Photoluminescence-Electrochemiluminescence Dual-Mode Sensor Arrays for Histidine and Its Metabolite Discrimination and Disease Identification.

Histidine (His) and its metabolite analysis is significant due to their vital roles in the diagnosis of diseases. In practical applications, simple and effective detection and discrimination of these metabolic species are still a great challenge due to their highly similar structures. Herein, photoluminescence (PL)-electrochemiluminescence (ECL) dual-mode sensor arrays consisting of a series of sensing elements were proposed for simultaneous quantitation and accurate discrimination of His and its four key metabolites (including histamine, imidazole-4-acetic acid, N-acetylhistamine, and imidazole propionate). The sensing elements of these sensor arrays were constructed by employing two solvent iridium(III) complexes ([Ir(pbz)2(DMSO)Cl] and [Ir(ppy)2(DMSO)Cl], pbz = 3-(2-pyridyl)benzoic acid, ppy = 2-phenylpyridine) with excellent PL and ECL performances as cross-responsive sensing units. Based on diverse coordination abilities of the two complexes with the imidazole group of the five targets, PL and ECL responses of each sensing unit can be enhanced to various degrees, which generate unique fingerprint patterns for the corresponding targets. Through principal component analysis, the multifarious patterns (two-, three-, and four-element sensor arrays) can be transformed into simple visualization modes, from which His and its four key metabolites can be effectively discriminated against each other. Moreover, the quantitation of an individual metabolic species at different concentrations and the recognition of the mixtures with different ratios were also accurately achieved. Notably, His and its four key metabolites in urine can also be successfully discriminated by the as-fabricated sensor arrays, and the patients with kidney diseases can be identified clearly, providing a promising way for disease diagnosis.

Near-Infrared Fluorescence Probe with a New Recognition Moiety for the Specific Detection of Cysteine to Study the Corresponding Physiological Processes in Cells, Zebrafish, and Arabidopsis thaliana.

Cysteine (Cys), as one of the biological thiols, is related to many physiological and pathological processes in humans and plants. Therefore, it is necessary to develop a sensitive and selective method for the detection and imaging of Cys in biological organisms. In this work, a novel near-infrared (NIR) fluorescent probe, Probe-Cys, was designed by connecting furancarbonyl, as a new recognition moiety, with Fluorophore-OH via the decomposition of IR-806. The use of the furan moiety is anticipated to produce more effective fluorescence quenching because of the electron-donating ability of the O atom. Probe-Cys has outstanding properties, such as a new recognition group, an emission wavelength in the infrared region at 710 nm, a linear range (0-100 µM), a low detection limit of 0.035 µM, good water solubility, excellent sensitivity, and selectivity without the interference of Hcy, GSH, and HS-. More importantly, Probe-Cys could achieve the detection of endogenous Cys by reacting with the stimulant 1,4-dimercaptothreitol (DTT) and the inhibitor N-ethylmaleimide (NEM) in HepG2 cells and zebrafish. Ultimately, it was successfully applied to obtain images of Arabidopsis thaliana, revealing that the content of Cys in the meristematic zone was higher than that in the elongation zone, which was the first time that the NIR fluorescence probe was used to obtain images of Cys in A. thaliana. The superior properties of the probe exhibit its great potential for use in biosystems to explore the physiological and pathological processes associated with Cys.

Automatic Electrochemiluminescence Method for the Detection of Cancerous Exosomes Incorporating Specific Aptamer-Magnetic Beads and Signal Nanoprobes.

