the deposition of highly fluorescent AgNCs. In the absence of NAD+, the ligation reaction does not occur, and the unsealed dumbbell-shaped template is digested into mononucleotides via cooperation of Exo III and Exo I. The destruction of the DNA template results in the agglomeration of AgNCs into silver nanoparticles with low fluorescence. The fluorescence enhancement depends on the ligation and digestion of the DNA template, allowing quantitative detection of NAD+ in the range of 0.5 nM–5000 nM with a detection limit of ∼0.25 nM.

•A simple method for ultrasensitive detection of Cr(VI) was developed.•A hollow sea urchin-like TiO2@Ag NPs was prepared with high SERS enhancement.•The method has a linearity in the range from 10 nM to 2 μM with a LOD of ~1.45 nM.•The method excellent selectivity and good application in water samples.As one of the most toxic heavy metals, hexavalent chromium (Cr(VI)) has long been a concern due to its threats to human health and the environment. In this work, we develop a sensitive surface-enhanced Raman scattering (SERS) sensor for highly specific detection of Cr(VI) using hollow sea urchin-like TiO2@Ag nanoparticles (NPs). The TiO2@Ag NPs are functionalized with glutathione (GSH) and used as substrates with 2-mercaptopyridine (2-MPy) as a Raman reporter for a recyclable SERS-active sensor, enabling ultrasensitive detection of Cr(VI). Excellent SERS signals of 2-MPy reporters are detected when GSH complexation with Cr(VI) causes aggregation of the TiO2@Ag NPs. The developed sensor exhibits good linearity in the range from 10 nM to 2 μM for Cr(VI) with a detection limit of ca. 1.45 nM. It features excellent selectivity to Cr(VI) over other interfering metal ions, and good application for quantitative analysis of Cr(VI) in water samples. Moreover, the proposed SERS sensor can be fully regenerated when exposed to UV light as a result of the self-cleaning ability of the substrates. In contrast to the traditional SERS detection, the present work shed new light on the design and synthesis of hierarchically self-assembled 3D substrate for SERS, catalysis and biosensor development.

•The large size of magnetic nanoparticles is easier to preparation and dispersion.•Cells coated by large Fe3O4@NH2 NPs (230 nm) yield better performance in phthalates degradation than small particles.•Cells coated by Fe3O4@NH2 NPs had extensive pH, temperature range and high tolerance towards hazardous substances.Phthalate acid esters (PAEs) are widely present in a variety of consumer products and they pose a public health concern due to the high mobility and hazards. In this study, we designed an immobilized method to eliminate phthalates by using Fe3O4@NH2 nanoparticles (NPs). The surface-modified NPs showed a good dispersion property, and the particle size was approximately 230 nm. The results showed that Acinetobacter sp. strain LMB-5 could be coated by the prepared magnetic particles allowing easy separation from solution by applying an external magnetic field. The coated cells possessed adaptability to a wide range of pH and high tolerance towards toxic substances. More importantly, when the temperature was elevated to 60 °C, the coated cells also exhibited remarkable degradability. There was no change in the main metabolites during the biodegradation between the free cells and immobilized ones through GC/MS analysis to detect the degradation intermediates. These results show that immobilization method using magnetic nanoparticles is a promising way for degradation of phthalates in application.

We present a new copper-mediated on–off switch for detecting either pyrophosphate (PPi) or alkaline phosphatase (ALP) based on DNA-scaffolded silver nanoclusters (DNA/AgNCs) templated by a single-stranded sequence containing a 15-nt polythymine spacer between two different emitters. The switch is based on three favorable properties: the quenching ability of Cu2+ for DNA/AgNCs with excitation at 550 nm; the strong binding capacity of Cu2+ and PPi; and the ability of ALP to transform PPi into orthophosphate (Pi). The change in fluorescence of DNA/AgNCs depends on the concentrations of Cu2+, PPi, and ALP. Copper(II) acts as a mediator to interact specifically with the Probe, while PPi and ALP convert the signal of the Probe by removing and recovering Cu2+, operating as an on–off switch. In the presence of Cu2+ only, DNA/AgNCs exhibit low fluorescence because the combination of Cu2+ and DNA template disturbs the precise formation of DNA/AgNCs. When PPi is added to the system containing Cu2+, free DNA template is obtained due to the stronger interaction of PPi and Cu2+, leading to a significant fluorescence increase (ON state) which depends on the concentration of PPi. Further addition of ALP results in the release of free Cu2+ via ALP-catalysis of hydrolysis of PPi into Pi, thereby returning the system to the low fluorescence OFF state. The switch allows the analysis of either PPi or ALP by observation of the fluorescence status, with the detection limit of 112.69 nM and 0.005 U/mL for PPi and ALP, respectively. The AgNCs on–off switch provides the advantages of simple design, convenient operation, and low experimental cost without need of chemical modification, organic dyes, or separation procedures.

It is well-known that proximity-dependent probes containing an analyte recognization site and a signal formation domain could be assembled specifically into a sandwich-like structure (probe–analyte–probe) via introducing an analyte. In this work, using the design for zirconium ion (Zr4+) detection as the model, we develop a novel and reliable proximity-dependent DNA-scaffolded silver nanocluster (DNA/AgNC) probe for Zr4+ detection via target-induced emitter proximity. The proposed strategy undergoes the two following processes: target-mediated emitter pair proximity as target recognition implement and the synthesis of DNA/AgNCs with fluorescence as a signal reporter. Upon combination of the rationally designed probe with Zr4+, the intact templates were obtained according to the –PO32−−Zr4+−PO32−– pattern. The resultant structure with an emitter pair serves as a potent template to achieve highly fluorescent DNA/AgNCs. To verify the universality of the proposed proximity-dependent DNA/AgNC probe, we extend the application of the proximity-dependent probe to DNA and adenosine triphosphate (ATP) detection by virtue of a specific DNA complementary sequence and ATP aptamer as a recognition unit, respectively. The produced fluorescence enhancement of the DNA/AgNCs in response to the analyte concentration allows a quantitative evaluation of the target, including Zr4+, DNA, and ATP with detection limits of ∼3.00 μM, ∼9.83 nM, and ∼0.81 mM, respectively. The proposed probe possesses good performance with simple operation, cost-effectiveness, good selectivity, and without separation procedures.

Lysine acylation is a dynamic, reversible post-translational modification that can regulate cellular and organismal metabolism in bacteria. Acetylome has been studied well in bacteria. However, to our knowledge, there are no proteomic data on the lysine malonylation in prokaryotes, especially in actinomycetes, which are the major producers of therapeutic antibiotics. In our study, the first malonylome of the erythromycin-producing Saccharopolyspora erythraea was described by using a high-resolution mass spectrometry-based proteomics approach and high-affinity antimalonyllysine antibodies. We identified 192 malonylated sites on 132 substrates. Malonylated proteins are enriched in many biological processes such as protein synthesis, glycolysis and gluconeogenesis, the TCA cycle, and the feeder metabolic pathways of erythromycin synthesis according to GO analysis and KEGG pathway analysis. A total of 238 S/T/Y/H-phosphorylated sites on 158 proteins were also identified in our study, which aimed to explore the potential cross-talk between acylation and phosphorylation. After that, site-specific mutations showed that malonylation is a negative regulatory modification on the enzymatic activity of the acetyl–CoA synthetase (Acs) and glutamine synthetase (Gs). Furthermore, we compared the malonylation levels of the two-growth state to explore the potential effect of malonylation on the erythromycin biosynthesis. These findings expand our current knowledge of the actinomycetes malonylome and supplement the acylproteome databases of the whole bacteria.

Iron is essential to microorganisms for its important biological function but could be highly toxic in excess. We have used genome-wide transcriptional analysis in Fe3+-treated (4 mM) Bacillus subtilis to reveal the effect of excess Fe3+ on B. subtilis and characterized the potential pathways involved in Fe3+ stress tolerance. A total of 366 and 400 genes were identified as significantly up-regulated and down-regulated, respectively. We found excess Fe3+ had four major influences on B. subtilis: Fe3+ resulted in oxidative stress and induced genes involved in oxidative stress resistance including the SigB-regulated genes, but the PerR regulon was not inducible in Fe3+-mediated oxidative stress except zosA; Fe3+ significantly disturbed homeostasis of Mn2+ and Zn2+, and the mechanism was proposed in this article; the acidity of Fe3+-induced genes involved in acid consuming and production of bases and shifted B. subtilis to carbon starvation state; Fe3+-induced genes related to membrane remodeling (bkd operon), which prevents Fe3+’s incorporation to membrane lipids. Moreover, Fe3+ repressed the stringent control response, consistent with the induction of stringent control in iron limitation, demonstrating that iron might be a signal in stringent control of B. subtilis. This study was the first to provide a comprehensive overview of the genetic response of B. subtilis to ecxess Fe3+.

