Highly fluorescent streptavidin-coated CdSe nanoparticles: preparation in water, characterization, and micropatterning

CdSe quantum dots (QDs) with a high fluorescence quantum yield of 25% and a narrow size distribution were synthesized in a single step in water using glutathione as a stabilizing molecule. The exceptional optical properties enabled for the first time the detection of in-water-prepared single quantum dots at room temperature. For application as fluorescent bioanalytical probes, the QDs were coated with streptavidin. These QDs self-assemble with high contrast on micropatterned biotin while preserving their optical properties and their capability to bind in addition biotinylated molecules, a prerequisite for the development of novel supramolecular structures and bioassays.     

Quantum dot-based immunosensor for the detection of prostate-specific antigen using fluorescence microscopy

A sensitive optical method based on quantum dot (QD) technology is demonstrated for the detection of an important cancer marker, total prostate-specific antigen (TPSA) on a disposable carbon substrate surface. Immuno-recognition was carried out on a carbon substrate using a sandwich assay approach, where the primary antibody (Ab)-protein A complex covalently bound to the substrate surface, was allowed to capture TPSA. After the recognition event, the substrate was exposed to the biotinylated secondary Abs. After incubation with the QD streptavidin conjugates, QDs were captured on the substrate surface by the strong biotin-streptavidin affinity. Fluorescence imaging of the substrate surface illuminated the QDs, and provided a very sensitive tool for the detection of TPSA in undiluted human serum samples with a detection limit of 0.25 ng/mL. The potential of this method for application as a simple and efficient diagnostic strategy for immunoassays is discussed.     

谷胱甘肽稳定的高性能CdTe/CdS核壳型量子点制备及应用于荧光免疫检测米醋中痕量黄曲霉毒素B1

谷胱甘肽作稳定剂水相合成CdTe/CdS核壳型量子点,以EDC/NHS为活化剂对黄曲霉毒素B1(AFB1)抗体进行量子点标记,然后用牛血清蛋白封闭抗体。通过对量子点和标记抗体性能的研究发现,CdTe/CdS核壳型量子点荧光的强度和稳定性较裸壳的CdTe量子点分别提高了4倍和2倍以上。由于谷胱甘肽碳链较长,量子点对抗体尤其是活性位点处的空间构型影响减少,从而改善了量子点标记抗体的稳定性和活性,CdTe/CdS标记的AFB1抗体与AFB1免疫前后荧光强度变化显示抗体至少可以稳定6 d。基于谷胱甘肽稳定的高性能CdTe/CdS量子点,建立了一种荧光免疫检测黄曲霉毒素B1的新方法。AFB1浓度在0.68～40 pmol/L之间荧光强度与浓度呈线性关系,相关系数(R2)为0.9914,检出限为0.3 pmol/L。方法已成功应用于米醋样品中痕量黄曲霉毒素B1的测定。     

Fabrication of immunosensor microwell arrays from gold compact discs for detection of cancer biomarker proteins

A simple method is reported to fabricate gold arrays featuring microwells surrounding 8-electrodes from gold compact discs (CDs) for less than $0.2 per chip. Integration of these disposable gold CD array chips with microfluidics provided inexpensive immunoarrays that were used to measure a cancer biomarker protein quickly at high sensitivity. The gold CD sensor arrays were fabricated using thermal transfer of laserjet toner from a computer-printed pattern followed by selective chemical etching. Sensor elements had an electrochemically addressable surface area of 0.42 mm(2) with RSD <2%. For a proof-of-concept application, the arrays were integrated into a simple microfluidic device for electrochemical detection of cancer biomarker interleukin-6 (IL-6) in diluted serum. Capture antibodies of IL-6 were chemically linked onto the electrode arrays and a sandwich immunoassay protocol was developed. A biotinylated detection antibody with polymerized horseradish peroxidase labels was used for signal amplification. The detection limit of IL-6 in diluted serum was remarkably low at 10 fg mL(-1) (385 aM) with a linear response with log of IL-6 concentration from 10 to 1300 fg mL(-1). These easily fabricated, ultrasensitive, microfluidic immunosensors should be readily adapted for sensitive detection of multiple biomarkers for cancer diagnostics.     

Patterning of quantum dot bioconjugates via particle lithography

We present a simple technique to fabricate hexagonally ordered quantum dot bioconjugate (QDBC) dot arrays on glass coverslips. We used particle lithography to create periodic holes in a layer of methoxy-poly(ethylene glycol)-silane and then adsorbed QDBCs into the holes. To demonstrate the versatility of this technique, we made separate periodic arrays of quantum dots (QDs) conjugated to three different biologically important molecules: biotin, streptavidin, and anti-mouse IgG. The diameters of the regions where the QDBCs adsorbed were 500-600 nm and independent of the QDBC patterned. The site density of the QDBCs in the patterned holes could be varied by simply adjusting the coating concentration of the QDBC solution. We demonstrate the applicability of these substrates by designing a QDBC-based binding assay with a working concentration range of several orders of magnitude and a sub-picomolar detection limit.     