Exosomes, as an emerging biomarker, have exhibited remarkable promise in early cancer diagnosis. Here, a highly sensitive, selective, and automatic electrochemiluminescence (ECL) method for the detection of cancerous exosomes was developed. Specific aptamer-(EK)4 peptide-tagged magnetic beads (MBs-(EK)4-aptamer) were designed as a magnetic capture probe in which the (EK)4 peptide was used to reduce the steric binding hindrance of cancerous exosomes with a specific aptamer. One new universal ECL signal nanoprobe (CD9 Ab-PEG@SiO2ϵRu(bpy)32+) was designed and synthesized by using microporous SiO2 nanoparticles as the carrier for loading ECL reagent Ru(bpy)32+, polyethylene glycol (PEG) layer, and anticluster of differentiation 9 antibody (CD9 Ab). A "sandwich" biocomplex was formed on the surface of the magnetic capture probe after mixing the capture probe, target exosomes, and ECL signal nanoprobe, and then it was introduced into an automated ECL analyzer for rapid and automatic ECL measurement. It was found that the designed signal nanoprobe shows a 270-fold improvement in the signal-to-noise ratio than that of the ruthenium complex-labeled CD9 antibody signal probe. The relative ECL intensity was proportional to MCF-7 exosomes as a model in the range of 102 to 104 particle/µL, with a detection limit of 11 particle/µL. Furthermore, the ECL method was employed to discriminate cancerous exosomes based on fingerprint responses using the designed multiple magnetic capture probes and the universal ECL signal nanoprobe. This work demonstrates that the utilization of a designed automated ECL tactic using the MBs-(EK)4-aptamer capture probe and the CD9 Ab-PEG@SiO2ϵRu(bpy)32+ signal nanoprobe will provide a unique and robust method for the detection and discrimination of cancerous exosomes.

Iridium(III) solvent complex-based electrogenerated chemiluminescence method for the detection of 3-methylhistidine in urine.

3-Methylhistidine (3-MeHis) is increasingly used as an indicator of muscle protein breakdown. The development of a sensitive, simple, and non-invasive method for 3-MeHis assay is important in clinical practice. Herein, a sensitive, simple, and non-invasive electrogenerated chemiluminescence (ECL) method was proposed for the quantitation of 3-MeHis in urine by using an iridium(III) solvent complex ([Ir(dfppy)2(DMSO)Cl], dfppy = 2-(2,4-difluorophenyl)pyridine, Ir-DMSO) as a signal reagent. The photoluminescence (PL) and ECL responses of Ir-DMSO to 3-MeHis were studied. The ECL intensity of Ir-DMSO was enhanced in the presence of 3-MeHis because of the coordination recognition between Ir-DMSO and the imidazole group of 3-MeHis. Based on the enhancement of ECL intensity, 3-MeHis can be sensitively detected in the range of 5 to 25 µM. The detection limit was 0.4 µM. This is the first report of an ECL method for the quantitation of 3-MeHis. Further, to investigate the feasibility of the Ir-DMSO-based ECL method in practical applications, the developed ECL method was applied for 3-MeHis assay in urine samples of 28 healthy volunteers and 2 patients. The urine samples from patients hospitalized with obesity and kidney disease and healthy individuals were distinguished by the ECL responses of Ir-DMSO. The proposed ECL method based on the coordination recognition between iridium(III) solvent complex and the imidazole group of 3-MeHis allows inexpensive, fast, non-invasive, and sensitive detection of 3-MeHis in urine, which is promising for assessing large volumes of patients for routine analysis in clinical practices.

Modified exfoliated graphene functionalized with carboxylic acid-group and thionine on a screen-printed carbon electrode as a platform for an electrochemical enzyme immunosensor.

An enzyme immunoassay was developed based on the coulometric measurement of immunoglobulin M (IgM) against Hantaan viruses (HTNV) by using virus-like particles (VLPs) as recognition molecules. The surface functionalization of screen-printed carbon electrodes (SPCEs) was achieved through paste-exfoliated graphene that was modified with a COOH group and a thionine mediator through supramolecular-covalent scaffolds, on SPCEs by using the binder contained in the ink. After the covalent immobilization of the antibody, the sensor was used for the sandwich enzyme immunoassay of IgM against HTNV. By using HTNV VLPs as the second recognization molecules, the resulting sensor efficiently monitored the reaction of IgM against HTNV and anti-IgM antibody with high specificity. By attaching HTNV nucleocapsid protein antibody conjugate with horseradish peroxidase (HRP) onto VLPs, the signal response of the assay was derived from the coulometric measurement of H2O2 reduction mediated by thionine on the electrode surface after the application of a potential (- 0.2 V vs. Ag/AgCl). The ratio of charges measured before or after H2O2 addition was used to quantify IgM because these charges could be used as background charges or total charges, respectively. The ratio exhibited good agreement with IgM concentration within a range 0.1 to 1000 pg mL-1, and a detection limit of 0.06 pg mL-1 was obtained. The assay demonstrated high sensitivity and specificity toward HTNV-specific IgM in serum.