In cells of all domains of life, reversible lysine acetylation modulates the function of proteins involved in central cellular processes such as metabolism. In this study, we demonstrate that the nitrogen regulator GlnR of the actinomycete Saccharopolyspora erythraea directly regulates transcription of the acuA gene (SACE_5148), which encodes a Gcn5-type lysine acetyltransferase. We found that AcuA acetylates two glutamine synthetases (GlnA1 and GlnA4) and that this lysine acetylation inactivated GlnA4 (GSII) but had no significant effect on GlnA1 (GSI-β) activity under the conditions tested. Instead, acetylation of GlnA1 led to a gain-of-function that modulated its interaction with the GlnR regulator and enhanced GlnR–DNA binding. It was observed that this regulatory function of acetylated GSI-β enzymes is highly conserved across actinomycetes. In turn, GlnR controls the catalytic and regulatory activities (intracellular acetylation levels) of glutamine synthetases at the transcriptional and posttranslational levels, indicating an autofeedback loop that regulates nitrogen metabolism in response to environmental change. Thus, this GlnR-mediated acetylation pathway provides a signaling cascade that acts from nutrient sensing to acetylation of proteins to feedback regulation. This work presents significant new insights at the molecular level into the mechanisms underlying the regulation of protein acetylation and nitrogen metabolism in actinomycetes.

We have developed a label-free method for sequence-specific DNA detection based on surface plasmon enhanced energy transfer (SPEET) process between fluorescent DNA/AgNC string and gold nanoparticles (AuNPs). DNA/AgNC string, prepared by a single-stranded DNA template encoded two emitter-nucleation sequences at its termini and an oligo spacer in the middle, was rationally designed to produce bright fluorescence emission. The proposed method takes advantage of two strategies. The first one is the difference in binding properties of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) toward AuNPs. The second one is SPEET process between fluorescent DNA/AgNC string and AuNPs, in which fluorescent DNA/AgNC string can be spontaneously adsorbed onto the surface of AuNPs and correspondingly AuNPs serve as “nanoquencher” to quench the fluorescence of DNA/AgNC string. In the presence of target DNA, the sensing probe hybridized with target DNA to form duplex DNA, leading to a salt-induced AuNP aggregation and subsequently weakened SPEET process between fluorescent DNA/AgNC string and AuNPs. A red-to-blue color change of AuNPs and a concomitant fluorescence increase were clearly observed in the sensing system, which had a concentration dependent manner with specific DNA. The proposed method achieved a detection limit of ∼2.5 nM, offering the following merits of simple design, convenient operation, and low experimental cost because of no chemical modification, organic dye, enzymatic reaction, or separation procedure involved.Keywords: DNA detection; fluorescence enhancement; fluorescent silver nanoclusters; gold nanoparticle; surface plasmon enhanced energy transfer;

We developed a novel colorimetric method for rapid detection of biogenic amines based on arylalkylamine N-acetyltransferase (aaNAT). The proposed method offers distinct advantages including simple handling, high speed, low cost, good sensitivity and selectivity.

Developing simple and rapid methods for sequence-specific microRNA (miRNA) analysis is imperative to the miRNA study and use in clinical diagnosis. We have developed a colorimetric method for miRNA detection based on duplex-specific nuclease (DSN)-assisted signal amplification coupled to the aggregation of gold nanoparticles (AuNPs). The proposed method involves two processes: target-mediated probe digestion by a DSN enzyme and probe-triggered AuNP aggregation as a switch for signal output. The reaction system consists of a rationally designed probe complex formed by two partly complementary DNA probes, and two sets of different oligonucleotide-modified AuNPs with sequences complementary to a DNA probe in the probe complex. In the presence of target miRNA, the probe complex is invaded, resulting in the formation of a miRNA-probe heteroduplex as the substrate of the DSN enzyme, and releasing the other probe to link to the AuNPs. The proposed method allows quantitative detection of miR-122 in the range of 20 pM to 1 nM with a detection limit of ∼16 pM, and shows an excellent ability to discriminate single-base differences. Moreover, the detection assay can be applied to accurately quantify miR-122 in cancerous cell lysates which is in excellent agreement with the results from a commercial miRNA detection kit. This method is simple, cost-effective, highly selective, and free of dye label and separation procedures.

Developing label-free molecular beacon (MB)-based methods for DNA detection has been of great significance in bioanalysis because of their simplicity, low cost, and specificity. In this work, we have developed a novel DNA-scaffolded silver nanocluster linear molecular beacon (AgNC-LMB)-based strategy for sequence-specific DNA detection via exonuclease III (Exo III)-assisted signal amplification. The proposed method involves two processes: target-mediated digestion by Exo III and the synthesis of AgNC-LMB as a switch for signal output. Upon hybridization of the rationally designed probe with target, Exo III, removes nucleotides from 3′ terminus of the probe. The resultant fragment acts as a favorable template to form highly fluorescent DNA/AgNCs. The resulting fluorescence enhancement of the AgNCs provides a quantitative readout proportional to the target concentration in the range of 5 to 300 nM with an LOD of ∼3.2 nM. This method is simple, cost-effective, highly selective, and free of modification or separation.

We have developed a novel type of intensely fluorescent DNA-templated silver nanoclusters (DNA/AgNCs), which is in the form of the intergrowth of a Ag emitter pair. In a representative mode, the Ag emitter pair with enhanced fluorescence was prepared by a single-stranded DNA template containing emitter-nucleation sequences at its termini and a spacer in the middle. Our work explicitly shows that the spacer length controls the fluorescence intensity of the forming Ag emitter pair, the spacer composition confers the secondary structure of the DNA template to express the optical and morphological properties of the Ag emitter pair, and the combination of the Ag emitter pair plays a significant role in the formation of fluorescent Ag emitter pair intergrowth. Notably, the produced Ag emitter pair exhibits superior luminescent features with a quantum yield of ∼16.3%, high fluorescence enhancement ratio of ∼850 fold, and rapid synthesis within ∼15 min. We have identified two processes of plasmonic resonance and ligand-to-metal–metal charge transfer (LMMCT) which are responsible for the production of the enhanced fluorescence in the Ag emitter pair in the intergrowth state. These findings offer new insight into the experimental and theoretical research of luminescent silver nanomaterials, which in our view render broad applicability of AgNCs for sensing and other applications.

•We presented a new signal amplification strategy for DNA detection.•The method is based on isothermal self-amplification ability of T7 RNA polymerase to produce targets-like molecules.•The method keeps the advantages of simplicity, rapidity, and specificity stemmed from MB, and stability of enzymatic reaction.Developing molecular beacon (MB)-based method for DNA detection has been of great interest to many researchers because of its intrinsic advantages of simplicity, rapidity, and specificity. In this work, we have developed a novel MB-based method for isothermal detection of sequence-specific DNA via T7 RNA polymerase-aided target regeneration strategy. The proposed method involves three primary processes of target-mediated ligation by T4 DNA ligase, transcription reaction by T7 RNA polymerase, and MB switch for signal output. Upon the hybridization with DNA target, a rationally designed MB and a pair of primers encoded with T7 promoter sequence were ligated via the formation of a phosphodiester bond by T4 DNA ligase. The resultant joint fragment acted as template to initiate T7 RNA polymerase-mediated transcription reaction. Correspondingly, a great amount of RNA strands complementary to MB and partial primers were transcribed to initiate new cyclic reactions of MB switch, ligation, and transcription. With such signal amplification strategy of the regeneration of target-like RNA fragments, our proposed assay achieved a detection limit as low as ∼10 pM, which was ∼3 orders of magnitude lower than the traditional MB-based method with a recognition mechanism in 1:1 stoichiometric ratio between MB and target molecule.