Biotinylation of quantum dots for application in fluoroimmunoassays with biotin-avidin amplification

A competitive microplate fluoroimmunoassay was developed for the determination of human serum albumin in urine. It is based on the use of biotinylated CdTe quantum dots (QDs) whose synthesis is optimised in terms of storage stability, purification, and signal-to-noise ratio. The bioconjugated QDs were characterised by gel chromatography and gel electrophoresis. Storage stability and quantum yield were investigated. The excitation/emission wavelengths are 360/620?nm. The immunoassay of human serum albumin in urine has a working range from 1.7 to 10?μg.mL^?1, and the limit of detection is 1.0?μg.mL^?1.

Quantum Dot-Based Biosensor for the Detection of Human T-Lymphotropic Virus-1

A method was developed to identify human T-lymphotropic virus-1 (HTLV-1) using cadmium–tellurium quantum dots. Two probes including the biotin-labeled acceptor and NH2-reporter probes with target DNA were hybridized. The resulted sandwich complex was immobilized on a well containing streptavidin. The quantum dot solution was added to the sandwich complex, conjugated with the amine group of reporter probe, and emission spectra of the quantum dots were recorded. The biosensor response was linear with HTLV-1 concentrations from 10 pg/µl to 0.24?ng/µl, with a detection limit of 19.5 pg/µl. The assay may be successfully used for detection of long nucleic acids.     

Detection of proteins cross-linked within galactoside polyacrylate-based hydrogels by means of a quantum dot fluororeagent

Protein toxins have been immobilized in a galactoside polyacrylate hydrogel in a microarray format. The large pore size and solution-like environment of these novel hydrogels allow for easy penetration of large proteins and detection reagents. Confocal microscopy provided three-dimensional visualization of dye-labeled toxins cross-linked within the gel and of streptavidin-coated quantum dot (QD) fluorophores used to visualize the toxins after incubation with biotinylated anti-toxin antibodies. Fluorescence microscopy was utilized to visualize arrays of toxins detected by a biotinylated antibody and then exposure to streptavidin-conjugated QDs. The intensity of the QD fluorescence was quantified, and binding to two toxins on three types of hydrogels was examined.     

碳量子点/壳聚糖复合物的制备及用于槲皮素检测

以柠檬酸三钠、11-氨基十一烷、聚乙二醇400为碳源,利用微波法制备了碳量子点,将其与壳聚糖反应,制备出碳量子点/壳聚糖复合物。采用荧光、紫外、红外光谱等对碳量子点和碳量子点/壳聚糖复合物进行表征,探究了温度、时间、缓冲溶液及pH对体系荧光强度的影响。在pH 7.6的硼酸—硼砂缓冲介质中,槲皮素可使碳量子点/壳聚糖复合物发生荧光猝灭,其猝灭程度与槲皮素浓度呈良好的线性关系,据此建立了碳量子点/壳聚糖荧光猝灭法测定槲皮素的新方法,方法线性范围为4~40μmol/L,相关系数为0.9940,检出限为0.5μmol/L。方法已应用于测定本地甜瓜中槲皮素的含量。     

实时生物分子相互作用分析技术用于链霉亲和素-生物素化抗体的层层组装研究

使用生物分子相互作用分析（Biomolecular interaction analysis,BIA)技术实时监测了在链霉素和素表面层层组装亲和素－生物素化抗体多层膜的过程，结果表明，通过链霉素和素与生物素之间的强亲和作用，能够在表面形成均一的多层膜，并用实时BIA技术求得了每层蛋白质的表面浓度，对于生物素化抗体，单层吸附表面浓度为1.32ng/mm^2；对于链霉亲和素，单层吸附表面浓度为2.93ng/mm^2。同时对蛋白质在表面的排列状态进行了探讨。     

Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins

Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag–BirA system. Through the high streptavidin (SA)–biotin interaction, the divalent biotinylated APs were clustered in the SA–biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ–AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable for the ZZ protein.     

