广东兰科植物多样性保育现状

为了使广东省的兰科植物及其遗传多样性得到有效的保育, 保存我国重要野生植物资源, 在2017-2019年间, 采用样线和样地相结合的调查手段、专家快速评估和野外调查相结合的评估技术以及Wilcoxon符号秩检验和Friedman检验的统计方法, 对广东省自然分布的兰科植物进行了全面的调查和濒危等级评估, 并对其在广东省自然保护区中的就地保育情况和全国植物园中的迁地保育情况进行了综合分析。结果表明, 广东省分布有兰科植物80属235种, 其中广东特有种20种; 广东兰科植物受威胁物种有186种, 其中极危11种、濒危114种、易危61种; 就地保育的兰科植物有111种, 迁地保育的兰科植物有156种, 就地和迁地共同保育的兰科植物有96种, 保育的有效程度较低; 另外, 就地、迁地、就地和迁地共同保育的兰科植物之间没有体现出明显的差异, 保育工作缺乏选择性和针对性。基于此, 我们建议广东兰科植物的保育工作应重视基础数据的收集和持续的野外监测、提高保育物种的数量、优化迁地保育物种的选择性和针对性、完善迁地保育和就地保育之间的协同性, 同时也应重视立法和公众教育, 并构建广东兰科植物保育的网络系统。     

An array of field-effect nanoplate SOI capacitors for (bio-)chemical sensing

An array of individually addressable nanoplate field-effect capacitive (bio-)chemical sensors based on an SOI (silicon-on-insulator) structure has been developed. The isolation of the individual capacitors was achieved by forming a trench in the top Si layer with a thickness of 350 nm. The realized sensor array allows addressable biasing and electrical readout of multiple nanoplate EISOI (electrolyte-insulator-silicon-on-insulator) capacitive biosensors on the same SOI chip as well as differential-mode measurements. The feasibility of the proposed approach has been demonstrated by realizing sensors for the pH and penicillin concentration detection as well as for the label-free electrical monitoring of polyelectrolyte multilayers formation and DNA (deoxyribonucleic acid)-hybridization event. A potential change of ～ 120 mV has been registered after the DNA hybridization for the sensor immobilized with perfectly matched single-strand DNA, while practically no signal changes have been observed for a sensor with fully mismatched DNA. The realized examples demonstrate the potential of the nanoplate SOI capacitors as a new basic structural element for the development of different types of field-effect biosensors.     

试论植物园功能变迁与中国国家植物园体系建设

植物资源是自然生态系统的基本组成部分, 是经济社会可持续发展的重要物质来源, 植物多样性是关系到国家生态安全和生物安全的战略资源。就地保护和迁地保护是植物多样性保护的两种主要方法, 构建以国家公园为主体的自然保护地体系是就地保护的主要形式, 构建以国家植物园为引领的植物园体系是迁地保护的主要形式, 二者相辅相成, 共同形成我国较为完整的植物多样性保护体系。通过建设国家植物园体系对我国植物多样性进行迁地保护, 同时开展科学研究、园林展示、科普教育和资源开发利用, 对深入推进生态文明建设和高质量发展具有重要意义。本文回顾了植物园的功能变迁、全球和中国植物园分布与数量以及植物迁地保护现状,讨论了植物园与植物迁地保护的关系, 在此基础上, 提出了我国国家植物园的定义及设立标准, 进而讨论了建设国家植物园体系的意义、挑战、统筹迁地保护和就地保护等问题, 最后提出了我国国家植物园体系的建设目标、管理体制、空间布局和认证等方面的建议, 以期为我国的国家植物园体系建设提供参考。     

Highly specific label-free protein detection from lysed cells using internally referenced microcantilever sensors

We report the investigation of label-free protein detection directly from lysed cells using microcantilever sensors. The integration of an internally referenced microcantilever sensor combined with peptide aptamer technology enables scalable and label-free detection of proteins from a complex biological environment (e.g. cell lysate). The internally referenced microcantilever sensor was found to be effective in minimizing both the effects of thermal drift and non-specific binding interactions with the backside of the cantilever, thereby allowing protein detection in a complex biological background. Highly specific peptide aptamers are used to modify the cantilever surface to specifically detect less than 80nM CDK2 protein from yeast cell lysate. This binding of CDK2 on the microcantilever generates a tensile surface stress of average magnitude equal to 70+/-22mN/m. Similar experiments conducted with quartz crystal microbalance (QCM) technology are consistent with the response observed using microcantilever sensors.     