Label-Free Electrogenerated Chemiluminescence Aptasensing Method for Highly Sensitive Determination of Dopamine via Target-Induced DNA Conformational Change.

A label-free electrogenerated chemiluminescence (ECL) aptasensing method for highly sensitive determination of dopamine (DA) was developed based on target-induced DNA conformational change. After anti-DA specific aptamer, as molecular recognition element, was hybridized with a capture ss-DNA (complementary with the aptamer), the formed double-strand DNA (ds-DNA) was self-assembled onto the surface of a gold electrode, and then Ru(phen)32+, as ECL reagent, was intercalated into ds-DNA to form an ECL biosensing platform. In the presence of DA, DA bound with its aptamer and target-induced DNA conformational change occurred, resulting in the dissociation of ds-DNA, the release of intercalated Ru(phen)32+ from the electrode surface, and the decrease of ECL intensity. For comparison, an ECL aptamer-based biosensing method using an ECL reagent-labeled aptamer was also developed for DA assay based on target-induced DNA conformational change. Because of the increase in the amount of ECL reagent into ds-DNA over that of the single-site ECL reagent-labeled aptamer, an obvious increase of ECL intensity was found at the ds-DNA modified electrode over the aptamer modified electrode. DA can be sensitively detected with a lower detection limit of 0.05 nM in the range from 0.1 to 100 nM. With the recognition of the aptamer for DA, DA can be selectively and sensitively detected in artificial cerebrospinal fluid and serum samples without interference from common small molecules. This work demonstrates that the combination of the direct transduction of specific recognition of DA and its aptamer into an ECL signal with Ru(phen)32+ intercalated ds-DNA amplification provides a promising strategy for the development of a simple, sensitive, and selective method for DA assay, which is of great importance in neurochemical assays and clinical diagnosis.

Tracking the Dissolution Surface Kinetics of a Single Fluorescent Cyclodextrin Metal-Organic Framework by Confocal Laser Scanning Microscopy.

The understanding of the dissolution processes of solids is important for the design and synthesis of solids in a controlled and precise manner and for predicting their fate in the aquatic environment. We report herein single-particle-based confocal laser scanning microscopy (CLSM) for tracking the dissolution surface kinetics of a single fluorescent cyclodextrin metal-organic framework (CD-MOF). As a proof of concept, CD-MOF containing fluorescein, named as CD-MOF⊃FL, was synthesized by encapsulating fluorescein into the interior of CD-MOF via a vapor diffusion method and used as a single-particle dissolution model because of its high FL efficiency and unique structure. The morphology of CD-MOF⊃FL and the distribution of fluorescein within CD-MOF⊃FL were characterized. The growth and dissolution processes of CD-MOF⊃FL at the single-particle level were visualized and quantified for the first time by recording the change of the fluorescence emission. Three processes, including nucleation, germination growth, and saturation stage, were found in the growth of CD-MOF⊃FL, and the growth kinetics followed Avrami's model. The dissolution rate at the face of a single CD-MOF⊃FL crystal was slower than that of its arris, and the dissolution rate of the CD-MOF⊃FL crystal was increased with the increase of the water amount in methanol solution. The dissolution process of the CD-MOF⊃FL crystal was a competitive process of erosion and diffusion in different methanol aqueous solutions, and the dissolution kinetics followed the Korsmeyer-Peppas model. These results offer new insights into the nature of dissolution kinetics of CD-MOF⊃FL and provide new venues for the quantitative analysis of solid dissolution and growth at the single-particle level.

Disposable capillary-fill device for the determination of proteases incorporating elimination of light-shielding from the magnetic beads with cleavage of the electrogenerated chemiluminescence label-tagged peptide probe.

