Direct detection of genomic DNA by enzymatically amplified SPR imaging measurements of RNA microarrays

A novel surface enzymatic reaction scheme that amplifies the optical response of RNA microarrays to the binding of complementary DNA is developed for the direct detection and analysis of genomic DNA. The enzyme RNase H is shown to selectively and repeatedly destroy RNA from DNA-RNA heteroduplexes on gold surfaces; when used in conjunction with the label-free technique of surface plasmon resonance (SPR) imaging, DNA oligonucleotides can be detected at a concentration of 1 fM. This enzymatically amplified SPR imaging methodology is then utilized to detect and identify the presence of the TSPY gene in human genomic DNA without PCR amplification.     

Hydrogel droplet microarrays with trapped antibody-functionalized beads for multiplexed protein analysis

Antibody microarrays are a powerful tool for rapid, multiplexed profiling of proteins. 3D microarray substrates have been developed to improve binding capacity, assay sensitivity, and mass transport, however, they often rely on photopolymers which are difficult to manufacture and have a small pore size that limits mass transport and demands long incubation time. Here, we present a novel 3D antibody microarray format based on the entrapment of antibody-coated microbeads within alginate droplets that were spotted onto a glass slide using an inkjet. Owing to the low concentration of alginate used, the gels were highly porous to proteins, and together with the 3D architecture helped enhance mass transport during the assays. The spotting parameters were optimized for the attachment of the alginate to the substrate. Beads with 0.2 μm, 0.5 μm and 1 μm diameter were tested and 1 μm beads were selected based on their superior retention within the hydrogel. The beads were found to be distributed within the entire volume of the gel droplet using confocal microscopy. The assay time and the concentration of beads in the gels were investigated for maximal binding signal using one-step immunoassays. As a proof of concept, six proteins including cytokines (TNFα, IL-8 and MIP/CCL4), breast cancer biomarkers (CEA and HER2) and one cancer-related protein (ENG) were profiled in multiplex using sandwich assays down to pg mL(-1) concentrations with 1 h incubation without agitation in both buffer solutions and 10% serum. These results illustrate the potential of beads-in-gel microarrays for highly sensitive and multiplexed protein analysis.     

SPR imaging for label-free multiplexed analyses of DNA N-glycosylase interactions with damaged DNA duplexes

Base excision repair (BER) is the major mechanism for the correction of damaged nucleobases resulting from the alkylation and oxidation of DNA. The first step in the BER pathway consists of excision of the abnormal base by several specific DNA N-glycosylases. A decrease in BER activity was found to be related to an increased risk of carcinogenesis and aging. To investigate BER activities we set up a new device for DNA repair analysis based on surface plasmon resonance imaging (SPRi). Oligonucleotides bearing an abnormal nucleoside, namely 8-oxo-7,8-dihydro-2'-deoxyguanosine and (5'S)-5',8-cyclopurine-2'-deoxynucleoside, were grafted by a pyrrole electro-copolymerization process on a glass prism coated with a gold layer. The latter label-free DNA sensor chip permits the detection of N-glycosylase/AP-lyase activity as well as the binding of repair proteins to DNA damage without cleavage activity. Thus, the Fapy DNA N-glycosylase (Fpg) protein is shown as expected to bind and then cleave its natural substrate, namely 8-oxo-7,8-dihydro-guanine, together with the resulting abasic site. Using the current SPR imaging-based DNA array we observed an original binding activity of Fpg towards the (5'S)-5',8-cyclodAdenosine residue. These results altogether show that SPR imaging may be used to simultaneously and specifically detect recognition and excision of several damaged DNA nucleobases, and constitutes an interesting technique to screen inhibitors of DNA repair proteins.     

Multi-analyte SPR immunoassays for environmental biosensing of pesticides

Multi-analyte detection of environmentally relevant pesticides is performed by using a two-channelled surface plasmon resonance (SPR) biosensor. The special design of the SPR instrument allows the determination of several analytes (DDT, chlorpyrifos and carbaryl) via different immobilization formats. First, simultaneous pesticide monitoring is possible by flowing chlorpyrifos, carbaryl or DDT samples separately over each channel of the SPR system, wherein their corresponding recognition element was previously immobilized. The second approach is based on the multiple and combined immobilization of several analyte recognition elements on the sensing surface of one individual flow cell. In this format, the analysis time for all three pesticides varied from 40 to 60 min depending on the number of regeneration cycles. In most cases, similar detection limits were attained for the target analyte irrespective of the assay format, with sensitivity values at the nanogram per litre level (18–50 ng L⁻¹). The assay reproducibility was proved through the repeated use of the same sensor surface for over more than 200 assay cycles, whereas the absence of biosensor response to non-related analytes showed the specificity and reliability of the analysis. The SPR instrument, including optics, electronics and microfluidics, is already commercialised by the company SENSIA, SL.     

