An electrochemical biosensor for the amplification of thrombin activity by perylene-mediated photoinitiated polymerization.

BACKGROUND: Thrombin, a coagulation system protease, is a key enzyme involved in the coagulation cascade and has been developed as a marker for coagulation disorders. However, the methods developed in recent years have the disadvantages of complex operation, long reaction time, low specificity and sensitivity. Meanwhile, thrombin is at a lower level in the pre-disease period. Therefore, to accurately diagnose the disease, it is necessary to develop a fast, simple, highly sensitive and specific method using signal amplification technology. RESULTS: We designed an electrochemical biosensor based on photocatalytic atom transfer radical polymerization (photo-ATRP) signal amplification for the detection of thrombin. Sulfhydryl substrate peptides (without carboxyl groups) are self-assembled to the gold electrode surface via Au-S bond and serve as thrombin recognition probes. The substrate peptide is cleaved in the presence of thrombin to generate -COOH, which can form a carboxylate-Zr(IV)-carboxylate complex via Zr(IV) and initiator (α-bromophenylacetic acid, BPAA). Subsequently, an electrochemical biosensor was prepared by introducing polymer chains with electrochemical signaling molecules (ferrocene, Fc) onto the electrode surface by photocatalytic (perylene, Py) mediated ATRP using ferrocenylmethyl methacrylate (FMMA) as a monomer. The concentration of thrombin was evaluated by the voltammetric signal generated by square wave voltammetry (SWV), and the result showed that the biosensor was linear between 1.0 ng/mL âˆ¼ 10 fg/mL, with a lower detection limit of 4.0 fg/mL (∼0.1 fM). Moreover, it was shown to be highly selective for thrombin activity in complex serum samples and for thrombin inhibition screening. SIGNIFICANCE: The biosensor is an environmentally friendly and economically efficient strategy while maintaining the advantages of high sensitivity, anti-interference, good stability and simplicity of operation, which has great potential for application in the analysis of complex samples.

Accurate assembly of thiophene-bridged titanium-oxo clusters with photocatalytic amine oxidation activity.

Designing and synthesizing well-defined crystalline catalysts for the photocatalytic oxidative coupling of amines to imines remains a great challenge. In this work, a crystalline dumbbell-shaped titanium oxo cluster, [Ti10O6(Thdc)(Dmg)2(iPrO)22] (Ti10, Thdc = 2,5-thiophenedicarboxylic acid, Dmg = dimethylglyoxime, iPrOH = isopropanol), was constructed through a facile one-pot solvothermal strategy and treated as a catalyst for the photocatalytic oxidative coupling of amines. In this structure, Thdc serves as the horizontal bar, while the {Ti5Dmg} layers on each side act as the weight plates. The molecular structure, light absorption, and photoelectrochemical properties of Ti10 were systematically investigated. Remarkably, the inclusion of the Thdc ligand, with the assistance of the Dmg ligand, broadens the light absorption spectrum of Ti10, extending it into the visible range. Furthermore, the effective enhancement of charge transfer within the Ti10 was achieved with the successful incorporation of the Thdc ligand, as opposed to PTC-211, where terephthalic acid replaces the Thdc ligand, while maintaining consistency in other aspects of Ti10. Building on this foundation, Ti10 was employed as a heterogeneous molecular photocatalyst for the catalytic oxidative coupling reaction of benzylamine (BA), demonstrating very high conversion activity and selectivity. Our study illustrates that the inclusion of ligands derived from Thdc enhances the efficiency of charge transfer in functionalized photocatalysts, significantly influencing the performance of photocatalytic organic conversion.

Ultrasensitive detection of miRNA-21 by click chemistry and fluorescein-mediated photo-ATRP signal amplification.

