Label-free characterisation of oligonucleotide hybridisation using reflectometric interference spectroscopy

The potential of a label-free detection method, reflectometric interference spectroscopy (RIfS), for temperature-dependent DNA hybridisation experiments (for example in single nucleotide polymorphism (SNP) analysis) is investigated. Hybridisations of DNA, peptide nucleic acid (PNA), and locked nucleic acid (LNA) to a single stranded DNA were measured for several temperatures, and the melting curves and temperatures were calculated from the changes in optical thickness obtained. These measurements were performed by hybridising surface-immobilised single stranded oligomers with their complementary ssDNA or with ssDNA containing SNPs at different temperatures. DNA was compared to its analogue oligomers PNA and LNA due to their stability against nuclease. A comparison of melting temperatures demonstrated the higher binding affinities of the DNA analogues. Moreover, a continuous melting curve was obtained by first hybridising the functionalised surface with its complementary DNA at room temperature and then heating up in-flow. Measurement of the continuous melting curve was only possible due to the insensitivity of the RIfS method towards temperature changes. This is an advantage over other label-free detection methods, which are based on determining the refractive index.Dedicated to the memory of Wilhelm Fresenius.     

Labelless impedance immunosensor based on polypyrrole-pyrolecarboxylic acid copolymer for hCG detection

In this work, a sensitive label-free impedimetric hCG-immunosensor was constructed by using a commercial screen-printing carbon ink electrode (namely disposable electrochemical printed chip) as the basis. The carbon ink electrode of DEP chip is modified first by deposition of polypyrrole-pyrole-2-carboxylic acid copolymer and thence hCG antibody immobilization via the COOH groups of pyrrole-2-carboxylic acid, which can serve as a linker for covalent biomolecular immobilization. The experimental results exposed that the designed immunosensor is more sensitive than other previously reported immunosensors, in the case of detection limit and linear range for antigen detection. With optimal fabrication parameters, the detection limit for α-hCG was 2.3 pg/mL in 10 mM phosphate buffer saline (PBS) solution containing 1% bovine serum albumine (BSA). Moreover, the use of inexpensive DEP chip as a basis for these immunosensors will allow for simple instrumentation, disposable and portable at low cost. This work also demonstrates a new approach to develop a sensitive and label-free impedimetric immunosensor based on screen-printed electrode for applications in clinical diagnosis.     

Recent Advances in Plasmonic Nanostructures Applied for Label-free Single-cell Analysis

Cellular heterogeneity presents a major challenge in understanding the relationship between cells of particular genotype and response in disease. In order to characterize the cell-to-cell differences during the biochemical processes, single-cell analysis is necessary. Profiting from the unique localized surface plasmon resonance (LSPR) and Mie scattering, plasmonic nanostructures have revealed stable and adjustable scattering signals, avoiding photobleaching, blinking and autofluorescence phenomenon. These characterizations are propitious to the dynamic trace and biological image of single living cells. In this review, we discuss the recent advances in plasmonic nanostructures applied for label-free detection and monitoring of target cells at single-cell level by using three different techniques, surface-enhanced Raman scattering (SERS), surface-enhanced Infrared absorption spectroscopy (SEIRAS), and dark-field microscopy. Various avenues to design plasmonic probes combining spectra and imaging for single-cell analysis are demonstrated as well. We hope this review can highlight the superiority of plasmonic nanostructures in single cellular analysis, and further motivate the development of label-free cell analysis technique to elucidate cellular diversity and heterogeneity.     

A label-free and self-assembled electrochemical biosensor for highly sensitive detection of cyclic diguanylate monophosphate (c-di-GMP) based on RNA riboswitch

Cyclic diguanylate monophosphate (c-di-GMP) is an important second messenger that regulates a variety of complex physiological processes involved in motility, virulence, biofilm formation and cell cycle progression in several bacteria. Herein we report a simple label-free and self-assembled RNA riboswitch-based biosensor for sensitive and selective detection of c-di-GMP. The detectable concentration range of c-di-GMP is from 50 nM to 1 μM with a detection limit of 50 nM.     