•We presented a Z-folding paper microfluidic with flow control for pathogen detection.•The device can be easily fabricated via wax printing and applied by twice folding.•The device used for ATP and Salmonella detection with detect limit of 1 μM and 2.6×107 CFU/mL.A cost-effective microfluidic paper analysis device (μPAD) was developed with a special Z-folding design for controlling the fluidic flowing and substrate transportation. This presented μPAD can be easily fabricated through wax printing by using a solid ink printer which deposits wax onto the surface of a chromatographic paper, and then baked on a hotplate by penetrating the molten wax into the paper to create a hydrophobic barrier. After μPAD fabrication, liquid control and substrate transportation can be easily carried out by twice folding the μPAD following Z shape. The Z folding made two separated reagent holding zone connected while the detection reaction occurred with the connection. In this paper, a pathogens detection indicated by ATP quantification was took as a proof-in-principle application of using this presented μPAD, the limit of detection (LOD) was 1 μM for ATP detection and 2.6×107 CFU/mL for Salmonella live cell detection, which showed a great potential for Point-of-Care Testing (POCT) applications.

•We presented a low-cost paper microfluidic for different type of chemical detection.•The device used for simultaneous multiplex determination of metal ions and antibiotic.•The device can be easily fabricated and the detection processes are very simple and fast.It is difficult to carry out multiple detection of different type of chemicals because of the different chemical microenvironment requirements. Herein, a low-cost and simple paper-based microfluidic device integrated with fluorescence labeled single-stranded DNA (ssDNA) functionalized graphene oxide sensor was developed for the multiplex determination of different types of chemical contaminants in food. In this work, Cy5 labeled corresponding functional ssDNA for different analytes associated with graphene oxide (ssDNA-GO) were employed as core detection sensors to sensitively report the presence of the different type of chemicals as well as enlarge the chemical compatibility, which made it possible to simultaneous detect multiple chemicals under a same chemical microenvironment. Paper microfluidic device can be easily fabricated and paper substrate also facilitated the integration of ssDNA-GO sensors via physical absorption. This device has been successfully applied in multiplex detection of heavy metal mercury (II) ion (Hg2+) and silver (I) ion (Ag+) and aminoglycoside antibiotics residues in food. It also provided enormous potential for applications of environmental monitoring and clinical diagnosis.

The regulatory mechanisms underlying the uptake and utilization of multiple types of carbohydrates in actinomycetes remain poorly understood. In this study, we show that GlnR (central regulator of nitrogen metabolism) serves as a universal regulator of nitrogen metabolism and plays an important, previously unknown role in controlling the transport of non-phosphotransferase-system (PTS) carbon sources in actinomycetes. It was observed that GlnR can directly interact with the promoters of most (13 of 20) carbohydrate ATP-binding cassette (ABC) transporter loci and can activate the transcription of these genes in response to nitrogen availability in industrial, erythromycin-producing Saccharopolyspora erythraea. Deletion of the glnR gene resulted in severe growth retardation under the culture conditions used, with select ABC-transported carbohydrates (maltose, sorbitol, mannitol, cellobiose, trehalose, or mannose) used as the sole carbon source. Furthermore, we found that GlnR-mediated regulation of carbohydrate transport was highly conserved in actinomycetes. These results demonstrate that GlnR serves a role beyond nitrogen metabolism, mediating critical functions in carbon metabolism and crosstalk of nitrogen- and carbon-metabolism pathways in response to the nutritional states of cells. These findings provide insights into the molecular regulation of transport and metabolism of non-PTS carbohydrates and reveal potential applications for the cofermentation of biomass-derived sugars in the production of biofuels and bio-based chemicals.

Via a molecular caliper p19 protein, we have developed an amplified fluorescence polarization method for rapid microRNA detection. This proposed assay has several intrinsic features including rapidity, simplicity, and accuracy.

By fluorescence enhancement of a proximity-dependent DNA-scaffolded silver nanocluster probe pair and exonuclease III-mediated signal amplification, we present a new fluorescence turn-on mode and its application for specific DNA detection.

The ligation-mediated polymerase chain reaction (PCR) method is widely applied for detecting short-length DNA target. The primary principle of this method is based on the linkage of two separated DNA probes as PCR templates via simultaneous hybridization with DNA target by DNA ligase. Even before taking into account low ligation efficiency, a 1:1 stoichiometric ratio between DNA target and the produced PCR template would put an intrinsic limitation on the detection sensitivity. In order to solve this problem, we have developed an improved ligation-mediated PCR method. It is designed such that a transcription reaction by T7 RNA polymerase is integrated into the ligation reaction. In this way, the produced joint DNA strand composed by two DNA probes can be used as a template both in the transcription reaction and the following PCR process. Then a great number of RNA strands containing the same sequence as DNA target are transcribed to act as a target to initiate new cyclic reactions of ligation and transcription. The results indicate that our proposed method can improve the detection sensitivity by ∼2 orders of magnitude compared with the conventional ligation-mediated PCR method.

A simple and facile approach was developed for monitoring EcoRI endonuclease activity and inhibition, in which a hairpin-like DNA containing restriction cutting site for EcoRI endonuclease acts as the sensing element and Hoechst dyes as the signal indicator in a label-free format.

A label-free fluorescence assay was developed for light-up detection of ATP in a visual-readout format based on the aptamer-directing fluorescence of Hoechst dyes.

•We generated a novel fluorescent sensor for 2-oxoglutarate detection.•This sensor exhibited great sensitivity, selectivity, and kinetics.•2OG dynamics in E. coli cells were reported in real time with this sensor.2-Oxoglutarate (2OG) is an important currency stands at the crossroad between carbon and nitrogen metabolism. Recent research found that 2OG acts as a signal in the regulation of nitrogen metabolism in prokaryote. While in eukaryotic cells, 2OG is also attractive since tricarboxylic acid cycle (TCA cycle) in tumor cells was found to undergo metabolic alterations such as the Warburg effect. A method of tracing this key metabolite 2OG at the cellular level is highly desirable. In order to visualize and monitor 2-oxoglutarate metabolism in single living cells, we developed a novel sensor by inserting the functional 2OG-binding domain GAF of the NifA protein into YFP. This sensor was found to be highly specific to 2OG. Following binding of 2OG, fluorescence intensity of the sensor increased with increasing 2OG concentration and reached a 1.5-fold maximum fluorescence signal change (F/F0−1), kinetics of fluorescence signal upon 2OG association with sensor was fast, the dynamic response range of the mOGsor sensors was 100 µM–100 mM. Dissociation between sensor and 2OG was verified both in vitro and in vivo. This sensor reported cellular 2OG dynamics in E. coli cells in real time upon different nutrition challenges and manifested the differences in 2OG pool accumulation and consumption rate.

Quorum sensing (QS) is involved in many important biological functions such as luminescence, antibiotic production, and biofilm formation. The autoinducer N-(3-oxo-hexanoyl)-l-homoserine lactone (3OC6HSL) plays a significant role in the QS system of the marine bacterium Vibrio fischeri. Tracing 3OC6HSL would be significant in studies related to QS signal transduction. Traditional detection of QS signaling molecules has relied on bacterial reporter strains and high-performance liquid chromatography, which are time consuming and have low sensitivity. Because 3OC6HSL binding to LuxR from V. fischeri causes a conformational change, we developed a genetically encoded biosensor based on Förster resonance energy transfer (FRET) by inserting LuxR between the FRET pair YFP/CFP. The detection limit of the sensor was 100 μM. We attained an optimized sensor with 70 % Δratio increase by screening different hydrophobic linkers, and demonstrated the feasibility of this sensor for visualizing 3OC6HSL both in vitro and in vivo.

In this study, the fluorescence of Hoechst dyes is significantly lit up by addition of our designed MB probe, forming a complex of “molecular beacon”-hosted Hoechst dyes (HMB). Combined with this property, a novel graphene oxide (GO)-based fluorescent “on/off” switch was developed to visually follow bioassay utilizing HMB as signal indicators and GO as an excellent energy acceptor to efficiently quench the fluorescence of HMB in a label-easy format. We have demonstrated its application for label-easy fluorescence “turn on” detection of sequence-specific DNA and “turn off” detection of exonuclease with sensitivity and selectivity in a single step in homogeneous solution. Compared to traditional molecular beacons, the proposed design is cost-effective and simple to prepare without fluorescence labeling or chemical modification.Keywords: biosensing; exonuclease; graphene; Hoechst; molecular beacon;

Via the base-stacking hybridization strategy, we have developed a universal, one-step real-time quantitative PCR assay for sensitive and selective detection of microRNAs. This proposed assay has several intrinsic features including rapid response, low cost, simple handling procedures, etc.