The development of a convenient and efficient assay using miRNA-21 as a lung cancer marker is of great importance for the early prevention of cancer. Herein, an electrochemical biosensor for the detection of miRNA-21 was successfully fabricated under blue light excitation using click chemistry and photocatalytic atom transfer radical polymerization (photo-ATRP). By using hairpin DNA as a recognition probe, the electrochemical sensor deposits numerous electroactive monomers (ferrocenylmethyl methacrylate) on the electrode surface under the reaction of photocatalyst (fluorescein) and pentamethyldiethylenetriamine, thereby achieving signal amplification. This biosensor is sensitive, precise and selective for miRNA-21, and is highly specific for RNAs with different base mismatches. Under optimal conditions, the biosensor showed a linear relationship in the range of 10 fM ∼1 nM (R2 = 0.995), with a detection limit of 1.35 fM. Furthermore, the biosensor exhibits anti-interference performance when analyzing RNAs in serum samples. The biosensor is based on green chemistry and has the advantages of low cost, specificity and anti-interference ability, providing economic benefits while achieving detection objectives, which makes it highly promising for the analysis of complex samples.

Mn-MOF catalyzed multi-site atom transfer radical polymerization electrochemical sensing of miRNA-21.

A green electrochemical biosensor was developed based on metal-organic framework (MOF)-catalyzed atom transfer radical polymerization (ATRP) for quantifying miRNA-21, used as the proof-of-concept analyte. Unlike conventional ATRP, Mn-PCN-222 (PCN, porous coordination network) could be used as an alternative for green catalyst to substitute traditional catalysts. First, poly (diallyldimethylammonium chloride) (PDDA) was fixed on the surface of the indium tin oxide (ITO) electrode, and then the Mn-PCN-222 was linked to ITO electrode via electrostatic binding with PDDA. Next, aminated ssDNA (NH2-DNA) was used to modify the electrode further by amide reaction with Mn-PCN-222. Then, the recognition and hybridization of NH2-DNA with miRNA-21 prompt the generation of DNA-RNA complexes, which further hybridize with Fc-DNA@ß-CD-Br15 and permit the initiator to be immobilized on the electrode surface. Accordingly, ß-CD-Br15 could initiate the polymerization of ferrocenylmethyl methacrylates (FcMMA) under the catalysis of MOF to complete the ATRP reaction. FcMMA presented a distinct electrochemical signal at ~ 0.33 V. Taking advantage of the unique multi-site properties of ß-CD-Br15 and the efficient catalytic reaction induced by Mn-PCN-222, ultrasensitive detection of miRNA-21 was achieved with a detection limit of 0.4 fM. The proposed electrochemical biosensor has been applied to the detection of miRNA-21 in serum samples. Therefore, the proposed strategy exhibited potential in early clinical biomedicine.

Solvent-directed multiple correspondence fluorescent probe for highly selective and sensitive detection of Cu2+ and Mg2.

A solvent-directed, new Schiff base multiple correspondence fluorescent probe, (E)-2-(2-hydroxybenzylidene) hydrazine-1-carboxamid (L), was synthesized for selective sensing of Cu2+ and Mg2+ ions. L showed excellent selectivity and high sensitivity toward Cu2+ in "turn off" mode with a detection limit of 40.5 nM in 10 mM, pH = 7.0 PBS buffer. Contrary to that, when acetonitrile was used as the solvent, L exhibited highly selective and sensitive fluorescence sensing ability for Mg2+ in "turn on" mode with a detection limit of 9.5 nM. L can coordinate to Cu2+ and Mg2+ in a 1:1 molar ratio, respectively, evidenced by Job's plot analysis. Their binding modes were investigated by NMR, IR and XPS spectroscopies. Moreover, the satisfied results were obtained when L was used to detect Cu2+ and Mg2+ in real water samples.

Cyanocobalamin (VB12) Bionic Enzyme-Assisted Photocatalytic Chain-Growth Polymerization for Detection of Lung Cancer Biomarkers.