基于Pd/COF-LZU1非标记型C-反应蛋白免疫传感器的研制

采用溶剂热法, 通过有机单体合成了一种亚胺键连接的共价有机框架材料(COF-LZU1); 在常温常压条件下, 通过后合成的方法将贵金属钯(Ⅱ)引入到COF材料中, 合成了复合材料Pd/COF-LZU1, 该材料具有优良的催化性能. 利用Pd/COF-LZU1多孔复合材料将C-反应蛋白(CRP)抗体(anti-CRP)固定在玻碳电极表面, 构建了一种非标记型CRP免疫传感器. 当抗体与抗原发生免疫反应时, 形成的免疫复合物会阻碍电化学探针[Fe(CN)₆]^4-/3-的电子传递, 降低其响应电流, 从而实现CRP的快速检测. 采用交流阻抗和差示脉冲伏安法(DPV)考察了免疫传感器的电化学特性, 同时考察了测试底液的pH值、 抗原培育时间和抗体固定浓度等实验条件对传感器性能的影响. 在最优的实验条件下, 采用DPV法对CRP进行检测的线性范围为5~180 ng/mL, 检出限为1.66 ng/mL, 线性相关系数为0.992.     

Red carbon dots as label-free two-photon fluorescent nanoprobes for imaging of formaldehyde in living cells and zebrafishes

Direct,in situ selective detection of intracellular formaldehyde(FA)is of great significance for understanding its function in FA-related diseases.Herein,red carbon dots(RCD)are reported as label-free two-photon fluorescent nanoprobes for detecting and imaging of FA.Upon addition of FA,the-NH2 groups of RCD could quickly and specially react with aldehydes to form Schiff base and then the strong fluorescence of RCD with blue-shift emission is recovery due to the destruction of the hydrogen bond interaction between RCD and water.In addition,the nanoprobes exhibit outsta nding photo stability,rapid response(<1 min),high sensitivity(~9.9μmol/L)and excellent selectivity toward FA over other aldehyde group compounds.Notably,owing to the good cell-membrane permeability and biocompatibility,as well as the large two-photon absorption cross-section,the as-prepared RCD can be used as label-free nanoprobes for selectively detecting and imaging FA in living cells and zebrafishes through one-photon and two-photon excitation.Moreover,RCD could stain the tissue of zebrafishes at depths interval of up to 240μm under two-photon excitation.This research implied that RCD are promising tools for directly and in situ imaging FA in vivo,thus providing critical insights into FA-related pathophysiological processes.     

氧化锆/聚亚甲基蓝修饰电极检测CaMV35S启动子基因

制备了基于氧化锆(ZrO_2)/聚亚甲基蓝(PMB)修饰电极的无标记DNA传感器,用于转基因植物CaMV35S启动子基因的检测。探针DNA(ssDNA)通过ZrO_2和DNA的磷酸基的相互作用修饰到电极表面,以PMB氧化峰的示差脉冲伏安响应为检测信号,传感器和完全互补的DNA片段杂交后,PMB的氧化峰电流明显降低,当和完全不匹配的DNA片段杂交时,峰电流无明显变化。对于完全互补的DNA片段,在2.0×10~(-12)~2.0×10~(-8) mol/L浓度范围内峰电流的变化值和浓度的对数成良好的线性关系,检测限为4.1×10~(-13) mol/L(S/N=3)。所制备的传感器具有良好的稳定性、再生性和重现性,用于样品检测,结果令人满意。     

Minireview: Recent Advances in Surface-Enhanced Raman Scattering-Based Nucleic Acid Detection with Application to Pathogen Diagnosis

Surface-enhanced Raman scattering (SERS) is a promising analytical tool in nanoscale detection because of its high sensitivity and selectivity. This review focuses on recent advances in SERS-based detection of DNA and RNA. First, nanostructure-based SERS-active substrates are introduced. Using label-free and labeled SERS, target biomolecules such DNA, RNA and microRNA have been successfully detected. Finally, applications in pathogen diagnosis are discussed. The prospects and challenges of SERS-based bioanalysis are highlighted.     