Stereoselective synthesis of light-activatable perfluorophenylazide-conjugated carbohydrates for glycoarray fabrication and evaluation of structural effects on protein binding by SPR imaging

A series of light-activatable perfluorophenylazide (PFPA)-conjugated carbohydrate structures have been synthesized and applied to glycoarray fabrication. The glycoconjugates were structurally varied with respect to anomeric attachment, S-, and O-linked carbohydrates, respectively, as well as linker structure and length. Efficient stereoselective synthetic routes were developed, leading to the formation of the PFPA-conjugated structures in good yields over few steps. The use of glycosyl thiols as donors proved especially efficient and provided the final compounds in up to 70% total yield with high anomeric purities. PFPA-based photochemistry was subsequently used to generate carbohydrate arrays on a polymeric surface, and surface plasmon resonance imaging (SPRi) was applied for evaluation of carbohydrate-protein interactions using the plant lectin Concanavalin A (Con A) as a probe. The results indicate better performance and equal efficiency of S- and O-linked structures with intermediate linker length.     

A highly stable RNA aptamer probe for the retinoblastoma protein in live cells

Although RNA aptamers can show comparable or better specificity and affinity to antibodies and have the advantage of being able to access different live cell compartments, they are often much less stable in vivo. We report here the first aptamer that binds human retinoblastoma protein (RB) and is stable in live cells. RB is both a key protein in cell cycle control and also a tumour suppressor. The aptamer was selected from an RNA library against a unique 12-residue helical peptide derived from RB rather than the whole protein molecule. It binds RB with high affinity (K_d = 5.1 ± 0.1 nM) and is a putative RNA G-quadruplex structure formed by an 18-nucleotide sequence (18E16 - GGA GGG UGG AGG GAA GGG), which may account for its high stability. Confocal fluorescence microscopy of live cells transfected with the aptamer shows it is stable intracellularly and efficient in entering the nucleus where an analogous antibody was inaccessible. The findings demonstrate this aptamer is an advanced probe for RB in live cell applications.

An RNA G-quadruplex aptamer, specific for the human retinoblastoma protein (RB) and highly stable inside cells, is selected and its application to live cell probing of the protein illustrated.     

Design and synthesis of monofunctionalized, water-soluble conjugated polymers for biosensing and imaging applications

Water-soluble conjugated polymers with controlled molecular weight characteristics, absence of ionic groups, high emission quantum yields, and end groups capable of selective reactions of wide scope are desirable for improving their performance in various applications and, in particular, fluorescent biosensor schemes. The synthesis of such a structure is described herein. 2-Bromo-7-iodofluorene with octakis(ethylene glycol) monomethyl ether chains at the 9,9'-positions, i.e., compound 4, was prepared as the reactive premonomer. A high-yielding synthesis of the organometallic initiator (dppe)Ni(Ph)Br (dppe = 1,2-bis(diphenylphosphino)ethane) was designed and implemented, and the resulting product was characterized by single-crystal X-ray diffraction techniques. Polymerization of 4 by (dppe)Ni(Ph)Br can be carried out in less than 30 s, affording excellent control over the average molecular weight and polydispersity of the product. Quenching of the polymerization with [2-(trimethylsilyl)ethynyl]magnesium bromide yields silylacetylene-terminated water-soluble poly(fluorene) with a photoluminescence quantum efficiency of 80%. Desilylation, followed by copper-catalyzed azide-alkyne cycloaddition reaction, yields a straightforward route to introduce a wide range of specific end group functionalities. Biotin was used as an example. The resulting biotinylated conjugated polymer binds to streptavidin and acts as a light-harvesting chromophore to optically amplify the emission of Alexa Fluor-488 chromophores bound onto the streptavidin. Furthermore, the biotin end group makes it possible to bind the polymer onto streptavidin-functionalized cross-linked agarose beads and thereby incorporate a large number of optically active segments.     