A label-free assay is reported in this work for the detection of DNA with enhanced sensitivity using complex DNA structures (DNA tetrahedrons) based on the biolayer interferometry. The DNA tetrahedrons help to amplify the optical signals of the biolayer interferometry, thus improving the detection limit of DNA by about 100-fold. We further demonstrated that this method could be expanded to ATP detection by taking advantage of the target-dependent adaptability of aptamers. It appears to us that this new label-free assay promises new opportunities for developing novel biolayer interferometry assays.Keywords: aptamer; DNA detection; DNA tetrahedron; label-free; signal amplification, dip-and-read;

A molecular beacon (MB)-like DNA hairpin biosensor based on the reversible directing fluorescence of Hoechst dyes was developed for fluorescent detection of Hg2+ and biothiols and furthermore for logic operation.

In this study, we have developed a novel strategy to highly sensitize the luminescence of terbium(III) (Tb3+) using a designed guanine/thymine-rich DNA (5′-[G3T]5-3′) as an antenna ligand, in which [G3T]5 improved the luminescence of Tb3+ by 3 orders of magnitude due to energy transfer from nucleic acids to Tb3+ (i.e., antenna effect). Furthermore, label-free probes for the luminescent detection of biothiols, Ag+, and sequence-specific DNA in an inexpensive, simple, and mix-and-read format are presented based on the [G3T]5-sensitized luminescence of Tb3+ (GTSLT). The long luminescence lifetime of the probes readily enables time-resolved luminescence (TRL) experiments. Hg2+ can efficiently quench the luminescence of Tb3+ sensitized by [G3T]5 (Tb3+/[G3T]5); however, biothiols are readily applicable to selectively grab Hg2+ for restoration of the luminescence of Tb3+/[G3T]5 initially quenched by Hg2+, which can be used for “turn on” detection of biothiols. With the use of cytosine (C)-rich oligonucleotide c[G3T]5 complementary to [G3T]5, the formed [G3T]5/c[G3T]5 duplex cannot sensitize the luminescence of Tb3+. However, in the presence of Ag+, Ag+ can combine the C base of c[G3T]5 to form C–Ag+–C complexes, leading to the split of the [G3T]5/c[G3T]5 duplex and then release of [G3T]5. The released [G3T]5 acts as an antenna ligand for sensitizing the luminescence of Tb3+. Therefore, the Tb3+/[G3T]5/c[G3T]5 probe can be applied to detect Ag+ in a “turn on” format. Moreover, recognition of target DNA via hybridization to a molecular beacon (MB)-like probe (MB-[G3T]5) can unfold the MB-[G3T]5 to release the [G3T]5 for sensitizing the luminescence of Tb3+, producing a detectable signal directly proportional to the amount of target DNA of interest. This allows the development of a fascinating label-free MB probe for DNA sensing based on the luminescence of Tb3+. Results and methods reported here suggest that a guanine/thymine-rich DNA-sensitized luminescence probe of Tb3+ represents a new opportunity for versatile background-free biosensing applications.

MicroRNA (miRNA) has become an ideal biomarker candidate for cancer diagnosis, prognosis, and therapy. In this study, we have developed a novel one-step method for sensitive and specific miRNA detection via enzymatic signal amplification and demonstrated its practical application in biological samples. The proposed signal amplification strategy is an integrated “biological circuit” designed to initiate a cascade of enzymatic polymerization reactions in order to detect, amplify, and measure a specific miRNA sequence by using the isothermal strand-displacement property of a mesophilic DNA polymerase together with the nicking activity of a restriction endonuclease. The circuit is composed of two molecular switches operating in series: the nicking endonuclease-assisted isothermal polymerization reaction activated by a specific miRNA and the strand-displacement polymerization reaction designed to initiate molecular beacon-assisted amplification and signal transduction. The hsa-miR-141 (miR-141) was chosen as a target miRNA because its level specifically elevates in prostate cancer. The proposed method allowed quantitative sequence-specific detection of miR-141 in a dynamic range from 1 fM to 100 nM, with an excellent ability to discriminate differences among miR-200 family members. Moreover, the detection assay was applied to quantify miR-141 in cancerous cell lysates. The results are in excellent agreement with those from the reverse transcription polymerase chain reaction method. On the basis of these findings, we believe that this proposed sensitive and specific assay has great potential as a miRNA quantification method for use in biomedical research and clinical diagnosis.

A universal silver-nanocluster method coupled with target-triggered isothermal exponential amplification reaction (TIEAR) is developed for light-up fluorescent detection of multiple microRNAs (miRNAs) in a label-free format. Taking advantage of the interesting feature of the fine-tuned emission spectrum of fluorescent DNA-scaffolded silver nanoclusters (DNA/AgNCs), our proposed assay is designed such that the different miRNA targets are transferred to the different oligonucleotide reporters via the TIEAR, in which unimolecular DNAs designed for different targets are employed as the amplification templates, polymerases and nicking enzymes as mechanical activators and miRNA targets as the trigger. The produced oligonucleotide reporters act as templates for the synthesis of multicolor DNA/AgNC probes, which correspond to different target inputs. This proposed method has been well validated on the multiplex detection of miRNAs and DNAs, as well as in practical application.

We have developed a facile and label-free method for sequence-specific microRNA detection using fluorescent dsDNA-templated copper nanoclusters as signal indicators. It is designed such that, the miRNA targets are transferred to the oligonucleotide reporters and act as the scaffold for the synthesis of fluorescent copper nanoclusters via an isothermal exponential amplification reaction, in which the unimolecular DNA designed for miRNA targets is employed as the amplification template with polymerases and nicking enzymes as mechanical activators. Under the selected conditions, the proposed assay allowed sensitive and selective detection of miRNAs with a linear range from 1 pM to 10 nM.

A novel label-free electrochemical impedance spectroscopy (EIS) biosensor for direct cancer cell detection based on the interaction between carbohydrate and lectin has been developed with good sensitivity and selectivity. In the present work, concanavalin A (Con A), a mannose specific lectin, was immobilized on a gold disk electrode to fabricate the Con A sensor. This sensor was incubated with the cancer cell sample, and the binding of cancer cells with Con A resulted in a change of charge transfer resistance (Rct). EIS measurement was employed to measure the impedance change which reveals the concentration of cancer cells. This method has been successfully applied in human liver cancer cell Bel-7404 for direct and sensitive detection with a detection limit of 234 cells/mL. This method could be extended to carry out multi-component diagnosis applications, thus providing enormous potential for applications of cancer monitoring and therapy.Highlights► Label-free electrochemical impedance spectroscopy biosensor for direct detection of cancer cells. ► Practical application for human liver cancer cell Bel-7404 determination with detection limit of 234 cells/mL. ► Enormous potential for applications of cancer monitoring and therapying.

It is well-known that microRNAs (miRNAs) have become an ideal class of biomarker candidates for clinical diagnosis of cancers, thus sensitive and selective detection of microRNAs is of great significance in understanding biological functions of miRNAs, early-phase diagnosis of cancers, as well as discovery of new targets for drugs. In this work, we have developed a sensitive method for microRNAs detection based on isothermal exponential amplification-assisted generation of catalytic G-quadruplex DNAzyme, and demonstrated its practical application in biological sample of cell lysate. The assay involves a combination of polymerase strand extension, single-strand nicking and catalytic reaction of G-quadruplex/hemin complex. It is designed such that, the target miRNA initiates the efficient synthesis of two kinds of short oligonucleotide fragments in the continuous cycle of the polymerization, nicking and displacement reactions, by means of thermostable polymerase and nicking endonuclease. One fragment has the same sequence as the target miRNA, except that the deoxyribonucleotides and thymine replace the ribonucleotides and uridine in the miRNA, to activate new cyclic chain reactions of polymerization, nicking and displacement reactions as the target miRNA. The other is the signal molecule of horseradish peroxidase (HRP)-mimicking G-quadruplex DNAzyme. With such designed signal amplification processes, the proposed assay showed a quantitative analysis of sequence-specific miRNAs in a wide range from 1 fM to 100 nM with a low detection limit of 1 fM. Moreover, this assay demonstrated excellent differentiation ability for the mismatch miRNAs targets and good performance in biological samples.Highlights► This paper presents a signal amplification method for nucleic acid detection. ► The amplification method is based on isothermal exponential amplification-assisted generation of catalytic G-quadruplex DNAzyme.► This paper demonstrates its practical application for microRNAs determination with detect limit of 1.0 fM.