Effect of phospholipid insertion on arrayed polydiacetylene biosensors

Micro-arrayed polydiacetylene (PDA) vesicles mixed with phospholipids on glass slides were prepared for label-free detection of Escherichia coli. When E. coli bound to its antibodies chemically attached to polydiacetylene, the fluorescence of the vesicles was dramatically increased. The insertion of dimyristoyl phosphatidylcholine (DMPC) in the vesicles drastically reduced the response time for the fluorescence changes. Vesicles with 20-30% DMPC provided optimal results for bacterial detection. Fourier transform infrared (FTIR) spectra analysis suggested that DMPC insertion decreased the strength of hydrogen bonding among the amide and carboxylic acid groups of the polydiacetylene vesicles. Reduced bonding strength resulted in less rigid structure of the polydiacetylene polymer, allowing more rapid detection upon molecular recognition.     

Fluorescence Determination of Merucury(II) Using a Thymine Aptamer

A label-free thymine-rich sequence and a molecular beacon were synthesized to construct a highly sensitive and selective fluorescence probe for the determination of mercury(II). The aptamer of the thymine-rich sequence selectively bonded with mercury(II) with an accompanying change in the fluorescence intensity of the molecular beacon due to the higher affinity of the aptamer with mercury(II). The limit of detection was 12.7 nanomolar, and a linear relationship was obtained between the fluorescence and mercury(II) concentrations up to 1 micromolar. The assay was highly selective for the mercury(II) and not significantly affected by other metal ions.     

A gold@silica core–shell nanoparticle-based surface-enhanced Raman scattering biosensor for label-free glucose detection

The gold nanostar@silica core–shell nanoparticles conjugated with glucose oxidase (GOx) enzyme molecules have been developed as the surface-enhanced Raman scattering (SERS) biosensor for label-free detection of glucose. The surface-immobilized GOx enzyme catalyzes the oxidation of glucose, producing hydrogen peroxide. Under laser excitation, the produced H₂O₂ molecules near the Au nanostar@silica nanoparticles generate a strong SERS signal, which is used to measure the glucose concentration. The SERS signal of nanostar@silica∼GOx nanoparticle-based sensing assay shows the dynamic response to the glucose concentration range from 25 μM to 25 mM in the aqueous solution with the limit of detection of 16 μM. The sensing assay does not show any interference when glucose co-exists with both ascorbic acid and uric acid. The sensor can be applied to a saliva sample.     

Label-free detection of C-reactive protein using reflectometric interference spectroscopy-based sensing system

Reflectometric interference spectroscopy (RIfS) is a label-free, time-resolved technique, and suitable for detecting antibody–antigen interaction. This work describes a continuous flow biosensor for C-reactive protein (CRP), involving an effective immobilization method of a monoclonal antibody against CRP (anti-CRP) to achieve highly sensitive RIfS-based detection of CRP. The silicon nitride-coated silicon chip (SiN chip) for the RIfS sensing was first treated with trimethylsilylchloride (TMS), followed by UV-light irradiation to in situ generation of homogeneous silanols on the surface. Following amination by 3-aminopropyltriethoxysilane, carboxymethyldextran (CMD) was grafted, and subsequently, protein A was immobilized to create the oriented anti-CRP surface. The immobilization process of protein A and anti-CRP was monitored with the RIfS system by consecutive injections of an amine coupling reagent, protein A and anti-CRP, respectively, to confirm the progress of each step in real time. The sensitivity was enhanced when all of the processes were adopted, suggesting that the oriented immobilization of anti-CRP via protein A that was coupled with the grafted CMD on the aminated surface of TMS-treated SiN chip. The feasibility of the present sensing system was demonstrated on the detection of CRP, where the silicon-based inexpensive chips and the simple optical setup were employed. It can be applied to other target molecules in various fields of life science as a substitute of surface plasmon resonance-based expensive sensors.     