A novel point-of-care testing (POCT) method for the determination of proteases was developed for the first time using a designed disposable capillary-fill device based on the cleavage of electrogenerated chemiluminescence (ECL)-label-tagged peptide probes and enabling elimination of the light-shielding from the magnetic beads (MBs). As a proof-of-principle, prostate-specific antigen (PSA) was taken as a model analyte, and streptavidin-coated magnetic beads bound with ruthenium-complex-tagged specific peptide (biotin-HSSKLQK) were utilized as MB ECL probes. The capillary-fill device was designed to be divided into a reaction zone and detection zone. In the reaction zone, the bio-cleavage reaction between the PSA analyte with the peptide on the surface of the MB ECL probes occurred, while in the detection zone, ECL emission was produced by a screen-printed carbon electrode, Ag/AgCl reference electrode and carbon counter electrode. When the analyte PSA was introduced into the suspension of MB ECL probes in the reaction zone of the device, biocleavage of the peptide occurred, and the cleaved Ru1 part was released from the surface of the MB ECL probes. The capillary-filled device was tilted 90°, and with the aid of gravity, the solution containing the released Ru1 part flowed to the surface of the working electrode in the detection region of the device, while the MB ECL probes were fixed in the reaction zone by an external magnet. PSA can be determined by the ECL emission from the released Ru1 part in the presence of the co-reactant tri-n-propylamine at the detection zone. Under the optimal conditions, the developed ECL method showed a low detection limit of 0.12 ng mL-1 for PSA. This work demonstrates that the developed ECL biosensing approach can eliminate the MB light-shielding effect and quantify proteases with high sensitivity and selectivity, which could be easily extended to POCT-based ECL biosensing for other proteases.

Description of Hymenobacter sediminicola sp. nov., isolated from contaminated sediment.

A polyphasic taxonomic study was conducted on two Gram-negative, non-sporulating, non-motile bacterial strains, S2-20-2T and S2-21-1, isolated from a contaminated freshwater sediment in China. Comparative 16S rRNA gene sequence studies revealed a clear affiliation of two strains with Bacteroidetes, which showed the highest pairwise sequence similarities with Hymenobacter duratus BT646T (99.3%), Hymenobacter psychrotolerans Tibet-IIU11T (99.3%), Hymenobacter kanuolensis T-3T (97.6%), Hymenobacter swuensis DY53T (96.9%), Hymenobacter tenuis POB6T (96.8%), Hymenobacter seoulensis 16F7GT (96.7%), and Hymenobacter rigui KCTC 12533T (96.5%). The phylogenetic analysis based on 16S rRNA gene sequences showed that two strains formed a clear phylogenetic lineage with the genus Hymenobacter. Major fatty acids were identified as iso-C15:0, anteiso-C15:0, and summed feature 3 (C16:1 ω6c and/or C16:1 ω7c/t) and summed feature 4 (iso-C17:1 I and/or anteiso-C17:1 B). Major cellular polar lipids were identified as phosphatidylethanolamine, three unidentified aminolipids, an unidentified aminophosopholipid and an unidentified lipid. The respiratory quinone was detected as MK-7 and the genomic DNA G + C content was determined to be 57.9% (genome) for type strain S2-20-2T and 57.7 mol% (HPLC) for strain S2-21-1. The observed ANI and dDDH values between strain S2-20-2T and its closely related strains were 75.7-91.4% and 21.2-43.9%, respectively. Based on physiological, biochemical, genetic and genomic characteristics, we propose that strains S2-20-2T and S2-21-1 represent a novel species of the genus Hymenobacter, for which the name Hymenobacter sediminicola sp. nov. is proposed. The type strain is S2-20-2T (= CGMCC 1.18734T = JCM 35801T).

Electrochemiluminescence bioassay with anti-fouling ability for determination of matrix metalloproteinase 9 secreted from living cells under external stimulation.