•Novel luminescent gold nanoclusters (AuNCs) were synthesized utilizing vitamin B2 (riboflavin) as stabilizer by a simple, rapid and one-pot green procedure.•The riboflavin-AuNCs (Ri-AuNCs) probe can be used as a ratiometric indicator for Cu2+ with sensitivity, selectivity and even reversibility.•This is a new concept for performance of ratiometric assays demonstrated with a AuNCs-based fluorescent probe, which expands the application of AuNCs.Most of the copper (II) fluorescent probes are based on the measurement of fluorescence at a single wavelength, which may be influenced by variations in the sample environment. To the end, the ratiometric fluorescent measurement, which involves the simultaneous measurement of two fluorescence signals at different wavelengths followed by calculation of their intensity ratio, can effectively eliminate the adverse effects on fluorescence signals and give greater precision to the data analysis relative to single-channel detection. In this work, we prepared novel luminescent gold nanoclusters (AuNCs) utilizing vitamin B2 (riboflavin) as stabilizer by a simple, rapid and one-pot green (low-toxicity materials use) procedure. The as-prepared riboflavin-AuNCs (Ri-AuNCs) solution can be luminescent exhibiting two fluorescence emission peaks at 530 nm and around 840 nm with excitation at 375 nm, however, in the presence of Cu2+, the fluorescence of the Ri-AuNCs was found to be quenched at around 840 nm and enhanced at 530 nm by Cu2+. The resultant ratiometric fluorescent response can provide a novel sensory probe for the determination of Cu2+. The present probe had excellent selectivity in the presence of several cations. The probe revealed a detection limit of 0.9 μM of Cu2+. Moreover, our proposed probe can reversibly switch between the “on” and “off” states through the addition of Cu2+ and EDTA, which is reusable in practical application. Results and method reported here provide a unique strategy for performance of ratiometric assays demonstrated with a AuNCs-based fluorescent probe, which expands the application of AuNCs.

Two genes encoding β-glucosidase from Streptomyces coelicolor A3(2) were cloned and expressed in Escherichia coli BL21 (DE3). Two recombinant enzymes (SC1059 and SC7558) were purified and characterized. The molecular mass of the purified SC1059 and SC7558 as determined by SDS-PAGE agrees with the calculated values (51.0 and 52.2 kDa, respectively). Optimal temperature and pH for the two enzymes were both at 35 °C and 6.0. SC7558 exhibited to be much more active than SC1059 under optimal conditions, and it was recombined with ice nucleation protein which could anchor on the surface of the cell. The optimal temperature and pH of the recombinant cells were 55 °C and 8.0, respectively. The resultant cells were to be used as material for immobilized β-glucosidase, which is convenient to catalyze substrates in various complicated conditions.

Traditional molecular beacons, widely applied for detection of nucleic acids, have an intrinsic limitation on sensitivity, as one target molecule converts only one beacon molecule to its fluorescent form. Herein, we take advantage of the duplex-specific nuclease (DSN) to create a new signal-amplifying mechanism, duplex-specific nuclease signal amplification (DSNSA), to increase the detection sensitivity of molecular beacons (Taqman probes). DSN nuclease is employed to recycle the process of target-assisted digestion of Taqman probes, thus, resulting in a significant fluorescence signal amplification through which one target molecule cleaves thousands of probe molecules. We further demonstrate the efficiency of this DSNSA strategy for rapid direct quantification of multiple miRNAs in biological samples. Our experimental results showed a quantitative measurement of sequence-specific miRNAs with the detection limit in the femtomolar range, nearly 5 orders of magnitude lower than that of conventional molecular beacons. This amplification strategy also demonstrated a high selectivity for discriminating differences between miRNA family members. Considering the superior sensitivity and specificity, as well as the multiplex and simple-to-implement features, this method promises a great potential of becoming a routine tool for simultaneously quantitative analysis of multiple miRNAs in tissues or cells, and supplies valuable information for biomedical research and clinical early diagnosis.

A versatile molecular beacon (MB)-like probe was developed for multiplexed detection based on fluorescence polarization by target-induced allosteric effect and furthermore for resettable logic gate operation.

We have developed a simple and ultrasensitive E-DNA sensor based on the ssDNA-assisted cascade of a hybridization reaction mechanism to form a long concatamers structure to improve its sensitivity, significantly. The proposed sensor was applied to sequence-specific DNA and ATP detection. Experimental results showed a quantitative measurement with the detection limit as low as 1 aM for specific DNA and 10 fM for ATP.

A simple and reliable fluorescent DNA logic gate is developed by utilizing graphene oxide as a signal transducer and mercury ions and iodide as mechanical activators.

We have developed a novel molecular logic gate system based on the incorporation of aptamer-crosslinked hydrogels. Modified gold nanoparticles are used as the output signal, which is visible to the naked eye. This system is designed for AND and OR operations using two chemicals as stimulus inputs.

A simple and reliable fluorescent molecular beacon is developed utilizing DNA-templated silver nanoclusters as a signal indicator and adenosine triphosphate (ATP) and adenosine deaminase as mechanical activators.

MicroRNAs (miRNAs) play vital roles in a plethora of biological and cellular processes. The levels of miRNAs can be useful biomarkers for cellular events or disease diagnosis, thus the method for sensitive and selective detection of miRNAs is imperative to miRNA discovery, study, and clinical diagnosis. Here we develop a novel method to quantify miRNA expression levels as low as attomolar sensitivity by target-assisted isothermal exponential amplification coupled with fluorescent DNA-scaffolded AgNCs and demonstrated its feasibility in the application of detecting miRNA in real samples. The method reveals superior sensitivity with a detection limit of miRNA of 2 aM synthetic spike-in target miRNA under pure conditions (approximately 15 copies of a miRNA molecule in a volume of 10 μL) and can detect at least a 10 aM spike-in target miRNA in cell lysates. The method also shows the high selectivity for discriminating differences between miRNA family members, thus providing a promising alternative to standard approaches for quantitative detection of miRNA. This simple and cost-effective strategy has a potential of becoming the major tool for simultaneous quantitative analysis of multiple miRNAs (biomarkers) in tissues or cells and supplies valuable information for biomedical research and clinical early diagnosis.

A colorimetric assay has been developed for parallel detection of Cd2+, Ni2+ and Co2+ utilizing peptide-modified gold nanoparticles (P-AuNPs) as a sensing element based on its unique surface plasmon resonance properties. The functional peptide ligand, CALNNDHHHHHH, was self-assembled on gold nanoparticles (AuNPs) to produce P-AuNPs probe. The P-AuNPs probe could be used to simultaneously detect and showed different responses to the three ions Cd2+, Ni2+ and Co2+ in an aqueous solution based on the aggregation-induced color change of AuNPs. The method showed good selectivity for Cd2+, Ni2+ and Co2+ over other metal ions, and detection limit as low as 0.05 μM Cd2+, 0.3 μM Ni2+ or 2 μM Co2+. To simultaneously (or parallel) detect the three metal ions coexisting in a sample, EDTA and imidazole were applied to mask Co2+ and Ni2+ for detecting Cd2+, glutathione and EDTA were applied to mask Cd2+ and Co2+ for detecting Ni2+, and glutathione and imidazole were applied to mask Cd2+ and Ni2+ for detecting Co2+. Finally, the simple and cost-effective probe could be successfully applied for simultaneously detecting Cd2+, Ni2+, and Co2+ in river water. Because this novel P-AgNPs-based probe design offers many advantages, including simplicity of preparation and manipulation compared with other methods that employ specific strategies, the sensing system shows potential application in the developing region for monitoring water quality.

As an alternative to antibodies, aptamers have shown promising applications in diagnostics and therapeutics. However, different from antibodies, the chemical nature of nucleic acids allows easy synthesis and modification of aptamers. As a result, there are various feasible ways to engineer aptamers with extended bioavailability (e.g., stability and binding affinity in complex environments), regulating ability, and multi-functional properties. In this review, recent advances in rational design and novel functionalization of aptamers, especially DNA aptamers, is described. The broad spectrum of ways for aptamer engineering and applications is paving the way for the future evolution of bioanalytical and biomedical developments.

A simple and reliable colorimetric method for determination of phthalates is developed utilizing uridine-5′-triphosphate (UTP)-modified gold nanoparticles as a color indicator and Cu2+ as a cross-linker. The method demonstrates superior sensitivity with a detection limit of phthalates of ca. 0.5 ppm.

Single stranded DNA sequences can be detected by target assisted exonuclease III-catalyzed signal amplification fluorescence polarization (TAECA-FP). The method offers an impressive detection limit of 83 aM within one hour for DNA detection and exhibits high discrimination ability even against a single base mismatch.