对国家植物园体系建设“统筹原则”的一些见解

我国建设国家植物园体系所提出的“统筹就地保护与迁地保护相结合的原则”是进一步推动《生物多样性公约》在我国实施的一个重要新举措。这需要国家植物园对其不同功能植物栽培保存种群大小的统筹协调,需要与我国其他植物园的濒危植物迁地保护统筹协调并注重统筹提升我国植物迁地保护的有效性等。此外,由于我国一些植物园与国际顶级植物园几乎都是同步开展受严重威胁的濒危植物迁地保护及其科学研究,所取得的成果各有千秋,而且在保护的有效性上都存在着或多或少的问题,所以“国家植物园将对标世界顶级植物园”的提法是值得商榷的,我们的做法应是“他山之石,可以攻玉”。     

Plant species with extremely small populations (PSESP) in China: A seed and spore biology perspective

Approximately one fifth of the world's plants are at risk of extinction. Of these, a significant number exist as populations of few individuals, with limited distribution ranges and under enormous pressure due to habitat destruction. In China, these most-at-risk species are described as ‘plant species with extremely small populations’ (PSESP). Implementing conservation action for such listed species is urgent. Storing seeds is one of the main means of ex situ conservation for flowering plants. Spore storage could provide a simple and economical method for fern ex situ conservation. Seed and spore germination in nature is a critical step in species regeneration and thus in situ conservation. But what is known about the seed and spore biology (storage and germination) of at-risk species? We have used China's PSESP (the first group listing) as a case study to understand the gaps in knowledge on propagule biology of threatened plant species. We found that whilst germination information is available for 28 species (23% of PSESP), storage characteristics are only known for 8% of PSESP (10 species). Moreover, we estimate that 60% of the listed species may require cryopreservation for long-term storage. We conclude that comparative biology studies are urgently needed on the world's most threatened taxa so that conservation action can progress beyond species listing.     

Conducting polymer nanowires-based label-free biosensors

Label-free sensing technologies have recently attracted a great deal of interest for sensitive, rapid and facile analysis for applications in health care, environmental monitoring, food safety and homeland security. One-dimensional (1-D) nanostructures such as nanowires, configured as field-effect transistors (FETs)/chemiresistors that change conductance upon binding of charged macromolecules to receptors linked to the device surfaces are extremely attractive for label-free biosensors. Herein, we review recent advances in label-free biosensors based on conducting polymer nanowires based FET/chemiresistor. Specifically, we address the fabrication, functionalization, assembly/alignment and sensing applications of FET/chemiresistor based on these nanomaterials. The advantages and disadvantages of various fabrication, functionalization, and assembling procedures of these nanosensors are reviewed and discussed.     

In situ, in-liquid, all-electrical detection of Salmonella typhimurium using lead titanate zirconate/gold-coated glass cantilevers at any dipping depth

Most biosensing techniques are indirect, slow, and require labeling. Even though silicon-based microcantilever sensors are sensitive and label-free, they are not suitable for in-liquid detection. More recently lead zirconate titanate (PZT) thin-film-based microcantilevers are shown to be sensitive and in situ. However, they require microfabrication and must be electrically insulated. In this study, we show that highly sensitive, in situ, Salmonella typhimurium detection can be achieved at 90% relative humidity using a lead zirconate titanate (PZT)/gold-coated glass cantilever 0.7 mm long with a non-piezoelectric 2.7 mm long gold-coated glass tip by partially dipping the gold-coated glass tip in the suspension at any depth without electrically insulating the PZT. In particular, we showed that at 90% relative humidity and with a dipping depth larger than 0.8 mm the PZT/gold-coated glass cantilever showed virtually no background resonance frequency up-shift due to water evaporation and exhibited a mass detection sensitivity of Δm/Δf = −5 × 10⁻¹¹ g/Hz. The concentration sensitivities of this PZT/gold-coated glass cantilever were 1 × 10³ and 500 cells/ml in 2 ml of liquid with a 1 and 1.5 mm dipping depth, respectively, both more than two orders of magnitude lower than the infectious dose and more than one order of magnitude lower than the detection limit of a commercial Raptor sensor.     