An electrochemiluminescence (ECL) bioassay with high sensitivity and anti-fouling ability was developed for determination of matrix metalloproteinase 9 (MMP-9) secreted from living cells under external stimulation. A peptide with sequence of CLGRMGLPGK and a new cyclometalated iridium(III) complex bearing carboxyl group, (pq)2Ir(dcbpy) (pq = 2-phenylquinoline, dcbpy = 2,2'-bipyridyl-4,4'-dicarboxyli acid, abbreviated as Ir) were employed as molecular recognition substrate and ECL emitter, respectively. The peptide was labelled with the Ir to form Ir-peptide as ECL probe. Ir-peptide was self-assembled onto Nafion and gold nanoparticles (AuNPs) modified glassy carbon electrode (AuNPs/Nafion/GCE) and then both of 6-mercapto-1-hexanol (MCH) and zwitterionic peptide as blocking reagents were co-assembled on Ir-peptide/AuNPs/Nafion/GCE to form an anti-fouling ECL peptide-based biosensor. MMP-9 can be quantified in the range 1.0-50 ng·mL-1 with a detection limit of 0.50 ng·mL-1 based on the decreased ECL intensity. Relative standard derivation was 2.3% for six fabricated anti-fouling ECL peptide-based biosensors after reaction with 50 ng·mL-1 MMP-9. The anti-fouling ECL peptide-based biosensor can be used to monitor MMP-9 secreted from living cells under external stimulation. 96.0%-108.0% of recoveries were obtained in 60-diluted cell culture media. This study demonstrates that the ECL biosensor by the combination of iridium(III) complex-based sensitive ECL method and the anti-fouling interface provides a promising way for the determination of MMP-9 in biological sample, which is viable in clinical diagnosis and point-of-care test of protease.

Design of a New Hydrazine Moiety-Based Near-Infrared Fluorescence Probe for Detection and Imaging of Endogenous Formaldehyde In Vivo.

Formaldehyde (FA), the smallest molecular aldehyde with strong reducing properties, could regulate body homeostasis endogenously during physiological and pathological processes. The effective near-infrared (NIR) fluorescent probe is needed as a visualizer of FA in biologic organisms. In this work, a novel NIR fluorescent Probe-NHNH2 was designed on the basis of Probe-NH2 via introducing a strong nucleophilic hydrazine group, which can be used as a quenching and recognizing moiety for the detection of FA. With the treatment of FA, the hydrazine group of Probe-NHNH2 undergoes condensation and achieves a turn-on NIR fluorescence signal at a wavelength of 706 nm. The spectroscopic performance of Probe-NHNH2 for FA was evaluated, and it exhibited high sensitivity and selectivity for the detection of FA in solution. Moreover, compared to the amine moiety-based Probe-NH2, which our group reported, we found that hydrazine moiety-based Probe-NHNH2, exhibited a better reaction time of within 10 min and a lower detection limit of 0.68 µM, reflecting that the reaction of FA with hydrazine moiety is faster and more sensitive than that of FA with the amino group. More importantly, Probe-NHNH2 was successfully applied to real-time imaging of endogenous FA by reacting with effective stimulant tetrahydrofolate and scavenger sodium bisulfite in zebrafish and mice. It is expected that we can provide a new rapid, sensitive NIR fluorescence theoretical basis for FA detection and in vivo imaging applications.

Near-Infrared Fluorescence Probe with a New Recognition Moiety for Specific Detection and Imaging of Aldehyde Dehydrogenase Expecting the Identification and Isolation of Cancer Stem Cells.

Aldehyde dehydrogenase (ALDH) is a vital enzyme that converts aldehyde to acetic acid during alcohol metabolism. ALDH is also a cellular marker of cancer stem cells (CSCs), which plays an important role in cancer diagnosis and prognosis assessment. Therefore, there is a need to explore convenient, selective, and sensitive methods for the detection and imaging of ALDH. Because of the low background fluorescence and high penetration, near-infrared (NIR) fluorescent probes are powerful tools for the detection of ALDH. Until now, only one NIR fluorescent probe has been reported for detecting ALDH. Hence, we synthesized a novel NIR fluorescent probe, Probe-ALDH, by linking the new specific recognition moiety 4-hydroxymethyl benzaldehyde with NIR fluorophore AXPI. Compared with the existing ALDH fluorescent probes, Probe-ALDH has excellent properties, such as a new specific recognition moiety without the substitution of benzaldehyde, a simple synthesis method, emission wavelength in the NIR region, reaction time of only 30 min, and a detection limit as low as 0.03 U·mL-1, which is better than those of the previously reported probes. The probe effectively eliminates the interference from reactive oxygen species (ROS), amino acids, and amines. More importantly, the flow cytometry results showed that Probe-ALDH has great potential applications in the identification and isolation of CSCs. Ultimately, it was successfully applied to the imaging analysis of endogenous ALDH in HepG2 cells by the addition of inhibitor disulfiram. The excellent performance of Probe-ALDH makes it a promising candidate for drug discovery, cancer diagnosis, and so forth.