新型水溶性咪唑基硅量子点制备及用于果蔬中痕量铜的荧光检测

硅量子点因其极佳的亲生物性和光学性能成为纳米材料新宠,但传统硅量子点水溶性差限制了它的广泛应用。本实验以三甲基硅咪唑为硅前驱体采用水热法制备水溶性咪唑基硅量子点。相对于硼氢化钠、抗坏血酸、牛血清蛋白、半胱氨酸和柠檬酸,柠檬酸钠作为还原剂和稳定剂制得的硅量子点荧光发射最强。合成反应于220℃下可在2 h内完成,所制备的硅量子点水溶性好,平均粒径为2.6 nm,红外分析证实其表面存在游离的咪唑基。研究表明,硅量子点能与铜离子相互作用导致荧光强度的明显下降。考察不同温度下Cu2+对硅量子点荧光的猝灭行为,发现荧光猝灭程度随温度升高而增大。这说明荧光下降属于静态猝灭,即Cu2+与硅量子点上的咪唑基作用形成稳定配合物。此外,共振光散射分析还揭示荧光猝灭过程伴随着粒子团聚。基于硅量子点的荧光猝灭行为,建立了痕量铜的荧光检测方法。当Cu2+浓度在0.04~2400μmol/L之间,硅量子点的荧光强度随Cu2+浓度的增加而线性下降,检出限(S/N=3)达1.29×10-8 mol/L。本方法具有高的灵敏度、选择性和重现性,已应用于果蔬中痕量铜的荧光检测。     

Avidin: a natural bridge for quantum dot-antibody conjugates

We describe the preparation and characterization of bioinorganic conjugates in which luminescent semiconductor CdSe-ZnS core-shell nanocrystal quantum dots (QDs) were coupled to antibodies through the use of an avidin bridge adsorbed to the nanocrystal surface via electrostatic self-assembly. Avidin, a highly positively charged protein, was found to adsorb tightly to QDs modified with dihydrolipoic acid, which gives their surface a homogeneous negative charge. QD conjugation to biotinylated antibodies subsequently is readily achieved. Fluoroimmunoassays utilizing these antibody conjugated QDs were successful in the detection of protein toxins (staphylococcal enterotoxin B, cholera toxin). QD-antibody conjugates formed in such a facile manner permit their use as a common immuno reagent, and in the development of multianalyte detection.     

Microfluidic patterning of nanodisc lipid bilayers and multiplexed analysis of protein interaction

We report a microfluidic method for precisely patterning lipid bilayers and a multiplexed assay to examine the interaction between the lipids and protein analytes. The lipids were packaged into nanoscale lipid bilayer particles known as Nanodiscs and delivered to surfaces using microfluidic channels. Two types of lipids were used in this study: biontinylated lipids and phosphoserine lipids. The deposition of biotinylated lipids on a glass surface was confirmed by attaching streptavidin coated quantum dots to the lipids, followed by fluorescent imaging. Using this multiplexed grid assay, we examined binding of annexin to phosphoserine lipids, and compared these results to similar analysis performed by surface plasmon resonance.     

Inkjet-printed gold nanoparticle electrochemical arrays on plastic. Application to immunodetection of a cancer biomarker protein

Electrochemical detection combined with nanostructured sensor surfaces offers potentially low-cost, high-throughput solutions for detection of clinically significant proteins. Inkjet printing offers an inexpensive non-contact fabrication method for microelectronics that is easily adapted for incorporating into protein immunosensor devices. Herein we report the first direct fabrication of inkjet-printed gold nanoparticle arrays, and apply them to electrochemical detection of the cancer biomarker interleukin-6 (IL-6) in serum. The gold nanoparticle ink was printed on a flexible, heat resistant polyimide Kapton substrate and subsequently sintered to create eight-electrode arrays costing <0.2 euro per array. The inkjet-printed working electrodes had reproducible surface areas with RSD <3%. Capture antibodies for IL-6 were linked onto the eight-electrode array, and used in sandwich immunoassays. A biotinylated secondary antibody with 16-18 horseradish peroxidase labels was used, and detection was achieved by hydroquinone-mediated amperometry. The arrays provided a clinically relevant detection limit of 20 pg mL(-1) in calf serum, sensitivity of 11.4 nA pg(-1) cm(-2), and a linear dynamic range of 20-400 pg mL(-1).     

Fluorescence single-molecule counting assays for protein quantification using epi-fluorescence microscopy with quantum dots labeling

A single-molecule counting approach for quantifying the antibody affixed to a surface using quantum dots and epi-fluorescence microscopy is presented. Modifying the glass substrates with carboxyl groups provides a hydrophilic surface that reacts with amine groups of an antibody to allow covalent immobilization of the antibody. Nonspecific adsorption of single molecules on the modified surfaces was first investigated. Then, quantum dots were employed to form complexes with surface-immobilized antibody molecules and used as fluorescent probes for single-molecule imaging. Epi-fluorescence microscopy was chosen as the tool for single-molecule fluorescence detection here. The generated fluorescence signals were taken by an electron multiplying charge-coupled device and were found to be proportional to the sample concentrations. Under optimal conditions, a linear response range of 5.0 × 10⁻¹⁴-3.0 × 10⁻¹² mol L⁻¹ was obtained between the number of single molecules and sample concentration via a single-molecule counting approach.     

