聚合物在荧光检测领域的应用

聚合物是由一种或几种重复单体以共价键连接形成的大分子化合物, 它不仅能够保持单体的性质, 而且由于聚合后单体间的协同作用, 使其表现出独特的性能. 聚合物作为基础材料在荧光检测领域得到广泛应用. 聚合物通过氢键作用、 亲疏水作用及范德华力等分子间相互作用, 实现了对特定目标物的选择性识别; 通过信号转换和放大功能, 可以将分子识别作用转化为荧光信号; 可以作为骨架连接多个识别单元, 通过多价结合作用等提高识别目标物的能力, 或连接不同的功能单元, 构建多功能的分子器件. 本文对聚合物在荧光检测领域的应用进行了概述.     

Utilization of nanoparticle labels for signal amplification in ultrasensitive electrochemical affinity biosensors: A review

Nanoparticles with desirable properties not exhibited by the bulk material can be readily synthesized because of rapid technological developments in the fields of materials science and nanotechnology. In particular their highly attractive electrochemical properties and electrocatalytic activity have facilitated achievement of the high level of signal amplification needed for the development of ultrasensitive electrochemical affinity biosensors for the detection of proteins and DNA. This review article explains the basic principles of nanoparticle based electrochemical biosensors, highlights the recent advances in the development of nanoparticle based signal amplification strategies, and provides a critical assessment of the likely drawbacks associated with each strategy. Finally, future perspectives for achieving advanced signal simplification in nanoparticles based biosensors are considered.     

Signal amplification using functional nanomaterials for biosensing

Signal amplification based on biofunctional nanomaterials has recently attracted considerable attention due to the need for ultrasensitive bioassays and the trend towards miniaturized assays. The biofunctional nanomaterials can not only produce a synergic effect among catalytic activity, conductivity and biocompatibility to accelerate the signal transduction, but also provide amplified recognition events by high loading of signal tags, leading to a highly sensitive and specific biosensing. Most importantly, nanoscaled materials are in direct contact with the environment, which permits them to act as chemical and biological sensors in single-molecule detection of biomolecules. In this tutorial review, we will focus on recent significant advances in signal amplification strategies combining the cross-disciplines of chemistry, biology, and materials science, and highlight some elegant applications of biofunctional nanomaterials as excellent electronic or optical signal tags in ultrasensitive bioanalysis. The biofunctional nanomaterials-based biosensing opens a series of concepts for basic research and offers new tools for detection of trace amounts of a wide variety of analytes in clinical, environmental, and industrial applications.     

Fluorescence Signal Amplification Strategies Based on DNA Nanotechnology for miRNA Detection

In this review,the most recent progresses in the field of fluorescence signal amplification strategies based on DNA nanotechnology for miRNA are summarized.The types of signal amplification are given and the principles of amplification strategies are explained,including rolling circle amplification(RCA),catalytic hairpin assembly(CHA),hybridization chain reaction(HCR)and DNA walker.Subsequently,the application of these signal amplification methods in biosensing and bioimaging are covered and described.Finally,the challenges and the outlook of fluorescence signal amplification methods for miRNA detection are briefly commented.     

Polymerization-assisted signal amplification for electrochemical detection of biomarkers

We present a novel immunosensor by using polymerization-assisted signal amplification strategy coupled with electrochemical detection. A sandwich immunoassay process was used to immobilize a polymerization reaction center, the initiator-conjugated polyclonal prostate specific antigen (PSA) or polyclonal carcinoembryonic antigen (CEA) antibodies on the surface of the electrode. Activator generated electron transfer for atom transfer radical polymerization (AGET ATRP) subsequently triggered the local accumulation of glycidyl methacrylate (GMA) monomers. Growth of long chain polymers provided excess epoxy groups for electrochemical tags aminoferrocene (FcNH(2)) coupling, which in turn significantly increased the loading of the signal molecules and enhanced the electrochemical readouts. The detection limit was ～0.14 pg mL(-1) for PSA and ～0.10 pg mL(-1) for CEA in PBS buffers. The proposed immunosensor was highly sensitive, selective and has a good match to the clinical electrochemiluminescent method. This suggested that the polymerization-assisted immunosensing strategy could be used as an effective method to significantly enhance signal output of the sandwich immunoassays and acted as a promising platform for the clinical screening of cancer biomarkers.     