Highly Efficient Electrogenerated Chemiluminescence Quenching on Lipid-Coated Multifunctional Magnetic Nanoparticles for the Determination of Proteases.

This work reports a highly efficient electrogenerated chemiluminescence (ECL) quenching on lipid-coated multifunctional magnetic nanoparticles (MMNP) for the determination of proteases incorporating membrane-confined quenching with a specific cleavage reaction for the first time. A new ruthenium complex [Ru(bpy)2(ddcbpy)](PF6)2 (bpy = 2,2'-bipyridine, ddcbpy = 4,4'-didodecyl-carbonyl-2,2'-bipyridine with two hydrophobic long alkyl chains) was synthesized as a signal probe, while [cholesterol-(CH2)6-HSSKLQK(peptide)-ferrocene (quencher)] was designed as a specific peptide-quencher probe. The MMNP were prepared by inserting both the signal probe and the peptide-quencher probe into the cholesterol-phospholipid-coated Fe3O4 magnetic nanoparticles (Fe3O4 NP, ∼200 nm). When prostate specific antigen (PSA) taken as a model analyte was introduced into the suspension of MMNP, PSA cleaved the amide bond of SK in cholesterol-(CH2)6-HSSKLQK-Fc, and then the cleaved peptide-motif-Fc-quencher was deviated from the MMNP, resulting in the increase in the ECL intensity. It was found that the ECL quenching constant of [Ru(bpy)2(ddcbpy)]2+ on MMNP (KSV, NP/lipECL =2.68 × 107 M-1) is 137-folds higher than that on the lipid-coated electrode (KSV, lipECL=1.95 × 105 M-1) and 391-folds higher than that in the solution (KSV, aqECL =6.86 × 104 M-1). The ECL emission of Ru(bpy)32+ derivative-attached Fe3O4 NP was observed at ∼1.2 V, involving the tunnel-electron transfer pathway (TPA• + Ru(bpy)33+ = Ru(bpy)32+*). Based on the highly efficient ECL quenching of the ruthenium complex by ferrocene on the MMNP, a new ECL method was developed for PSA with a linear range from 0.01 to 1.0 ng/mL and a limit of detection of 3.0 pg/mL. This work demonstrates that the approach of ECL quenching by ferrocene on lipid-coated Fe3O4 NP is promising and could be easily extended to determine other proteases.

Highly Efficient Aggregation-Induced Enhanced Electrochemiluminescence of Cyanophenyl-Functionalized Tetraphenylethene and Its Application in Biothiols Analysis.

Exploring new electrochemiluminescence (ECL) luminophores with high ECL efficiency and good stability in aqueous solution is in great demand for biological sensing. In this work, highly efficient aggregation-induced enhanced ECL of cyanophenyl-functionalized tetraphenylethene (tetra[4-(4-cyanophenyl)phenyl]ethene, TCPPE) and its application in biothiols analysis were reported. TCPPE contains four 4-cyanophenyl groups covalently attached to the tetraphenylethene (TPE) core, generating a nonplanar three-dimensional twisted conformation structure. TCPPE nanoparticles (NPs) with an average size of 15.84 nm were prepared by a precipitation method. High ECL efficiency (593%, CdS as standard) and stable ECL emission (over one month) were obtained for TCPPE NPs in aqueous solution. The unique properties of TCPPE NPs could be ascribed to the efficient suppression of nonradiative transition, the decrease of the energy gap, and the increase of anionic radical stability, which were proved by theoretical calculation and electrochemical and fluorescence methods. Contrasting aggregation-induced ECL chromic emission was first observed for TCPPE NPs. As a proof-of-methodology, an ECL method was developed for three biothiol assays with detection limits of 6, 7, and 300 nM for cysteine, homocysteine, and glutathione, respectively. This work demonstrates that TCPPE NPs are promising ECL luminophores, and the incorporation of appropriate substituents into luminophores can improve ECL efficiency and radical stability.