Cobalt-mediated radical polymerization is noted for its great level of control over the polymerization of acrylic and vinyl esters monomers, even at high molar mass. Vitamin B12, a natural bionic enzyme cobalt complex, involves the conversion of organic halides to olefins through chain-growth polymerization. In this work, the notion of R-Co(III) free radical persistent free radical effect and vitamin B12 circulation were first reported for the perception of ultralow abundance of microRNA-21, a lung cancer biomarker. Indeed, most Co-containing catalytic reactions can occur under mild conditions due to their minimal bond dissociation of the C-Co bond, with blue light irradiation. Based on the intrinsic stability of the vitamin B12 framework and recycling of the catalyst, it is evident that this natural catalytic scheme has potential applications in medicinal chemistry and biomaterials. In addition, this strategy, combined with highly specific recognition probes and vitamin B12 circulation-mediated chain-growth polymerization, has a detection limit as low as 910 aM. Furthermore, it is sensitive for sensing in serum samples containing biomarkers and shows great potential for RNA selection and amplification sensing in clinical samples.

A novel fluorescence sensor for uranyl ion detection based on a dansyl-modified peptide.

It is of great significance to sensitively and selectively detect uranyl ion (UO22+) in environmental and biological samples due to the high risks of UO22+ to human health. However, such suitable sensors are still scarce. A novel fluorescence sensor based on a dansyl-modified peptide, Dansyl-Glu-Glu-Pro-Glu-Trp-COOH (D-P5), was efficiently synthesized by Fmoc solid phase peptide synthesis. As the first linear peptide-based fluorescence sensor for UO22+, D-P5 exhibited high selectivity and sensitivity to UO22+ over 27 metal ions (UO22+, Cr3+, Cu2+, Ba2+, Hg2+, Pb2+, Co2+, Ag+, Fe3+, Ca2+, K+, Mg2+, Mn2+, Na+, Ni2+, Cd2+, Zn2+, Al3+, Dy3+, Er3+, Gd2+, Ho3+, La3+, Lu3+, Pr3+, Sm3+, Tm3+) by a turn-off fluorescence response in 10 mM HEPES buffer (pH 6.3). The effects of anions such as S2-, NO3-, SO42- CO32-, HCOO-, antioxidant ascorbic acid and 4-nitrophenyl acetate on the selectivity for UO22+ detection were also studies. D-P5 sensor could be used for detecting UO22+ in a good linear relationship with concentration in the range of 0-8.0 µM with a low limit of detection of 83.2 nM. Furthermore, the interaction of the sensor with UO22+ was characterized by ESI-MS, IR, XPS and ITC measurements. The 1:1 binding stoichiometry between the sensor and UO22+ was measured by the job's plot and further verified by ESI-MS. The binding constant of the sensor with UO22+ was calculated to be 9.8 × 104 M-1 by modified Benesi-Hildebrand equation. ITC results showed that theΔHθ andΔSθ for the interaction of D-P5 with UO22+ were -(7.167 ± 1.25) kJ·mol-1 and 66.5 J·mol-1·K-1, respectively. Time-resolved fluorescence spectroscopy indicated that the mechanism of fluorescence quenching of D-P5 by UO22+ ion was static quenching process. In addition, this sensor displayed a good practicality for UO22+ detection in lake water sample without tedious sample pretreatment.

Ferritin-Enhanced Direct MicroRNA Detection via Controlled Radical Polymerization.

Accurate quantitative detection of tracing nucleic acids remains a great challenge in cancer genetic testing. It is crucial to propose a low-cost and highly sensitive direct gene detection method for cancer prevention and treatment. Herein, this work reports an ultrasensitive biosensor via a ferritin-enhanced atom-transfer radical polymerization (Ft-ATRP) process. Intriguingly, microRNA-21, an early marker of lung cancer, can be detected without being transcribed in advance by an innovative signal amplification strategy using ferritin-mediated aggregation of hydrophilic nitroxide radical monomers as an electrochemical biosensor. The sensor uses peptide nucleic acid probes modified on a gold electrode to accurately bind the target lung cancer marker in the sample, and then ferritin, which is naturally present in human blood, induces Ft-ATRP on the electrode surface under mild conditions. Many of 4-methacryloyloxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical (MATMP) monomers with electrochemical signals are combined into polymeric chains to be modified on target assays. The limit of detection (LOD) of microRNA-21 is as low as 6.03 fM, and the detection concentration ranges from 0.01 to 100 pM (R2 = 0.994). The RNA biosensor can realize great performance analysis of complicated samples in simple operation, in addition, the detection process used by the catalyst, polymers containing electrochemical signals, and the electrolyte solution all have good water solubility. The superior performance of the RNA biosensor demonstrates its potential to screen and identify lung cancer in target patients.