DNA-Based Artificial Receptors as Transmembrane Signal Transduction Systems for Protocellular Communication

The ability to reproduce signal transduction and cellular communication in artificial cell systems is significant in synthetic protobiology. Here, we describe an artificial transmembrane signal transduction through low pH-mediated formation of the i-motif and dimerization of DNA-based artificial membrane receptors, which is coupled to the occurrence of fluorescence resonance energy transfer and the activation of G-quadruplex/hemin-mediated fluorescence amplification inside giant unilamellar vesicles. Moreover, an intercellular signal communication model is established when the extravesicular H⁺ input is replaced by coacervate microdroplets, which activate the dimerization of the artificial receptors, and subsequent fluorescence production or polymerization in giant unilamellar vesicles. This study represents a crucial step towards designing artificial signalling systems with environmental response, and provides an opportunity to establish signalling networks in protocell colonies.     

Ultrasensitive Fluorescence Polarization Aptasensors Based on Exonuclease Signal Amplification and Polystyrene Nanoparticle Amplification

Here, we combine T7 exonuclease (T7 Exo) signal amplification and polystyrene nanoparticle (PS NP) amplification to develop novel fluorescence polarization (FP) aptasensors. The binding of a target/open aptamer hairpin complex or a target/single‐stranded aptamer complex to dye‐labeled DNA bound to PS NPs, or the self‐assembly of two aptamer subunits (one of them labeled with a dye) into a target/aptamer complex on PS NPs leads to the cyclic T7 Exo‐catalyzed digestion of the dye‐labeled DNA or the dye‐labeled aptamer subunit. This results in a substantial decrease in the FP value for the amplified sensing process. Our newly developed aptasensors exhibit a sensitivity five orders of magnitude higher than that of traditional homogeneous aptasensors and a high specificity for the target molecules. These distinct advantages of our proposed assay protocol make it a generic platform for the design of amplified aptasensors for ultrasensitive detection of various target molecules.     

Studies in some new initiator systems for vinyl polymerization. Part I. Molecular halogens or halates as one component

Several new initiating systems involving a molecular halogen, halates, or perhalate as one of the components and a reducing metal salt, ammonia, an amine or an organic compound as the other component, have been introduced for the aqueous (and in a few cases the non-aqueous) polymerization of methyl methacrylate. Some of the resulting polymers have been subjected to endgroup analysis by the application of the ultrasensitive dye techniques recently developed in our laboratory. Reducing metal salt—halogen-initiated polymers are found to incorporate hydroxyl and halogen endgroups, while polymers initiated by amine—halogen systems incorporate hydroxyl and halogen as well as amino endgroups. However, in the ammonia—halogen system polymers incorporate only halogen endgroups. On the basis of the results of the endgroup analysis of the polymers an attempt has been made to explain the probable initiation mechanism with regard to the nature and identity of the initiating species involved in such processes.     

Potential controlling highly-efficient catalysis of wheat-like silver particles for electrochemiluminescence immunosensor labeled by nano-Pt@Ru and multi-sites biotin/streptavidin affinity

The potential controlling silver catalysis for Ru(bpy)(3)(2+) electrochemiluminescence (ECL) signal at a special potential -0.4～1.25 V was newly developed as the new ECL signal amplification strategy for ultrasensitive protein detection. Firstly, the wheat-like deposited silver (DpAg) particles were modified on the bare glass carbon electrode (GCE) surface by cyclic voltammetry deposition to capture the primary antibodies and then bind the antigen analytes. Secondly, as a sandwich immunoreaction format, the secondary antibodies conjugated with the Ru(bpy)(3)(2+)-doped Pt (Pt@Ru) nanoparticles by the multi-sites biotin/streptavidin (SA) affinity can be captured onto the electrode surface to generate ECL signal. In the proposed Ru(bpy)(3)(2+) ECL system without any co-reactant, the detected ECL signal was amplified due to following multiple amplification strategies: (1) the ECL catalysis for Ru(bpy)(3)(2+) was performed by electro-inducing the DpAg particles to generate Ag(+) ion and controlled by the special potential. The catalyzer Ag(+) was produced near the electrode surface and reproduced by cyclic potential scan, which improved the catalytic efficiency. (2) The amount of the ECL signal probes linked to secondary antibodies were amplified by the adsorption of Pt nanoparticles and the multiple sites bridge linkage of biotin/SA. These new multiple signal amplification strategies made the proposed ECL immunosensor achieve ultrasensitive detection for model protein human IgG with a detection limit down to 3 pg mL(-1), which can be further extended to the detection of disease biomarkers.     