We have developed a novel graphene-based biosensing platform using peptides as probe biomolecules, and demonstrated its feasibility in the application of real-time monitoring of protease activity based on FRET between GO and dye-labeled peptides. This assay allows the rapid and accurate determination of enzyme kinetic parameters as well as inhibition constants.

Chiral recognition is among the important and special modes of molecular recognition. It is highly desirable to develop a simple, rapid, sensitive, and high-throughput routine assay for chiral recognition. In this study, we demonstrate that nucleotide-capped Ag nanoparticles (AgNPs) can be used as an ultrahigh efficiency enantioseparation and detection platform for d- and l-cysteine. The aggregation of AgNPs is selectively induced by an enantiomer of cysteine, which allowed the rapid colorimetric enantiodiscrimination of cysteine without any prior derivatization and specific instruments and left an excess of the other enantiomer in the solution, thus resulting in enantioseparation. This is the first application of a nucleotide-capped AgNP-based biosensing platform for chiral recognition and opens new opportunities for design of more novel enantiosensing strategies and enantiospecific adsorbents and expansion of its application in different fields.

A colorimetric assay has been developed for the simultaneous selective detection of silver(I) and mercury(II) ions utilizing metal nanoparticles (NPs) as sensing element based on their unique surface plasmon resonance properties. In this method, sulfhydryl group modified cytosine-(C)-rich ssDNA (SH-C-ssDNA) was self-assembled on gold nanoparticles (AuNPs) to produce the AuNPs–C-ssDNA complex, and sulfhydryl group modified thymine-(T)-rich ssDNA (SH-T-ssDNA) was self-assembled on silver nanoparticles (AgNPs) to produce the AgNPs–T-ssDNA complex. Oligonucleotides (SH-C-ssDNA or SH-T-ssDNA) could enhance the AuNPs or AgNPs against salt-induced aggregation. However, the presence of silver(I) ions (Ag+) in the complex of ssDNA–AuNPs would reduce the stability of AuNPs due to the formation of Ag+ mediated C–Ag+–C base pairs accompanied with the AuNPs color change from red to purple or even to dark blue. Moreover, the presence of mercury(II) ions (Hg2+) would also reduce the stability of AgNPs due to the formation of Hg2+ mediated T–Hg2+–T base pairs accompanied with the AgNPs color change from yellow to brown, then to dark purple. The presence of both Ag+ and Hg2+ will reduce the stability of both AuNPs and AgNPs and cause the visible color change. As a result, Ag+ and Hg2+ could be detected qualitatively and quantitatively by the naked eye or by UV-vis spectral measurement. The lowest detectable concentration of a 5 nM mixture of Ag+ and Hg2+ in the river water was gotten by the UV-vis spectral measurement.

A flexible nanoparticle-based sulfate assay is demonstrated in which the positively-charged gold nanoparticles (cysteamine-AuNPs) act as indicator. The aggregation of cysteamine-AuNPs is selectively induced by sulfate, which allows the rapid colorimetric sensing of sulfate without any precipitant, sample preparation and specific instruments. In this work, the cysteamine-AuNPs probe has been successfully applied to the colorimetric detection of sulfate and demonstrates superior sensitivity with a detection limit of sulfate of ∼50 ppb. A surprise finding is that the proposed probe can achieve the goal of real-time monitoring and translating a redox process into an appreciable color change via the aggregation of nanoparticles. This is a novel application of a positively-charged AuNPs-based nanoprobe for sulfate detection, kinetic study of the redox process, and opens up new opportunities for design of more novel colorimetric strategies and expansion of AuNPs-based application in different fields.

Novel luminescent silver nanoclusters (AgNCs) were synthesized utilizing DNA as templates by a simple, rapid and one-pot procedure. Luminescence studies indicated that these DNA-AgNCs exhibited strong emission with peak maximum at 624 nm. The fluorescence of the DNA-AgNCs was found to be quenched by Cu2+ enabling its detection with high sensitivity and selectivity.

The present work demonstrates a rapid, single-step and ultrasensitive label-free and signal-off electrochemical sensor for specific DNA detection with excellent discrimination ability for single-nucleotide polymorphisms, taking advantage of Exonuclease III (Exo III)-aided target recycling strategy to achieve signal amplification. Exo III has a specifical exo-deoxyribonuclease activity for duplex DNAs in the direction from 3′ to 5′ terminus, however its activity on the duplex DNAs with 3′-overhang and single-strand DNA is limited. In response to the specific features of Exo III, the proposed E-DNA sensor is designed such that, in the presence of target DNA, the electrode self-assembled signaling probe hybridizes with the target DNA to form a duplex in the form of a 3′-blunt end at signaling probe and a 3′-overhang end at target DNA. In this way, Exo III specifically recognizes this structure and selectively digests the signaling probe. As a result, the target DNA dissociates from the duplex and recycles to hybridize with a new signaling probe, leading to the digestion of a large amount of signaling probes gradually. A redox mediator, Ru(NH3)63+ (RuHex) is employed to electrostatically adsorbed onto signaling probes, which is directly related to the amount and the length of the signaling probes remaining in the electrode, and provides a quantitative measure of sequence-specific DNA with the experimentally measured (not extrapolated) detection limit as low as 20 fM. Moreover, this E-DNA sensor has an excellent differentiation ability for single mismatches with fairly good stability.

We have designed a versatile molecular beacon (MB)-like probe for the multiplex sensing of targets such as sequence-specific DNA, protein, metal ions and small molecule compounds based on the self-assembled ssDNA–graphene oxide (ssDNA–GO) architecture. The probe employs fluorescence “on/off” switching strategy in a single step in homogeneous solution. Compared to traditional molecular beacons, the proposed design is simple to prepare and manipulate and has little background interference, but still gives superior sensitivity and rapid response. More importantly, this ssDNA–GO architecture can serve as a universal beacon platform by simply changing the types of ssDNA sequences for the different targets. In this work, the ssDNA–GO architecture probe has been successfully applied in the multiplex detection of sequence-specific DNA, thrombin, Ag+, Hg2+ and cysteine, and the limit of detection was 1 nM, 5 nM, 20 nM, 5.7 nM and 60 nM, respectively. The results demonstrate that the ssDNA–GO architecture can be an excellent and versatile platform for sensing multiplex analytes, easily replacing the universal molecular beacon.

The application of comprehensive gene expression profiling technologies to compare wild and mutated microorganism samples or to assess molecular differences between various treatments has been widely used. However, little is known about the normal variation of gene expression in microorganisms. In this study, an Agilent customized microarray representing 4,106 genes was used to quantify transcript levels of five-repeated flasks to assess normal variation in Bacillus subtilis gene expression. CV analysis and analysis of variance were employed to investigate the normal variance of genes and the components of variance, respectively. The results showed that above 80% of the total variation was caused by biological variance. For the 12 replicates, 451 of 4,106 genes exhibited variance with CV values over 10%. The functional category enrichment analysis demonstrated that these variable genes were mainly involved in cell type differentiation, cell type localization, cell cycle and DNA processing, and spore or cyst coat. Using power analysis, the minimal biological replicate number for a B. subtilis microarray experiment was determined to be six. The results contribute to the definition of the baseline level of variability in B. subtilis gene expression and emphasize the importance of replicate microarray experiments.

The estrogenic compound 17β-estradiol (E2) is widely studied for its potential endocrine disruption effects. Due to the low level of E2 present in the environment, it is highly desirable to develop a sensitive and efficient separation and enrichment method for E2 analysis. In this paper, we proposed a novel E2 preconcentration method using anti-E2 aptamer-anchored isothiocyanate-modified beads (NCS beads). The glass beads are chemically modified with primary amino group, and then treated with phenylene diisothiocyanate (PDITC) to generate an isothiocyanate group, which is reactive towards the amine group. The amino-modified anti-E2 aptamer can be easily covalently immobilized onto the as-prepared NCS beads. The experimental results demonstrated that the aptamer affinity microbeads could selectively retain and separate E2 compound. The effects of the operation parameters on retention of E2, including washing condition, eluting condition, the number of beads, and the incubation time were investigated. Moreover, high-performance liquid chromatography with preconcentration of E2 on the aptamer affinity microbeads was applied to detect the E2 in the spiked water samples and obtained a good recovery.