Research Progress in Fluorescent Probes for Arsenic Species.

Arsenic is a toxic non-metallic element that is widely found in nature. In addition, arsenic and arsenic compounds are included in the list of Group I carcinogens and toxic water pollutants. Therefore, rapid and efficient methods for detecting arsenic are necessary. In the past decade, a variety of small molecule fluorescent probes have been developed, which has been widely recognized for their rapidness, efficiency, convenience and sensitivity. With the development of new nanomaterials (AuNPs, CDs and QDs), organic molecules and biomolecules, the conventional detection of arsenic species based on fluorescence spectroscopy is gradually transforming from the laboratory to the portable kit. Therefore, in view of the current research status, this review introduces the research progress of both traditional and newly developed fluorescence spectrometry based on novel materials for arsenic detection, and discusses the potential of this technology in the rapid screening and field testing of water samples contaminated with arsenic. The review also discusses the problems that still exist in this field, as well as the expectations.

Engineering Human Neuroglobin into a Cytochrome c-Like Protein with a Single Thioether Bond in Non-native State.

A double mutant of human H64M/V71C neuroglobin (Ngb) was engineered, which formed a single thioether bond as that in atypical cytochrome c, whereas the heme distal Met64 was oxidized to both sulfoxide (SO-Met) and sulfone (SO2 -Met). By contrast, no Cys-heme cross-link was formed in V71C Ngb with His64/His96 coordination, as shown by the X-ray crystal structure, which indicates that an open distal site facilitates the activation of heme iron for structural modifications.

Hemoglobin-catalyzed atom transfer radical polymerization for ultrasensitive electrochemical DNA detection.

The use of hemoglobin (Hb) to drive atom transfer radical polymerization (ATRP) process (Hb-ATRP) for detection of lung cancer related nucleic acid is firstly reported. Hb does not need to be treated prior to using indicating the potential for synthetic engineering in complex biological microenvironments without the need for in vitro techniques. Here, we report a new signal amplification strategy using Hb-mediated graft of nitronyl niroxide monoradical polymers as a signal-on electrochemical biosensor for ultralow level DNA highly selective detection. Building DNA biosensors includes: (i) the fixation of peptide nucleic acid (PNA) probe (no phosphate group) via the 5' terminus-SH; (ii) the modification of transition metal; (iii) Site-specific markers of Hb-ATRP promoter, and (iv) the grafting of polymers with electrochemical signal by Hb-ATRP process. Through the Hb-ATRP process of nitronyl nitroxide monoradical (TEMPO), the presence of a small amount of DNA can eventually result in calling a certain number of TEMPO redox tags. Obviously, the Hb-ATRP is a method of easy source of raw materials, simple operation and no need for complex equipment. The constructed biosensor, as expected, is highly selective and sensitive to target DNA. The detection limit can be calculated as 15.96 fM under optimal conditions. The excellent performance also shows that the constructed DNA biosensor is suitable for DNA screening and DNA concentration determination in complex sample matrix.

Naturally Occurring I81N Mutation in Human Cytochrome c Regulates Both Inherent Peroxidase Activity and Interactions with Neuroglobin.

Human cytochrome c (hCyt c) is a crucial heme protein and plays an indispensable role in energy conversion and intrinsic apoptosis pathways. The sequence and structure of Cyt c were evolutionarily conserved and only a few naturally occurring mutants were detected in humans. Among those variable sites, position 81 was proposed to act as a peroxidase switch in the initiation stages of apoptosis. In this study, we show that Ile81 not only suppresses the intrinsic peroxidase activity but also is essential for Cyt c to interact with neuroglobin (Ngb), a potential protein partner. The kinetic assays showed that the peroxidase activity of the naturally occurring variant I81N was enhanced up to threefold under pH 5. The local stability of the Ω-loop D (residues 70-85) in the I81N variant was decreased. Moreover, the Alphafold2 program predicted that Ile81 forms stable contact with human Ngb. Meanwhile, the Ile81 to Asn81 missense mutation abolishes the interaction interface, resulting in a ∼40-fold decrease in binding affinity. These observations provide an insight into the structure-function relationship of the conserved Ile81 in vertebrate Cyt c.