A combining signal amplification of atom transfer radical polymerization and redox polymerization for visual biomolecules detection

A novel combination of atom transfer radical polymerization (ATRP) and redox polymerization is here used to allow instrument‐free visualization of special biomolecules for which dynamic polymer growth is used in signal amplification. In this method, the convenient and mild redox polymerization‐assisted amplification with cerium ammonium (IV) nitrate as oxidant at the second stage was achieved by directly using the hydroxyl groups from poly(hydroxyethyl methacrylate) (PHEMA) synthesized via ATRP at the first stage. The brushed polymers poly(hydroxylethyl methacrylate)‐branched‐poly (acrylamide) (PHEMA‐branched‐PAM) prepared by successive ATRP and redox polymerization in situ drastically grew up at the detected biomolecules spot to improve the visibility of biomolecule and simplify the detection procedure. With the proposed strategy, the signal amplification of streptavidin (SA) as model detected biomolecule was investigated on two different substrates such as silicon wafer and gold, respectively. As a result, detection limit of SA was demonstrated on the gold substrates where binding of 1.0 ng/mL SA was differentiable from the background using ellipsometry. Moreover, binding of 0.5 nmol/L DNA led to visually distinguishable spots on the gold surface under mild condition. The proposed method exhibited an efficient amplification performance for molecules detection, and paved a new way for visual diagnosis of biomolecules. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 2791–2799     

Chemosensory performance of molecularly imprinted fluorescent conjugated polymer materials

Fluorescent conjugated polymers are an attractive basis for the design of low detection limit sensing devices owing to their intrinsic signal amplification capability. A simple and universal method to rationally control or fine-tune the chemodetection selectivity of conjugated polymer materials toward a desired analytical target would further benefit their applications. In a quest of such a method we investigated a general approach to cross-linked molecularly imprinted fluorescent conjugated polymer (MICP) materials that possess an intrinsic capability for signal transduction and have potential to enhance selectivity and sensitivity of sensor devices based on conjugated polymers. To study these capabilities, we prepared an MICP material for the detection of 2,4,6-trinitrotoluene and related nitroaromatic compounds. We found the imprinting effect in this material to be based on analyte shape/size recognition being substantial and generally overcoming other competing thermodynamically determined trends. The described molecularly imprinted fluorescent conjugated polymers show remarkable air stability and photostability, high fluorescence quantum yield, and reversible analyte binding and therefore are advantageous for sensing applications due to the ability to "preprogram" their detection selectivity through a choice of an imprinted template.     

Synthesis and Characterization of Water‐Soluble Conjugated Glycopolymer for Fluorescent Sensing of Concanavalin A

A neutral polyfluorene derivative that contains 20 mol % 2,1,3‐benzothiadiazole (BT) is synthesized by Suzuki cross‐coupling polymerization. A cationic conjugated polymer A and an α‐mannose‐bearing polymer B are subsequently obtained through different post‐polymerization methods. As a result of the charged pendant groups or sugar‐bearing groups attached to the polymer side chains, both A and B show good water‐solubility. The titration of Concanavalin A (Con A) into polymer aqueous solution leads to different fluorescent responses for polymers A and B . Polymer A does not show any obvious fluorescence change upon interaction with Con A, whereas polymer B shows fluorescence increase in BT emission intensity when Con A is added. This is because of the specific interaction between α‐mannose and Con A, which induces polymer aggregation, and then facilitates energy transfer from the phenylene–fluorene segments to the BT units. A practical calibration curve ranging from 1 nM to 250 nM is obtained by correlating the changes in BT emission intensity with Con A concentration. The advantage of polymer B ‐based Con A macromolecular probe is that it shows signal increase upon Con A recognition, which is significantly different from other conjugated polymer‐based fluorescence quenching assays.     