We have developed a novel multiplexed bead-based mesofluidic system (MBMS) based on the specific recognition events on the surface of a series of microbeads (diameter 250 μm) arranged in polydimethylsiloxane (PDMS) microchannels (diameter 300 μm) with the predetermined order and assembled an apparatus implementing automatically the high-throughput bead-based assay and further demonstrated its feasibility and flexibility of gene diagnosis and genotyping, such as β-thalassemia mutation detection and HLA-DQA genotyping. The apparatus, consisting of bead-based mesofluidic PDMS chip, liquid-processing module, and fluorescence detection module, can integrate the procedure of automated-sampling, hybridization reactions, washing, and in situ fluorescence detection. The results revealed that MBMS is fast, has high sensitivity, and can be automated to carry out parallel and multiplexed genotyping and has the potential to open up new routes to flexible, high-throughput approaches for bioanalysis.

Bleomycins (BLMs) are widely used in combination with chemotherapy for the treatment of a variety of cancers. The clinical application of BLMs is featured by the occurrence of sometimes fatal side effects, such as renal and lung toxicity, and the potential dose-limiting side effect of pulmonary fibrosis. Therefore, it is highly desirable to develop a sensitive method to quantitatively determine the BLM content in both pharmaceutical analysis and clinical samples, to make full use of therapeutic efficacy and to weaken its toxicity. Here, we proposed a simple, rapid, and convenient electrochemical assay for trace BLM detection. A reported DNA motif, as substrate for BLMs, is prepared to self-assemble onto the gold electrode to fabricate an electrochemical DNA (E-DNA) sensor, with a terminus tethered on the electrode surface and the other terminus labeled with ferrocenyl moiety as a signal reporter to form a stem-loop structure, giving an arise of remarkable faradaic current. In the presence of Fe(II)·BLM, the E-DNA sensor undergoes the irreversible cleavage event, which can be transduced into a significant decrease in current peak. This proposed sensor reveals an impressive sensitivity as low as 100 pM BLMs and exhibits a good performance as well as in serum sample. Considering the high sensitivity and specificity of this proposed sensor, as well as the cost-effective and simple-to-implement features of the electrochemical technique, we believe that this method shows distinct advantages over conventional methods and it is a promising alternative for the determination of trace amounts of BLMs in clinical samples.

An automated multicomponent mesofluidic system (MCMS) based on biorecognitions carried out on meso-scale glass beads in polydimethylsiloxane (PDMS) channels was developed. The constructed MCMS consisted of five modules: a bead introduction module, a bioreaction module, a solution handling module, a liquid driving module, and a signal collection module. The integration of these modules enables the assay to be automated and reduces it to a one-step protocol. The MCMS has successfully been applied toward the detection of veterinary drug residues in animal-derived foods. The drug antigen-coated beads (ϕϕ250 μm) were arrayed in the PDMS channels (ϕϕ300 μm). The competitive immunoassay was then carried out on the surface of the glass beads. After washing, the Cy3-labeled secondary antibody was introduced to probe the antigen–antibody complex anchored to the beads. The fluorescence intensity of each bead was measured and used to determine the residual drug concentration. The MCMS is highly sensitive, with its detection limits ranging from 0.02 (salbutamol) to 3.5 μg/L (sulfamethazine), and has a short assay time of 45 min or less. The experimental results demonstrate that the MCMS proves to be an economic, efficient, and sensitive platform for multicomponent detection of compound residues for contamination in foods or the environment.

Gold nanoparticles can be exploited to facilitate a highly sensitive and selective metal ion detection based on fluorescence anisotropy assay with metal ion-dependent DNA-cleaving DNAzyme. This assay allows rapid and accurate determination of metal ions in aqueous medium at room temperature. The method has been demonstrated for determination of Cu2+ and Pb2+ ions. The detection sensitivity can be significantly improved to 1 nM by using a “nanoparticle enhancement” approach. Moreover, the assay was also tested in 384-well plates for high-throughput routine determination of toxic metal ions in environmental samples. The method showed distinct advantages over conventional methods in terms of its potential sensitivity, specificity, and ability for rapid response.

The use of highly active β-agonists as growth promoters is not appropriate because of the potential hazard for human and animal health. To investigate the residue level of these β-agonists, hapten microarrays were employed for clenbuterol (CLB), ractopamine (RAC) and salbutamol (SAL) residue analysis. CLB, RAC and SAL conjugates were immobilized on the slides, which were precoated by agarose film to construct hapten microarrays, and then the corresponding monoclonal antibodies of these β-agonists and the standards or samples were introduced for indirect competitive immunoassay. Finally, Cy3-labeled secondary antibody was employed to indicate the antigen–antibody complex. The fluorescence intensity of each spot was imaged and recorded, and the calibration curve of each analyte was obtained by plot fluorescence intensity against different standard concentrations. Compared to the ELISA, the hapten microarray method was more sensitive, which got the detection limits 0.09 μg/L for CLB, 0.50 μg/L for RAC, and 0.01 μg/L for SAL. What's more, with the recovery rate between 96.5% and 106.4%, and the coefficient of variation below 10%, the proposed hapten microarray method was shown to be both quantitative and reproducible.

An effective dual-DNAzyme-based unimolecular probe design employing intramolecular signal transduction is demonstrated. The probe is composed of three domains: a DNA-cleaving DNAzyme, a substrate, and an HRP-mimicking DNAzyme. When the probe meets its target, cleavage of the substrate by the DNA-cleaving DNAzyme activates the HRP-mimicking DNAzyme, producing a colorimetric signal. The Cu2+-dependent DNAzyme engineered to demonstrate this design revealed a sensitivity corresponding to 65 ppb, which is sufficient to detect Cu2+ in drinking water. The new probe has excellent selectivity toward Cu2+. This three-component design is simple and easy to engineer. It may provide the basis for future development of other nucleic acid-based probes for toxicological and environmental monitoring.

We present a novel approach of single-nucleotide polymorphism (SNP) analysis in which allele-specific oligonucleotide hybridization is followed by non-gel capillary electrophoresis (ASOH–NGCE) in conjunction with laser-induced fluorescence (LIF). This allows rapid multiplex allelotyping and allele frequency estimation. This method, based on site separation of the hybridization duplexes, retains the simplicity and specificity of ASOH and the homogeneous feature of NGCE with poly(N,N-dimethylacrylamide) (PDMA) as a sieving medium. ASOH–NGCE can be applied to multiplex SNP loci genotyping with excellent separation of hybridization mixtures. Average relative standard deviations (RSDs) were low for within-day (1.10%) and between-day (2.41%) reproducibility. Moreover, the allele frequencies in pooled DNAs were accurately determined from peak areas and equilibrium dissociation constants. Our method was highly sensitive in detecting alleles with frequency as low as 1% and in distinguishing allele frequencies differing by 1% between pools. The average value of differences between real and estimated frequencies (accuracy) was only 0.004.

A novel microarray for the multiplex determination of heavy metal ions in aqueous solution based on DNAzymes has been developed with good sensitivity and selectivity. In the present work, metal ion dependent DNAzymes of copper (Cu-Enz) and lead (Pb-Enz) were first associated with their corresponding DNA substrates (Cu-Sub and Pb-Sub) immobilized on the surface of aldehyde-coated slides. After introducing the corresponding metal ions, the DNA cleavage of the substrates caused by the DNAzymes took place, resulting in a dramatic change in fluorescent signal intensity. The proposed microarray method, which can be used as a multi-component assay with high efficiency, combines the high sensitivity and selectivity of DNAzymes with the high throughput and parallel analysis of microarray technology. The method reveals a sensitivity corresponding to 0.6 ppb and 2 ppb for Cu2+ and Pb2+, respectively, which is sufficient to detect them in drinking water. This approach may find potential applications in environmental monitoring, food safety monitoring, clinical toxicology, waste treatment, the cosmetic industry and industrial process monitoring.

The glass bead is a new biochip support material for immobilization biomolecules, due to its independence and convenient rearrangement. In order to optimize the immobilization efficiency of oligonucleotides onto glass beads and obtain the highest hybridization efficiency, three commonly used coupling strategies have been studied for covalently attaching oligonucleotides onto large glass beads. Glass beads with 250 μm diameter were amino-silaned with 2% 3-aminopropyltrimethoxysilane (APTMS) and then reacted separately with glutaraldehyde, succinic anhydride and 1,4-phenylene diisothiocyanate (PDITC) to derive CHO beads, COOH beads and isothiocyanate-modified beads (NCS-Beads) accordingly. Afterwards, amino-terminal oligonucleotides were covalently attached onto the surface of beads achieved by three strategies mentioned above. The immobilization efficiency were studied to compare the three strategies, which turned out 2.55 × 1013 probes/cm2 for CHO-Beads, 3.21 × 1013 probes/cm2 for COOH beads and 6.68 × 1013 probes/cm2 for NCS beads. It meant that the immobilization efficiency based on NCS beads was most acceptable. And the method, developed by attaching amino-terminal oligonucleotides onto these cyanate active beads, could be regarded as an efficient one for immobilizing oligonucleotides onto a solid surface. Moreover, in this paper, the hybridization properties of NCS bead-based oligonucleotides have been studied by employing Cy5-tagged complementary oligonucleotides. It was found that the high probe density NCS beads led to low hybridization efficiency possibly due to the existence of steric crowding. In addition, the equilibrium binding constant KA was determined by employing Langmuir isotherm model, which was 7.0 × 106 M−1 for NCS beads with the density of 6.7 × 1013 probes/cm2. Furthermore, it only took 60 min to reach hybridization equilibrium. These large microspheres (>100 μm) can be employed in the mesofluidic systems for automated heterogeneous assays.