A new metal-free near-infrared fluorescent probe based on nitrofuran for the detection and bioimaging of carbon monoxide releasing molecule-2 in vivo.

As a stable donor for releasing controlled amounts of CO, carbon monoxide releasing molecule-2 (CORM-2) is a new type of therapeutic drug that contributes to exploring the pathophysiological effects of CO. The accurate detection of CORM-2 in biological systems is of great significance for controlling its dosage as a therapeutic drug and elucidating the reaction mechanisms of CO, but currently there is a lack of metal-free near-infrared fluorescent probes. Herein, a new metal-free near-infrared fluorescent probe based on nitrofuran which could selectively identify CORM-2 was designed and it has been successfully applied in living cells, zebrafish and mice. After reacting with CORM-2, both the color and fluorescence signal of the solution are restored, which is ascribed to the reduction of the nitro group. The spectroscopic probe DXPN shows high sensitivity to CORM-2 with a low detection limit of 87 nM and near-infrared fluorescence emission of 712 nm. Notably, this is the first time that paper chips are being used as a carrier to detect CORM-2 through fluorescence signals instead of the traditional liquid phase detection mode of fluorescent probes. These superior properties of the probe make it a promising and reliable tool for exploring the role played by CORM-2 in biological systems.

Sensitive and rapid determination of heat shock protein 70 using lateral flow immunostrips and upconversion nanoparticle fluorescence probes.

Heat shock protein 70 (Hsp70), belonging to the heat shock protein (HSP) family, is reported to be a potential diagnostic biomarker. In this work, a lateral flow immunostrip was fabricated for the sensitive and rapid determination of Hsp70 by the incorporation of fluorescence and upconversion nanoparticle probes. The upconversion nanoparticles (UCNPs, size ∼39 nm, λex = 980 nm; λem = 540 nm) consisting of a NaYF4:Yb/Er core and polyacrylic acid-modified shell were covalently coupled with Hsp70 antibodies to form the signal probe, which was characterized by dynamic light scattering and zeta potential analyses. The lateral flow assay (LFA) was constructed based on the sandwich-type immunoassay using a sample pad, a test pad, and an adsorption pad on a PVP backing. Hsp70 antibody, IgG antibody and the signal probe were separately dropped on the test zone, the control zone of the test pad, and the sample pad, respectively. In the sandwich LFA, since two antibodies bind to Hsp70 antigenic epitopes, i.e. specific binding, it provided superior specificity and high sensitivity, making it an ideal sensing platform for complex samples like serum Hsp70 samples. The important parameters for the preparation of the lateral flow immunostrips were optimized. Under the optimized conditions, Hsp70 can be detected using the increased fluorescence intensity of UCNPs with a wide linear range from 0.11 to 12 ng mL-1, low detection limit of 0.06 ng mL-1, small sample volume (120 µL), short assay time (15 min) and good reproducibility. The fluorescence method was successfully applied in the determination of Hsp70 in serum samples with good recovery. By combining the accessibility of the lateral flow immunostrips and upconversion nanoparticles, the fluorescence method can serve as a point-of-care testing method for protein assays with high sensitivity and fast detection.

Development and Applications of CRISPR/Cas9-Based Genome Editing in Lactobacillus.

Lactobacillus, a genus of lactic acid bacteria, plays a crucial function in food production preservation, and probiotics. It is particularly important to develop new Lactobacillus strains with superior performance by gene editing. Currently, the identification of its functional genes and the mining of excellent functional genes mainly rely on the traditional gene homologous recombination technology. CRISPR/Cas9-based genome editing is a rapidly developing technology in recent years. It has been widely applied in mammalian cells, plants, yeast, and other eukaryotes, but less in prokaryotes, especially Lactobacillus. Compared with the traditional strain improvement methods, CRISPR/Cas9-based genome editing can greatly improve the accuracy of Lactobacillus target sites and achieve traceless genome modification. The strains obtained by this technology may even be more efficient than the traditional random mutation methods. This review examines the application and current issues of CRISPR/Cas9-based genome editing in Lactobacillus, as well as the development trend of CRISPR/Cas9-based genome editing in Lactobacillus. In addition, the fundamental mechanisms of CRISPR/Cas9-based genome editing are also presented and summarized.