Fluorescence Study of Energy Transfer in PMMA Polymers with Pendant Oligo‐Phenylene‐Ethynylenes

A spectroscopic characterization of polymers containing rigid π‐conjugated oligo(phenyleneethynylene) chromophores as well as oligo(phenyleneethynylene) and methyl methacrylate is presented. The polymers exhibit molar masses of up to 15 000 g mol^?1 and a degree of polymerization between 22 and 80. Emission measurements of the monomeric and polymeric species show that radiative as well as nonradiative rates are influenced by the degree of polymerization due to intramolecular interactions of chromophores pendant to the polymer backbone. Time‐resolved emission anisotropy measurements suggest that energy migrates within the polymers. Steady‐state emission anisotropy measurements also point to energy migration. Additionally, two oligo(phenyleneethynylene)s with different sizes of the conjugated system are copolymerized in order to enable energy trapping due to energy transfer. The shortened energy‐donor fluorescence lifetime within the donor–acceptor copolymers suggest energy transfer. Depending on the degree of polymerization, dispersion of the donor fluorescence lifetime is observed.     

细胞内源性分子辅助荧光信号放大策略及细胞成像

细胞内原位信号放大策略是检测低丰度内源性目标物的有效手段, 但多数信号放大策略依赖于外源性辅助物, 不可避免地改变细胞内微环境, 进而对机体造成一定干扰. 针对此问题, 可利用细胞内源性物质(如金属离子、 核酸、 蛋白酶等)实现原位荧光信号放大, 对不同生物标志物进行荧光成像, 此方法对低丰度靶分子检测及成像具有重要意义. 本文对内源性物质辅助信号放大及细胞内荧光成像相关研究进行了归纳整理, 介绍了内源性核酸、 酶、 蛋白质、 三磷酸腺苷(ATP)和金属离子辅助信号放大策略, 并探讨了其信号放大机理; 总结了内源性物质辅助信号放大探针在低丰度物质检测及成像方面的研究进展; 最后展望了该策略在细胞成像方面的优势及应用前景.     

A Duplex Polymerization Strategy for General,Programmable and High-Resolution Nanopore-Based Sensing

Nanopore sensing is highly promising in single molecular analysis but their broad applications have been challenged by the limited strategies that can transduce a target-of-interest into a specific and anti-false/inference signal, especially for solid-state nanopores with relatively lower resolution and higher noise. Here we report a high-resolution signal-production concept named target-induced duplex polymerization strategy (DPS). Through linking the same or different duplex substrates (DSs) with a special linker (L) and an optional structure tag (ST), the DPS can generate target-specific DS polymers with highly controllable duration times, duration intervals and even distinguished secondary tagging currents. Experimentally, DPS mono-polymerization of single DS and co-polymerization of multiple DSs has verified the duration time of a DPS product is the sum of those for each DS monomer. Tetrahedron-DNA structures with different sizes are used as the STs to provide needle-like secondary peaks for further resolution enhancement and multiplex assay. With these examples DPS represents a general, programmable and advanced strategy that may simultaneously provide size-amplification, concentration amplification, and signal-specificity for molecular recognition. It is also promisingly in various applications regarding to single molecular investigation, such as polymerization degree, structure/side chain conformation, programmable multiplex decoding and information index.     

Ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon mediated circular strand displacement polymerization and hyperbranched rolling circle amplification

Using a cascade signal amplification strategy, an ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon (MB) mediated circular strand displacement polymerization (CSDP) and hyperbranched rolling circle amplification (HRCA) was proposed. The hybridization of MB probe to target DNA resulted in a conformational change of the MB and triggered the CSDP in the presence of bio-primer and Klenow fragment (KF exo⁻), leading to multiple biotin-tagged DNA duplex. Furthermore, the HRCA was implemented to product amounts of double-stranded DNA (ds-DNA) fragments using phi29 DNA polymerase via biotin-streptavidin interaction. After the product of HRCA binded numerous biotinylated detection probes, an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor exhibited excellent detection sensitivity and specificity with a log-linear response to target DNA from 0.01 fM to 10 pM as low as 8.9 aM. The proposed method allowed DNA detection with simplicity, rapidness, low cost and high specificity, which might have the potential for application in clinical molecular diagnostics and environmental monitoring.     