This paper presents an approach to simultaneously detect sulfamethazine, streptomycin, and tylosin in milk by indirect competitive multianalyte Fluorescence immunoassay (FIA). Microscope glass slides modified with agarose were used for the preparation of small molecule microarrays (SMMs). Bovine serum albumin (BSA) conjugates of the haptens were immobilized on glass slides. The system consists of four glass slides containing 96 wells formed by an enclosing hydrophobic mask, which precisely matches a standard microplate. All liquid handling and sample processing were fully automated as 96-wells ELISA format. Monoclonal antibodies against sulfamethazine, streptomycin, and tylosin allowed the simultaneous detection of the respective analytes. Antibody binding was detected by a second antibody labeled with Cy5 generating fluorescence, which was scanned with chip scanner. The detection limits for three analytes were 3.26 ng/ml (sulfamethazine), 2.01 ng/ml (streptomycin), and 6.37 ng/ml (tylosin), being far below the respective MRLs. The system proved to be the first SMM–FIA platform having the potential to test for numerous antibiotics in parallel, such being of considerable interest for the control of safety in the food industry.

A highly selective and sensitive mesofluidic immunoassay system based on competitive immunoassay in polydimethylsiloxane (PDMS) channels was developed. This immunoassay system was successfully applied to quantificationally detect chloramphenicol (CAP) in animal foods. The glass beads (⌀ 250 μm) were amino-silane modified, covalently precoated with chloramphenicol succinate, and then infused into the microchannels (⌀ 300 μm); the CAP molecules of samples or standards in flow solution competed for CAP antibody with the CAP immobilized on the beads. The CAP antigen−antibody complex anchored on the beads was probed by Cy5-labeled secondary antibody, and the fluorescence intensities of beads were employed to determine the concentration of CAP. In this system, the detection limit of CAP is 0.008 μg/L. The method reveals good recovery rates from 90 to 108% and coefficients of variance (CV) from 4.72 to 6.52%. The experimental results demonstrate that the bead-based mesofluidic system has high sensitivity and excellent performance. Indeed, this system can readily be operated automatically and expanded for multicomponent analysis. It is therefore an attractive alternative to conventional immunoassays in routine supervised domain application for contamination in foods or the environment.

DNA microarray technology has become powerful and popular in mutation/single nucleotide polymorphism (SNP) discovery and genotyping. However, this method is often associated with considerable signal noise of nonbiological origin that may compromise the data quality and interpretation. To achieve a high degree of reliability, accuracy, and sensitivity in data analysis, an effective normalization method to minimize the technical variability is highly desired. In the current study, a simple and robust normalization method is described. The method is based on introduction of a reference probe coimmobilized with SNP probes on the microarray for a dual-probe hybridization (DPH) reaction. The reference probe is used as an intraspot control for the customized microarrays. Using this method, the interassay coefficient of variation (CV) was reduced significantly by approximately 10%. After DPH normalization, the CVs and ranges of the ratios were reduced by two to five times. The relative magnitudes of variation of different sources were also analyzed by analysis of variance. Glass slides were shown to contribute the most to the variance, whereas sampling and residual errors had relatively modest contribution. The results showed that this DPH-based spot-dependent normalization method is an effective solution for reducing experimental variation associated with microarray genotyping data.

The human leukocyte antigen (HLA) class II system is strongly connected to immunological response and its compatibility between tissues is critical in transplantation. The simple robust typing analyses of HLA genes are extremely important. In this paper, we developed an approach based on microarray technology for genotyping of DQA gene. The microarrays were constructed with a total 31 unmodified 45-mer oligonucleotide. The second exon of DQA gene was amplified, and allowed to hybridize with the array. DQA genotypes were assigned by quantitative analysis of the hybridization results. The arrays were evaluated by DQA genotyping of nine reference samples and 120 clinical samples. The results demonstrate that the genotyping accuracy/concordance achieved 97.5% compared with the direct DNA sequencing. Although our methods did not perform high-resolution genotyping, it could be an alternative for serological typing in routine medical practice.

As an alternative to antibodies, aptamers have shown promising applications in diagnostics and therapeutics. However, different from antibodies, the chemical nature of nucleic acids allows easy synthesis and modification of aptamers. As a result, there are various feasible ways to engineer aptamers with extended bioavailability (e.g., stability and binding affinity in complex environments), regulating ability, and multi-functional properties. In this review, recent advances in rational design and novel functionalization of aptamers, especially DNA aptamers, is described. The broad spectrum of ways for aptamer engineering and applications is paving the way for the future evolution of bioanalytical and biomedical developments.

We have developed a simple and ultrasensitive E-DNA sensor based on the ssDNA-assisted cascade of a hybridization reaction mechanism to form a long concatamers structure to improve its sensitivity, significantly. The proposed sensor was applied to sequence-specific DNA and ATP detection. Experimental results showed a quantitative measurement with the detection limit as low as 1 aM for specific DNA and 10 fM for ATP.

We have developed a novel molecular logic gate system based on the incorporation of aptamer-crosslinked hydrogels. Modified gold nanoparticles are used as the output signal, which is visible to the naked eye. This system is designed for AND and OR operations using two chemicals as stimulus inputs.

A molecular beacon (MB)-like DNA hairpin biosensor based on the reversible directing fluorescence of Hoechst dyes was developed for fluorescent detection of Hg2+ and biothiols and furthermore for logic operation.

A simple and reliable fluorescent molecular beacon is developed utilizing DNA-templated silver nanoclusters as a signal indicator and adenosine triphosphate (ATP) and adenosine deaminase as mechanical activators.

A simple and reliable fluorescent DNA logic gate is developed by utilizing graphene oxide as a signal transducer and mercury ions and iodide as mechanical activators.

A label-free fluorescence assay was developed for light-up detection of ATP in a visual-readout format based on the aptamer-directing fluorescence of Hoechst dyes.

We developed a novel colorimetric method for rapid detection of biogenic amines based on arylalkylamine N-acetyltransferase (aaNAT). The proposed method offers distinct advantages including simple handling, high speed, low cost, good sensitivity and selectivity.

Via the base-stacking hybridization strategy, we have developed a universal, one-step real-time quantitative PCR assay for sensitive and selective detection of microRNAs. This proposed assay has several intrinsic features including rapid response, low cost, simple handling procedures, etc.

Via a molecular caliper p19 protein, we have developed an amplified fluorescence polarization method for rapid microRNA detection. This proposed assay has several intrinsic features including rapidity, simplicity, and accuracy.

By fluorescence enhancement of a proximity-dependent DNA-scaffolded silver nanocluster probe pair and exonuclease III-mediated signal amplification, we present a new fluorescence turn-on mode and its application for specific DNA detection.

A versatile molecular beacon (MB)-like probe was developed for multiplexed detection based on fluorescence polarization by target-induced allosteric effect and furthermore for resettable logic gate operation.

Single stranded DNA sequences can be detected by target assisted exonuclease III-catalyzed signal amplification fluorescence polarization (TAECA-FP). The method offers an impressive detection limit of 83 aM within one hour for DNA detection and exhibits high discrimination ability even against a single base mismatch.

We have developed a novel graphene-based biosensing platform using peptides as probe biomolecules, and demonstrated its feasibility in the application of real-time monitoring of protease activity based on FRET between GO and dye-labeled peptides. This assay allows the rapid and accurate determination of enzyme kinetic parameters as well as inhibition constants.

A simple and reliable colorimetric method for determination of phthalates is developed utilizing uridine-5′-triphosphate (UTP)-modified gold nanoparticles as a color indicator and Cu2+ as a cross-linker. The method demonstrates superior sensitivity with a detection limit of phthalates of ca. 0.5 ppm.