Immobilization of Escherichia coli for detection of phage T4 using surface plasmon resonance

Phage contamination is a very serious and unavoidable problem in modern fermentation industry.It is necessary to develop sensitive and rapid phage detection methods for the early detection of phage contamination.In the present work,a real-time,rapid,specific and quantitative phage T4 detection method based on surface plasmon resonance(SPR) technique has been introduced.Escherichia coli was immobilized onto the preformed MPA self-assembled monolayer(SAM) through the widely used EDC/NHS cross-linking reaction as the recognition element.The bacteria immobilization was verified efficiently through the electrochemical measurements and fluorescence microscopy observations.The specific adsorption was much stronger than the non-specific adsorption of phage T4 binding to the biosensor surface modified by E.coli,and the latter could be neglected.The detection sensitivity reached 1×10 7 PFU/mL within 10 min.Within the experimental phage concentrations,the linear correlation between the SPR response and the phage concentration was good.The results suggest that the SPR technique is a potentially powerful tool for the phage or other virus detections,as a label-free,real-time,and rapid method.     

Electrochemical aptasensor for the detection of vascular endothelial growth factor (VEGF) based on DNA-templated Ag/Pt bimetallic nanoclusters

In this paper, the DNA-templated Ag/Pt bimetallic nanoclusters were successfully synthesized using an optimized synthetic scheme. The obtained DNA-Ag/Pt NCs have an ultrasmall particle size and excellent distribution. The DNA-Ag/Pt NCs show intrinsic peroxidase-mimicking activity and can effectively catalyze the H₂O₂-mediated oxidation of a substrate, 3,3',5,5'-tetramethylbenzidine (TMB), to produce a blue colored product. Based on this specific property, we employed the aptamer of VEGF to design a label-free electrochemical biosensor for VEGF detection. Under the optimized experimental conditions, a linear range from 6.0 pmol/L to 20 pmol/L was obtained with a detection limit of 4.6 pmol/L. The proposed biosensor demonstrated its high specificity for VEGF and could directly detect the VEGF concentration in human serum samples of breast cancer patients with satisfactory results. This novel electrochemical aptasensor was simple and convenient to use and was cost-effective and label-free in design, and would hold potential applications in medical diagnosis and treatment.     

Label-free biosensing with lipid-functionalized gold nanorods

The fabrication of a label-free mass spectrometry and optical detection-based biosensor platform for the detection of low-abundance lipophilic analytes in complex mixtures is described. The biosensor consists of a lipid layer partially tethered to the surface of a gold nanorod. The effectiveness of the biosensor is demonstrated for the label-free detection of a lipophilic drug in aqueous solution and a lipopeptide in serum.     

Label-free molecular interaction determinations with nanoscale interferometry

Quantification of protein-protein and ligand-substrate interactions is central to understanding basic cellular function and for evaluating therapeutics. To mimic biological conditions, such studies are best executed without modifying the proteins or ligands (i.e., label-free). While tools for label-free assays exist, they have limitations making them difficult to fully integrate into microfluidic devices. Furthermore, it has been problematic to reduce detection volumes for on-channel universal analyte quantification without compromising sensitivity, as needed in label-free methods. Here we show how backscattering interferometry in rectangular channels (BIRC) facilitates label-free studies within picoliter volumes. The simple and unique optical train was based on rectangular microfluidic channels molded in poly(dimethylsiloxane) and low-power coherent radiation. Quantification of irreversible streptavidin-biotin binding and reversible protein A-human IgG Fc molecular interactions in a 225 pL detection volume was carried out label-free and noninvasively. Detection limits of 47 x 10(-15) mol of biotin reacted with surface-immobilized streptavidin were achieved. In the case of reversible interactions of protein A and the Fc fragment of human IgG, detection limits were determined to be 2 x 10(-15) mol of IgG Fc. These experiments demonstrate for the first time that (1) high-sensitivity universal solute quantification is possible using interferometry performed within micrometer-sized channels formed in inexpensive PDMS chips, (2) label-free reversible molecular interaction can be studied with femtomoles of solute, and (3) BIRC has the potential to quantify binding affinities in a high-throughput format.     