Quantum-dots based electrochemical immunoassay of interleukin-1α

We describe a quantum-dot (QD, CdSe@ZnS) based electrochemical immunoassay to detect a protein biomarker, interleukin-1α (IL-1α). QD conjugated with anti-IL-1α antibody was used as a label in an immunorecognition event. After a complete sandwich immunoreaction among the primary IL-1α antibody (immobilized on the avidin-modified magnetic beads), IL-1α, and the QD-labeled secondary antibody, QD labels were attached to the magnetic-bead surface through the antibody-antigen immunocomplex. Electrochemical stripping analysis of the captured QDs was used to quantify the concentration of IL-1α after an acid-dissolution step. The streptavidin-modified magnetic beads and the magnetic separation platform were used to integrate a facile antibody immobilization (through a biotin/streptavidin interaction) with immunoreactions and the isolation of immunocomplexes from reaction solutions in the assay. The voltammetric response is highly linear over the range of 0.5–50 ng ml⁻¹ IL-1α, and the limit of detection is estimated to be 0.3 ng ml⁻¹ (18 pM). This QD-based electrochemical immunoassay shows great promise for rapid, simple, and cost-effective analysis of protein biomarkers.     

Fluorescence Analysis in Microarray Technology

Fluorescence has been the preferred choice for data quantification in biomedical microarray formats since their earliest days. As much as the formats have grown and evolved over the years, the methods in optical analysis have become ever more sophisticated and complex in order to produce more and better output. This review will provide an insight into the most common methods and the state-of-the-art of all areas in microarray fluorescence analysis. Starting with an overview on microarray formats with a focus on their demands on the readout, the most common and useful organic fluorescent stains are discussed before proceeding on to other approaches; the use of semiconductor nanocrystals (quantum dots), polymer and silica nanoparticles and fluorescent proteins. Ways to enhance the intrinsically low signal on biochips have become increasingly important as they offer a sound approach towards the detection of low concentration sample content. The three main categories are presented: amplification using DNA, enzymes, and dendrimers. As much diversity as on the microarrays themselves can be found at the detection device. Standard optical microarray detectors, and non-standard methods using fluorescence anisotropy, fluorescence lifetime imaging (FLIM) and fluorescence resonance energy transfer (FRET), and their advantages and disadvantages are discussed.     

Adsorption and stability of streptavidin on cluster-assembled nanostructured TiOx films

The study of the adsorption of proteins on nanostructured surfaces is of fundamental importance to understand and control cell-surface interactions and, notably, cell adhesion and proliferation; it can also play a strategic role in the design and fabrication of nanostructured devices for postgenomic and proteomic applications. We have recently demonstrated that cluster-assembled nanostructured TiO x films produced by supersonic cluster beam deposition possess excellent biocompatibility and that these films can be functionalized with streptavidin, allowing the immobilization of biotinylated retroviral particles and the realization of living-cell microarrays for phenotype screening. Here we present a multitechnique investigation of the adsorption mechanisms of streptavidin on cluster-assembled TiO x films. We show that this nanostructured surface provides an optimal balance between adsorption efficacy and protein functionality. By using low-resolution protein arrays, we demonstrate that a layer of adsorbed streptavidin can be stably maintained on a cluster-assembled TiO x surface under cell culture conditions and that streptavidin retains its biological activity in the adsorbed layer. The adsorption mechanisms are investigated by atomic force microscopy in force spectroscopy mode and by valence-band photoemission spectroscopy, highlighting the potential role of the interaction of the exposed carboxyl groups on streptavidin with the titanium atoms of the nanostructured surface.     

硅烯量子点二聚物的等离激元激发

基于含时密度泛函理论,研究了随着间距改变时硅烯量子点二聚物的等离激元激发特性。沿垂直于硅烯所在平面方向激发时,在一定间距范围内,硅烯量子点二聚物中形成了长程电荷转移激发模式。参与长程电荷转移激发的π电子主要在两个量子点之间运动。该等离激元模式随着间隙的减小发生蓝移。此外,在不同间距时,体系中还有两个等离激元共振带,分别位于7和15 eV附近。沿平行于硅烯所在平面方向激发时,由于两个量子点之间的耦合,在低能