Sensitive electrochemiluminescence analysis of lung cancer marker miRNA-21 based on RAFT signal amplification.

An electrochemiluminescence approach based on surface-initiated reversible addition-fragmentation chain transfer (SI-RAFT) was developed for miRNA-21 detection for the first time. The SI-RAFT polymerization generates polymer chains with functional groups that are used to recruit luminol, enabling strong ECL signal output with low concentrations of miRNA-21, and greatly improving the detection sensitivity.

The X-ray crystal structure of human A15C neuroglobin reveals both native/de novo disulfide bonds and unexpected ligand-binding sites.

Human neuroglobin (Ngb) contains a heme group and three Cys residues (Cys46, Cys55, and Cys120) in the polypeptide chain. By introducing an additional Cys at position 15, the X-ray structure of A15C Ngb mutant was solved at a high resolution of 1.35 Å, which reveals the formation of both the native (C46C55) and the engineered (C15C120) disulfide bonds, likely playing a functional and structural role, respectively, according to the geometry analysis. Unexpectedly, 1,4-dioxane from the crystallization reagents was bound not only to the protein surface, but also to the heme distal pocket, providing insights into protein-ligand interactions for the globin and guiding the design of functional heme enzymes.

Coenzyme-catalyzed electroinitiated reversible addition fragmentation chain transfer polymerization for ultrasensitive electrochemical DNA detection.

Ultrasensitive detection of biomarkers at an early stage is generally limited by external influence factors such as high reaction temperature, complex operations, and sophisticated instruments. Here, we circumvent these problems by using nicotinamide adenine dinucleotide (NAD+) to control electroinitiated reversible addition fragmentation chain transfer (electro-RAFT) polymerization for biosensing that enables the detection of a few molecules of target DNA. In this coenzyme-catalyzed electro-RAFT polymerization, numerous ferrocenylmethyl methacrylate (FCMMA) as monomer with electrochemistry signal were linked to the biomarker on Au electrode. Afterwards, a strong oxidation peak appears at the potential of about 0.3 V that represents a typical oxidation potential of FCMMA. The sensitivity of this methodology was presented by detecting DNA from 10-1 to 104 fM concentration and detection limit (LOD) being down to 4.39 aM in 10 µL samples. This is lower by factors than detection limits of most other ultra-sensitive electrochemical DNA assays.

Ultrasensitive electrochemical detection of miRNA based on polymerization signal amplification.

The detection of trace tumor-related serum miRNA biomarkers is in great demand for the early diagnosis of cancer. Herein, for the first time, an electrochemical sensing platform based on atom transfer radical polymerization (ATRP) signal amplification strategy for ultrasensitive determination of the breast and prostate cancer marker miRNA-141 has been developed. The hairpin DNAs were immobilized on the benzoic acid modified electrode to capture the target miRNA-141, the recognition of miRNA-141 released thiol groups on the end of probes, followed by the association of ATRP initiators modified gold nanoparticles with thiol groups, and then triggered the polymerization on electrode surface, causing a great number of ferrocene (Fc) signal molecules grafted on the sensor interface. As a result, the electrochemical signal intensity of signal molecule has been greatly increased. The proposed biosensor has a linear range from 10 pM to 10 aM with a detection limit of 3.23 aM for miRNA-141, opening a new and promising path for ultrasensitive analysis of tumor-related miRNAs.

A highly selective and sensitive Zn2+ fluorescent sensor based on zinc finger-like peptide and its application in cell imaging.