Graft polymer synthesis by RAFT transfer-to

The synthesis of graft polymers via controlled polymerization techniques has enabled the facile modification of the surface properties of a variety of substrates. Three grafting methods are typically highlighted in the literature: grafting-to, grafting-from, and grafting-through. However, a fourth method exists when grafting is conducted using reversible addition-fragmentation chain transfer (RAFT) polymerization, which we refer to as transfer-to. Transfer-to differs from the other grafting strategies in the types of structural defects and impurities that arise during polymerization. This review addresses important considerations when conducting RAFT transfer-to, including RAFT chain transfer agent selection, monomer structure, and reaction conditions. In addition, we highlight key mechanistic differences between grafting-from and transfer-to and their effects on the structure and sample composition of the resulting graft polymers. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 2865–2876     

Optimization of the molecular orbital energies of conjugated polymers for optical amplification of fluorescent sensors

Cationic water-soluble poly(fluorene-co-phenylene)s with electron withdrawing or donating substituents on the conjugated backbone were designed and synthesized. Fluorescence resonance energy transfer (FRET) experiments between these conjugated polymers and dye-labeled single-stranded DNA (ssDNA-C*) reveal the importance of matching donor and acceptor orbital energy levels to improve the sensitization of C* emission. Quenching of polymer fluorescence with ssDNA-C* and differences in C* emission suggest involvement of photoinduced charge transfer (PCT) as an energy wasting mechanism. The HOMO and LUMO energy levels of the conjugated polymers and C serve as a preliminary basis to understand the competition between FRET and PCT. Dilution of C in polymer/ssDNA-C complexes by addition of ssDNA yields insight into C*...C self-quenching. Under optimized conditions, where there is no probe self-quenching and minimum PCT, efficient signal amplification is demonstrated despite poor spectral overlap between polymer and C.     

Cellobiose dehydrogenase modified electrodes: advances by materials science and biochemical engineering

The flavocytochrome cellobiose dehydrogenase (CDH) is a versatile biorecognition element capable of detecting carbohydrates as well as quinones and catecholamines. In addition, it can be used as an anode biocatalyst for enzymatic biofuel cells to power miniaturised sensor–transmitter systems. Various electrode materials and designs have been tested in the past decade to utilize and enhance the direct electron transfer (DET) from the enzyme to the electrode. Additionally, mediated electron transfer (MET) approaches via soluble redox mediators and redox polymers have been pursued. Biosensors for cellobiose, lactose and glucose determination are based on CDH from different fungal producers, which show differences with respect to substrate specificity, pH optima, DET efficiency and surface binding affinity. Biosensors for the detection of quinones and catecholamines can use carbohydrates for analyte regeneration and signal amplification. This review discusses different approaches to enhance the sensitivity and selectivity of CDH-based biosensors, which focus on (1) more efficient DET on chemically modified or nanostructured electrodes, (2) the synthesis of custom-made redox polymers for higher MET currents and (3) the engineering of enzymes and reaction pathways. Combination of these strategies will enable the design of sensitive and selective CDH-based biosensors with reduced electrode size for the detection of analytes in continuous on-site and point-of-care applications.     

RAFT试剂N-咔唑二硫代甲酸1,4-对二甲基苯双酯的合成及应用

以咔唑和对二氯甲基苯为原料, 合成了以咔唑为Z基团的双功能团RAFT聚合链转移试剂N-咔唑二硫代甲酸1,4-对二甲基苯双酯(PXCBD). 以PXCBD为链转移试剂, 以苯乙烯、丙烯酸甲酯及N,N-二丁基丙烯酰胺为单体, 考察了PXCBD在RAFT聚合中合成多嵌段共聚物上的应用, 并研究了PXCBD及由其合成的聚合物的荧光特性. 研究结果表明, PXCBD是一种性能优异的双功能团RAFT聚合链转移试剂, 可用于合成特殊结构并且带有荧光标识的功能高分子材料.