中国野生植物保护管理的政策、法律制度分析和建议

中国是全球生物多样性最丰富的国家之一。最近40年, 中国的植物多样性保护取得了巨大成就, 实施了多项政策和法律, 尤其是《野生植物保护条例》和《国家重点保护野生植物名录》先后颁布, 奠定了中国植物保护的法律和政策框架, 就地保护和迁地保护网络基本形成。但与生态文明建设的要求相比, 野生植物保护依然存在许多不足。本文系统回顾了中国野生植物保护管理的政策和法律制度, 从就地保护、迁地保护、开发利用活动管理三方面分析了其优缺点并提出建议; 重点对修订《野生植物保护条例》进行讨论并提出建议, 包括修订野生植物和人工培植的定义、优化对开发利用活动的管理程序、加强国际法和国内法的衔接、细化优化罚则等。     

Nanomechanics: Opening new frontiers in bio analyses and diagnostics

Micro-fabricated silicon cantilevers arrays offer a novel label-free approach where ligand-receptor binding interactions occurring on the sensor generate nanomechanical signals like bending or a change in mass that is optically detected in-situ. We report the detection of multiple unlabelled biomolecules simultaneously down to picomolar concentrations within minutes. Differential measurements including reference cantilevers on an array of eight sensors enables sequence-specifically detection of unlabelled DNA and is suitable to detect specific gene fragments within a complete genome (gene fishing). Expression of detection of inducible genes as well as the ultimate challenge: the detection of total RNA fragments in a unspecific back ground will be shown. Ligand-receptor binding interactions, such as antigen recognition will be presented. Antibody activated cantilevers with sFv (single chain fragments) which bind to the indicator proteins show a significant improved sensitivity which is comparable with SPR (Surface Plasmon Resonance). In addition this technology offers a brought variety of receptor molecules application such as e.g. membrane protein recognition, micro-organism detection, enantiomeric separation. New coating procedures, enlargement of the active surface area by dendritic molecules as well as improvement of the receptor-cantilever chemical bond will be presented. This new findings may lead to a novel individual diagnostic assay in a combined label-free GENOMIC and PROTEOMIC biomarker sensor (COMBIOSENS).     

The interplay between pH sensitivity and label-free protein detection in immunologically modified nano-scaled field-effect transistor

We present experimental results in order to establish a correlation between pH sensitivity of immunologically modified nano-scaled field-effect transistor (NS-ImmunoFET) with their sensing capacity for label-free detection. The NS-ImmunoFETs are fabricated from silicon-on-insulator (SOI) wafers and are fully-depleted with thickness of ~20 nm. The data shows that higher sensitivity to pH entails enhanced sensitivity to analyte detection. This suggests that the mechanism of analyte detection as pure electrostatic perturbation induced by antibody-analyte interaction is over simplified. The fundamental assumption, in existing models for field-effect sensing mechanism assumes that the analyte molecules do not directly interact with the surface but rather stand 'deep' in the solution and away from the dielectric surface. Recent studies clearly provide contradicting evidence demonstrating that antibodies lie down flat on the surface. These observations led us to propose that the proteins that cover the gate area intimately interact with active sites on the surface thus forming a network of interacting sites. Since sensitivity to pH is directly correlated with the amount of amphoteric sites, we witness a direct correlation between sensitivity to pH and analyte detection. The highest and lowest threshold voltage shift for a label-free and specific detection of 6.5 nM IgG were 40 mV and 2.3 mV for NS-ImmunoFETs with pH sensitivity of 35 mV/decade and 15 mV/decade, respectively. Finally, physical modeling of the NS-ImmunoFET is presented and charge of a single IgG protein at pH 6 is calculated. The obtained value is consistent with charge of IgG protein cited in literature.     

Extended-gate FET-based enzyme sensor with ferrocenyl-alkanethiol modified gold sensing electrode

We developed a field-effect transistor (FET)-based enzyme sensor that detects an enzyme-catalyzed redox-reaction event as an interfacial potential change on an 11-ferrocenyl-1-undecanethiol (11-FUT) modified gold electrode. While the sensitivity of ion-sensitive FET (ISFET)-based enzyme sensors that detect an enzyme-catalyzed reaction as a local pH change are strongly affected by the buffer conditions such as pH and buffer capacity, the sensitivity of the proposed FET-based enzyme sensor is not affected by them in principle. The FET-based enzyme sensor consists of a detection part, which is an extended-gate FET sensor with an 11-FUT immobilized gold electrode, and an enzyme reaction part. The FET sensor detected the redox reaction of hexacyanoferrate ions, which are standard redox reagents of an enzymatic assay in blood tests, as a change in the interfacial potential of the 11-FUT modified gold electrode in accordance with the Nernstian response at a slope of 59 mV/decade at 25 degrees C. Also, the FET sensor had a dynamic range of more than five orders and showed no sensitivity to pH. A FET-based enzyme sensor for measuring cholesterol level was constructed by adding an enzyme reaction part, which contained cholesterol dehydrogenase and hexacyanoferrate (II)/(III) ions, on the 11-FUT modified gold electrode. Since the sensitivity of the FET sensor based on potentiometric detection was independent of the sample volume, the sample volume was easily reduced to 2.5 microL while maintaining the sensitivity. The FET-based enzyme sensor successfully detected a serum cholesterol level from 33 to 233 mg/dL at the Nernstian slope of 57 mV/decade.     