New Multidrug Efflux Systems in a Microcystin-Degrading Bacterium Blastomonas fulva and Its Genomic Feature.

A microcystin-degrading bacterial strain, Blastomonas fulva T2, was isolated from the culture of a microalgae Microcystis. The strain B. fulva T2 is Gram-stain-negative, non-motile, aerobic, non-spore-forming and phototrophic. The cells of B. fulva T2 are able to grow in ranges of temperature from 15 to 37 °C, with a pH of 6 to 8 and a salinity of 0 to 1% NaCl. Here, we sequenced the complete genome of B. fulva T2, aiming to better understand the evolutionary biology and the function of the genus Blastomonas at the molecular level. The complete genome of B. fulva T2 contained a circular chromosome (3,977,381 bp) with 64.3% GC content and a sizable plasmid (145.829 bp) with 60.7% GC content which comprises about 3.5% of the total genetic content. A total of 3842 coding genes, including 46 tRNAs and 6 rRNAs, were predicted in the genome. The genome contains genes for glycolysis, citric acid cycle, Entner-Doudoroff pathways, photoreaction center and bacteriochlorophylla synthesis. A 7.9 K gene cluster containing mlrA, mlrB, mlrC and mlrD1,2,3,4 of microcystin-degrading enzymes was identified. Notably, eight different efflux pumps categorized into RND, ABC and MFS types have been identified in the genome of strain T2. Our findings should provide new insights of the alternative reaction pathway as well as the enzymes which mediated the degradation of microcystin by bacteria, as well as the evolution, architectures, chemical mechanisms and physiological roles of the new bacterial multidrug efflux system.

Electrogenerated Chemiluminescence Biosensing.

This Feature simply introduces the history and mechanism of classical electrogenerated chemiluminescence (ECL) systems for the detection of biomolecules, highlights new advances and emerging fields of the ECL biosensing with recent illustrative examples, and presents the challenges and perspectives of ECL biosensing.

Electrogenerated Chemiluminescence Immunoassays on Nanoelectrode Ensembles Platform with Magnetic Microbeads for the Determination of Carbohydrate Antigen.

This work reports a gold nanoelectrode ensembles (Au-NEE) platform taken as a disposable electrogenerated chemiluminescence (ECL) platform with immunomagnetic microbeads for ECL immunoassays for the first time. The peak-shaped voltammograms were obtained at the Au-NEE, attributed to the total diffusional overlap. The ECL intensity at Au-NEE was 12.9 folds in the Ru(bpy)32+-tri-n-propylamine (TPA) ECL system and 19.6 folds in the luminol-H2O2 system, compared with that at the Au macroelectrode using the normalized active area of the electrodes, mainly attributed to the diffusion overlap of the Au-NEE and the edge effect of the individual gold nanodisks of the Au-NEE. The ECL immunoassay on the Au-NEE platform with magnetic microbeads for the determination of cancer biomarkers was developed. Carbohydrate antigen 19-9 (CA 19-9) was chosen as a model analyte while CA 19-9 antibody on the magnetic microbeads was taken as the capture probe, and ruthenium complex-labeled CA 19-9 antibody was used as the signal probe. A "sandwich" bioconjugates on the magnetic beads were transferred onto the ECL platform, and then the ECL measurements were performed in TPA solution. The developed method showed that the ECL peak intensity was directly in proportion to the concentration of CA 19-9 in the range from 0.5 to 20 U/mL with a limit of detection of 0.4 U/mL. This work demonstrates that the Au-NEE can be employed as a useful disposable ECL platform with the merits of cheapness, low nonspecific adsorption and practical application. The proposed approach will open a new avenue in the point-of-care test for the determination of protein biomarkers.