Analysis of micro-contact printed protein patterns by SPR imaging with a LED light source

We demonstrate the characterization of mu-contact printed protein patterns and analysis of protein-protein interactions by two-dimensional (2-D) surface plasmon resonance imaging (SPRi). Advancements in SPRi image quality from employing a light emitting diode (LED) as the light source are described. We show that a LED offers an ideal point source that can eliminate interference artifacts and speckles found when using a laser source. The attainable thickness resolution in fixed-angle imaging is comparable to that of a monochromatic source, providing a solid foundation for quantitative analysis with the system. The SPR imaging technique reported here affords sub-nanometer thickness sensitivity and micrometer lateral resolution, allowing for convenient studies of biomolecular interactions and surface morphologies of ultrathin films. Spatially well-defined protein patterns of bacterial toxins were obtained by microcontact printing using a polydimethylsiloxane (PDMS) stamp on a functionalized self-assembled monolayer on Au. The influence of protein concentration in the inking solution on transfer efficiency was investigated, and a nonlinear correlation was observed between the solution concentration and the amount of protein immobilized on the surface. Quantitative analysis of protein interaction was performed with toxin-specific antibody, showing a concentration-dependent relationship that verifies the retention of biological activity of the protein after printing. The study demonstrates the feasibility and effectiveness of using LEDs as light sources in SPR imaging, opening doors for developing compact SPR instruments for direct, sensitive, and label-free detection of biohazardous molecules.     

Attomole microarray detection of microRNAs by nanoparticle-amplified SPR imaging measurements of surface polyadenylation reactions

Multiple microRNAs (miRNAs) are detected in a microarray format using a novel approach that combines a surface enzyme reaction with nanoparticle-amplified SPR imaging (SPRI). The surface reaction of poly(A) polymerase creates poly(A) tails on miRNAs hybridized onto locked nucleic acid (LNA) microarrays. DNA-modified nanoparticles are then adsorbed onto the poly(A) tails and detected with SPRI. This ultrasensitive nanoparticle-amplified SPRI methodology can be used for miRNA profiling at attomole levels.     

Ultrabright AIEdots with tunable narrow emission for multiplexed fluorescence imaging

AIEgen doped fluorescent nanodots (AIEdots) have attracted lots of attention, due to their superior characteristics as fluorescent probes, such as excellent photostability, large Stokes shift, high brightness and tunable emission. Unfortunately, most of the currently available AIEdots exhibit broad emission bandwidth, which limits their applications in multiplexed fluorescence imaging and detection. In this work, the strategy of designing and fabricating narrow emissive AIEdots (NE-AIEdots) with tunable wavelengths was presented by constructing a light-harvesting system with high energy transfer efficiency. Efficient intra-particle energy transfer from highly doped AIEgens, serving as the light-harvesting antenna, to the lightly doped narrow emissive fluorophore, resulted in high brightness and narrow emission. The emission band of NE-AIEdots with the full-width-at-half-maximum varied from 18 to 36 nm was 3–6.3 times narrower than that of traditional AIEdots. The single-particle brightness of NE-AIEdots was over 5-times that of commercial quantum dots under the same excitation and collection conditions. Taking advantage of the superior performance of these NE-AIEdots, multiplexed fluorescence imaging of lymph nodes in living mice was realized, which supported the future applications of NE-AIEdots for in vivo multiplexed labeling and clinical surgery.

AIEdots with high brightness and narrow emission bandwidth were developed for multiplexed in vitro and in vivo fluorescence imaging.     

A label-free electrochemical RNA aptamer for selective detection of theophylline

In this work, we report a novel electrochemical RNA aptamer for the selective detection of theophylline. Firstly, gold nanoparticles were electrodeposited on the surface of glassy carbon (GC) electrode to form a gold nanoparticles modified electrode. Secondly, the designed single-stranded RNA (ssRNA) was immobilized on gold nanoparticles through a thiol linker as a probe RNA. Then, the complement stranded RNA, which can combine with the probe ssRNA to form a double-stranded RNA (dsRNA) with a recognition unit of theophylline, was linked on the probe RNA through a hybrid reaction in the presence of theophylline. Doxorubicin was selected as an electrochemical indicator. The proposed RNA aptamer presents an excellent selectivity for the detection of theophylline. The detectable concentration range of theophylline is from 2.0 to 50.0 μM with a limit of detection of 1.2 μM.     