基于双模型融合的大熊猫头部图像分割

在大熊猫(Ailuropoda melanoleuca)的迁地保护和种群饲养管理中,及时、快速地进行个体识别和行为监测,对其健康管理具有至关重要的作用。圈养大熊猫健康状况通常由专门的饲养人员肉眼观测,人力成本高、效率低并且缺乏时效性。基于图像的动物个体识别与行为分析技术效率高、时间成本低,已经成为新的监测发展趋势。已有研究提出,通过大熊猫面部图像的检测和分析,可实现个体识别和行为分类。但该方法依然存在检测精度不足导致识别准确率难以提升的问题。本文提出一种基于YOLOv3和Mask R-CNN的双模型融合方法,实现了大熊猫头部图像分割和精准检测。包含3个部分：YOLOv3完成头部检测,Mask R-CNN完成大熊猫轮廓分割,然后将两个模型的输出进行交并比融合。结果显示,头部检测准确率为82.6%,大熊猫轮廓分割准确率为95.2%,总体头部轮廓分割准确率为87.1%。该方法对大熊猫头部图像的识别率和分割准确率高,为大熊猫的个体识别、性别分类提供了帮助,为行为分析提供了技术参考。     

Detection of DNA hybridization and extension reactions by an extended-gate field-effect transistor: characterizations of immobilized DNA-probes and role of applying a superimposed high-frequency voltage onto a reference electrode

As we have already shown in a previous publication [Kamahori, M., Ihige, Y., Shimoda, M., 2007. Anal. Sci. 23, 75-79], an extended-gate field-effect transistor (FET) sensor with a gold electrode, on which both DNA probes and 6-hydroxyl-1-hexanethiol (6-HHT) molecules are immobilized, can detect DNA hybridization and extension reactions by applying a superimposed high-frequency voltage to a reference electrode. However, kinetic parameters such as the dissociation constant (K(d)(s)) and the apparent DNA-probe concentration (C(probe)(s)) on a surface were not clarified. In addition, the role of applying the superimposed high-frequency voltage was not considered in detail. In this study, the values of K(d)(s) and C(probe)(s) were estimated using a method involving single-base extension reaction combined with bioluminescence detection. The value of K(d)(s) on the surface was 0.38 microM, which was about six times that in a liquid phase. The value of C(probe)(s), which expressed the upper detection limit for the solid phase reaction, was 0.079 microM at a DNA-probe density of 2.6 x 10(12)molecules/cm(2). We found that applying the superimposed high-frequency voltage accelerated the DNA molecules to reach the gold surface. Also, the distance between the DNA-probes immobilized on the gold surface was controlled to be over 6 nm by applying a method of competitive reaction with DNA probes and 6-HHT molecules. This space was sufficient to enable the immobilized DNA-probes to lie down on the 6-HHT monolayer in the space between them. Thus, the FET sensor could detect DNA hybridization and extension reactions by applying a superimposed high-frequency voltage to the DNA-probes density-controlling gold surface.     

场效应晶体管生物传感器在生物医学检测中的应用研究进展*

场效应晶体管生物传感器因其灵敏度高、分析速度快、无标记、体积小、操作简单等特点而受到了很多关注,广泛应用于DNA、蛋白质、细胞、离子等生物识别物的检测。近年来,更有纳米材料和微电子技术在传感器设计中提高传感器的传感性能,场效应晶体管生物传感器朝着高灵敏、微型化、快速化以及多功能化的方向以令人惊叹的速度发展。研究场效应晶体管生物传感器工作原理,阐述近年来场效应晶体管生物传感器在生物医学检测领域中最新的研究进展与应用,探讨场效应晶体管生物传感器克服各种缺陷的应对策略,为该传感器在未来生物医学检测中的开发提供参考。     

Multiplexed detection of cardiac biomarkers in serum with nanowire arrays using readout ASIC