Label-free characterization of cell adhesion using reflectometric interference spectroscopy (RIfS)

Reflectometric interference spectroscopy (RIfS) is a label-free, time-resolved technique for detecting interactions of molecules immobilized on a surface with ligands in solution. Here we show that RIfS also permits the detection of the adhesion of tissue culture cells to a functionalized surface in a flow system. Interactions of T cells with other leukocytes or epithelial cells of blood vessels are crucial steps in the regulating immune response and inflammatory reactions. Jurkat T cell leukemia cells rapidly attached to a transducer functionalized with a monoclonal antibody directed against the T cell receptor (TCR)/CD3 complex, followed by activation-dependent cell spreading. RIfS curves were obtained for the Jurkat derivative JCaM 1.6 (which lacks the key signaling protein Lck), cells preincubated with cytochalasin D (an inhibitor of actin polymerization), and for surfaces functionalized with an antibody directed against the coreceptor CD28. These curves differed with respect to the maximum signal and the initial slope of the increase in optical thickness. The testing of chemical inhibitors, cell surface molecules and gene products relevant to a key event in T cell immunity illustrates the potential of label-free techniques for the analysis of activation-dependent cell-surface contacts. The first two authors contributed equally to this paper     

Nanoparticle-enhanced sensitivity of a nanogap-interdigitated electrode array impedimetric biosensor

Interdigitated electrode (IDE) arrays with nanometer-scale gaps have been utilized to enhance the sensitivity of affinity-based detection. The geometry of nanogap IDEs was first optimized on the basis of simulations of the electric field and current density. It was determined that the gap (G) between the electrodes was the most important geometric parameter in determining the distribution and strength of the electric field and the current density compared to the width (W) and height (H) of the IDEs. Several devices were materialized and analyzed for their sensitivity to the electrochemical environment using faradic electrochemical impedance spectroscopy (EIS) as the detection technique. Nanogap optimized IDEs were then employed as biosensors for the label-free, affinity-based detection of antitissue transglutaminase antibodies (αtTG-Abs), a biomarker for the detection of autoimmune disorder celiac sprue, triggered by ingesting gluten. The label-free biosensor assay was found to be less sensitive compared to on-chip ELISA. Gold nanoparticles (GNPs) were then employed to improve the sensitivity of the nanogap IDE-based biosensor. With GNPs, the transducer sensitivity increased by 350% over that of label-free detection. The suitability of nanogap IDEs as biosensor transducers for EIS in label-free and GNP-labeled formats was established. The immunobiosensor assay detection sensitivity with the GNPs was found comparable to ELISA.     

Label-free sensing of pH and silver nanoparticles using an “OR” logic gate

Many natural phenomena are associated with the presence of two or more separate variables. We report here an “OR” DNA logic gate based on a luminescent platinum(II) switch-on probe for silver nanoparticles and pH, both of which may be considered putative indicators of pollution. The modulation of metal complex/double-stranded DNA complex phosphorescence by Ag⁺ and H⁺ was used to construct a simple, rapid and label-free method for the label-free detection of pH and nanomolar Ag⁺ ions and nanoparticles in aqueous solutions with high selectivity.     

基于金纳米颗粒生长的液晶生物传感器检测酪氨酸

基于酶介导金纳米颗粒(AuNPs)生长构建了液晶生物传感器, 并用于检测酪氨酸(Tyr). 将酪氨酸酶(TR)固定于经戊二醛活化的二甲基十八烷基(3-[三甲氧基硅烷] 丙基)氯化铵/3-氨丙基三乙氧基硅烷(DMOAP/APTES)混合自组装修饰的玻片表面, 当向玻片表面滴加含Tyr的生长溶液时, TR催化Tyr羟基化为左旋多巴(L-Dopa), L-Dopa还原生长溶液中的AuCl₄^-生成AuNPs并沉积于玻片表面, 导致玻片表面地貌发生变化, 这一变化能诱导液晶取向发生变化进而调控透光量, 从而实现对Tyr的检测, 且检测浓度可低至6×10^-7 mol/L.