Developing new chemosensors for detection of Zn2+ has attracted great attentions because of the important roles of Zn2+ in biological systems, and it will produce toxic effects with an excessive intake of zinc ion. Metalloproteins are often used as an effective template for the design and development of peptide-based fluorescent sensors. In this study, we designed a new and simple ratiometric fluorescent sensor for Zn2+, which was based on a zinc finger-like peptide and labeled with a dansyl group, i.e., Dansyl-His-Gln-Arg-Thr-His-Trp-NH2 (D-P6), by using solid phase peptide synthesis (SPPS). The dimeric peptide has a high affinity for Zn2+ overothermetalions, as indicated by spectroscopic studies, as well as molecular modeling. Remarkably, the sensor exhibited a highly selective and sensitive ratiometric fluorescent response to Zn2+ by fluorescent resonance energy transfer effect between tryptophan residue and fluorophore dansyl group, with a very low detection limit of 33 nM in aqueous solution. Furthermore, the sensor displayed a very low biotoxicity, which allows successful detection of Zn2+ in living HeLa cells. We believe that the new sensor may have potential applications in biological science.

Conversion of Human Neuroglobin into a Multifunctional Peroxidase by Rational Design.

Protein design has received much attention in the last decades. With an additional disulfide bond to enhance the protein stability, human A15C neuroglobin (Ngb) is an ideal protein scaffold for heme enzyme design. In this study, we rationally converted A15C Ngb into a multifunctional peroxidase by replacing the heme axial His64 with an Asp residue, where Asp64 and the native Lys67 at the heme distal site were proposed to act as an acid-base catalytic couple for H2O2 activation. Kinetic studies showed that the catalytic efficiency of A15C/H64D Ngb was much higher (∼50-80-fold) than that of native dehaloperoxidase, which even exceeds (∼3-fold) that of the most efficient native horseradish peroxidase. Moreover, the dye-decolorizing peroxidase activity was also comparable to that of some native enzymes. Electron paramagnetic resonance, molecular docking, and isothermal titration calorimetry studies provided valuable information for the substrate-protein interactions. Therefore, this study presents the rational design of an efficient multifunctional peroxidase based on Ngb with potential applications such as in bioremediation for environmental sustainability.

Ultrasensitive mercury ion and biothiol detection based on Dansyl-His-Pro-Gly-Asp-NH2 fluorescent sensor.

Mercury is an environmental contaminant, which is highly toxic even at extremely low concentrations. Long-term accumulation of mercury in human body will damage the central nervous system or digestive tract system. Here, a new fluorescent chemical sensor Dansyl-His-Pro-Gly-Asp-NH2 (D-P4) was synthesized for the determination of Hg2+. The D-P4 sensor exhibits excellent selectivity and sensitivity to Hg2+ in aqueous solution with a 'turn-off' fluorescence response. Furthermore, D-P4-Hg system displays a good 'turn-on' fluorescence response to biothiols. The calculated binding constant for the 1:1 complex of D-P4 with Hg2 + is 1.07 × 105 M-1, which also confirms the high affinity of D-P4 for Hg2+. Results indicate that the detection limit of D-P4 for Hg2+ is 61.0 nM, and that of D-P4-Hg system for Cys is as low as 80.0 nM.

A Highly Selective and Sensitive Peptide-Based Fluorescent Ratio Sensor for Ag.

A fluorescence ratio sensor based on dansyl-peptide, Dansyl-Glu-Cys-Glu-Glu-Trp-NH2 (D-P5), was efficiently synthesized by Fmoc solid phase peptide synthesis. The sensor exhibits high selectivity and sensitivity for Ag+ over 16 metal ions in 100 mM sodium perchlorate and 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer solution by fluorescence resonance energy transfer. The 1:1 binding stoichiometry of the sensor and Ag+ is measured by fluorescence ratio response and the job's plot. The dissociation constant of the sensor with Ag+ was calculated to be 6.4 × 10-9 M, which indicates that the sensor has an effective binding affinity for Ag+. In addition, the limit of detection of the sensor for Ag+ was determined to be 80 nM, which also indicates that the sensor has a high sensitivity to Ag+. Result showed that the sensor is an excellent Ag+ sensor under neutral condition. Furthermore, this sensor displays good practicality for Ag+ detection in river water samples without performing tedious sample pretreatment, as well as for silver chloride detection.