Early detection of cardiac biomarkers for diagnosis of heart attack is the key to saving lives. Conventional method of detection like the enzyme-linked immunosorbent assay (ELISA) is time consuming and low in sensitivity. Here, we present a label-free detection system consisting of an array of silicon nanowire sensors and an interface readout application specific integrated circuit (ASIC). This system provides a rapid solution that is highly sensitive and is able to perform direct simultaneous-multiplexed detection of cardiac biomarkers in serum. Nanowire sensor arrays were demonstrated to have the required selectivity and sensitivity to perform multiplexed detection of 100 fg/ml troponin T, creatine kinase MM, and creatine kinase MB in serum. A good correlation between measurements from a probe station and the readout ASIC was obtained. Our detection system is expected to address the existing limitations in cardiac health management that are currently imposed by the conventional testing platform, and opens up possibilities in the development of a miniaturized device for point-of-care diagnostic applications.     

A CMOS label-free DNA sensor using electrostatic induction of molecular charges

This paper reports a label-free biosensor for the detection of DNA hybridization. The proposed biosensor measures the surface potential on oligonucleotide modified electrodes using a direct charge accumulation method. The sensor directly and repeatedly measures the charges induced in the working electrode, which correspond to intrinsic negative charges in immobilized molecules. The sensor achieves an improved signal-to-noise ratio (SNR), through the oversampling effect of accumulation for charges and the differential architecture. The sensor also shows stable, robust, and reproducible measurement independent of slight changes in the reference voltage, unlike previous ion-sensitive field effect transistors (ISFETs), providing the benefits of choosing a wide variety of reference electrode materials. The proposed device is integrated with working electrodes, a reference electrode and readout circuits into one package via a 0.35 μm complementary metal-oxide-semiconductor (CMOS) process. The sensor achieves a detectable range of 88.3 dB and a detection limit of 36 μV for surface potential. It is demonstrated that the sensor successfully achieves specific detection of oligonucleotide sequences derived from the H5N1 avian influenza virus. The experiments show a limit of detection of 100 pM and include a single-base mismatch test in 18-mer oligonucleotides.     

CMOS-compatible, label-free silicon-nanowire biosensors to detect cardiac troponin I for acute myocardial infarction diagnosis

A label-free biosensor for electrical detection of cardiac troponin I (cTnI), a highly sensitive and selective biomarker of acute myocardial infarction (AMI), is demonstrated using silicon nanowire (SiNW) based field-effect transistors (FETs). The FET devices were fabricated by a complementary metal oxide semiconductor (CMOS) compatible top-down approach to define the SiNW followed by tetramethylammonium hydroxide (TMAH) wet etching. Electrical characterizations of the SiNW FET revealed an ambipolar conduction characteristic with an on/off ratio of 10(5)-10(6). CTnI monoclonal antibodies were then covalently immobilized on the SiNW surfaces. By integrating with a homemade biosensor measurement system, the biosensor exhibited rapid and sensitive response to cTnI proteins. The current response showed a nature of logarithm relationship against the cTnI concentration from 46 ng/mL down to 0.092 ng/mL. Moreover, an anti-interference capability of the fabricated biosensor was also assessed. By utilizing the top-down fabrication method, this work provides an efficient way for the cTnI proteins detection with an enormous potential of mass-production, which definitely facilitate the practical applications.     

An Enzymic Method of Preparing Plant Chromosomes for In Situ Hybridization

A method of preparing chromosomes from plant root tips for in situ hybridization with tritiated DNA is described. The technique relies on the enzymic hydrolysis of plant cell walls with a pectinase-cellulase mixture. It is shown that, despite the enzymic mixture possessing nuclease activity, there is no detectable degradation of DNA within fixed root tips. To demonstrate the suitability of this method of preparing plant chromosomes for in situ hybridization, a cloned repetitive DNA sequence has been hybridized to Allium sativum chromosomes. Chromosomes prepared using this technique also can be readily G-banded.     

长江流域林木资源的重要性及种质资源保护

中国长江流域有着丰富的林木资源, 包含极高水平的物种多样性、特有性和遗传多样性。根据考古证据, 在旧石器和新石器时代长江文明早期的孕育与发展中, 林木在食物、能源、工具、建筑和舟船中的应用起到了关键作用。现在, 长江流域和珠江流域逐渐成为国内木材供给的热点地区。面对木材供给总量不足和大径级木材结构性短缺问题, 长江流域林木资源将是未来国内木材安全的重要保障。这使得林木种质资源的保护更加迫切。针对长江流域林木种质资源保护存在的家底不清和保存体系不完善问题, 我们应尽快完成林木种质资源的全面调查和重要树种的多样性分析; 完善原地、异地和设施保存相结合的保存体系。