Label-free detection of protein binding with multisine SPR microchips

Label-free techniques such as surface plasmon resonance (SPR) have used a step-response excitation method to characterize the binding of two biochemical entities. A major drawback of the step response technique is its high susceptibility to thermal drifts and noise which directly determine the minimum detectable binding mass. In this paper we present a new frequency-domain method based on the use of multisine chemical excitation that is much less sensitive to these disturbances. The multisine method was implemented in a PDMS microfluidic chip using a dual channel, dual multiplug chemical signal generator connected to functionalized and reference SPR binding spots. Kinetic constants for the reaction are extracted from the characteristics of the sense spot response versus frequency. The feasibility of the technique was tested using a model system of Carbonic Anhydrase-II analyte and amino-benzenesulfonamide ligand. The experimental signal to noise ratio (SNR) for the multisine measurement is about 32 dB; 7 dB higher than that observed with the single step-response method, while the overall measurement time is twice as long as the step method.     

Design, preparation and testing of suitable probe-receptors for RNA biosensing

Absolute measurements of a given RNA in a cheap, easy, rapid and reproducible manner using biosensors technology could overcome many of the operative and analytical limits of conventional molecular biology methods. To this end, an integrated approach for the design, synthesis, and connection of RNA probes to the transducing surface of a microgravimetric biosensor has been developed. Suitable probes to be used as the bioreceptors in RNA biosensor were successfully designed by using a purposely developed computational method whose selection criteria are based on the accessibility of target region to probe, on pairing stability of probe-target duplex and on the uniqueness of selected targets over all known expressed sequences from a genome data base. Automated chemical synthesis of selected probes was performed and the oligonucleotides produced were covalently conjugated to the sensing surface of a quartz microbalance. The microgravimetric sensor was tested in a flow chamber by measuring the variation of resonance frequency due to the binding of synthetic target substrates. Specific dose dependent binding was observed. Furthermore, the binding of a transcribed full-length mRNA substrate was successfully monitored under similar conditions.     

Optimization of aptamer microarray technology for multiple protein targets

Aptamer-based microarrays for the quantitation of multiple protein analytes have been developed. A multiplex aptamer microarray was generated by printing two RNA aptamers (anti-lysozyme and anti-ricin) and two DNA aptamers (anti-IgE and anti-thrombin) on to either streptavidin (SA) or neutravidin (NA)-coated glass slides. However, substantial optimization was required in order to ensure the simultaneous function of the aptamer:analyte pairs. The effects of protein labeling, assay buffer, surface coating, and immobilization chemistry and orientation were investigated. A single buffer (PBS buffer containing 5 mM MgCl₂ and 0.1% Tween 20) was found to work well with all the aptamers, even though this was not the buffer originally used in their selection, while neutravidin-coated slides yielded a lower detection limit, wider detection range, and more uniform background than streptavidin-coated slides. Incubation with Cy3-labeled proteins yielded sensitive, target-specific, and dose-dependent responses to each protein. Target protein concentrations as low as 72 pg/mL (5 pM, lysozyme), 15 ng/mL (0.5 nM, ricin), 1.9 ng/mL (0.01 nM, IgE), and 170 ng/mL (5 nM, thrombin) could be detected. These results show that aptamer arrays can potentially be used with numerous proteins in parallel, furthering the notion that aptamer arrays may be useful in proteomics.     

Click chemistry facilitates formation of reporter ions and simplified synthesis of amine-reactive multiplexed isobaric tags for protein quantification

We report the development of novel reagents for cell-level protein quantification, referred to as Caltech isobaric tags (CITs), which offer several advantages in comparison with other isobaric tags (e.g., iTRAQ and TMT). Click chemistry, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), is applied to generate a gas-phase cleavable linker suitable for the formation of reporter ions. Upon collisional activation, the 1,2,3-triazole ring constructed by CuAAC participates in a nucleophilic displacement reaction forming a six-membered ring and releasing a stable cationic reporter ion. To investigate its utility in peptide mass spectrometry, the energetics of the observed fragmentation pathway are examined by density functional theory. When this functional group is covalently attached to a target peptide, it is found that the nucleophilic displacement occurs in competition with formation of b- and y-type backbone fragment ions regardless of the amino acid side chains present in the parent bioconjugate, confirming that calculated reaction energetics of reporter ion formation are similar to those of backbone fragmentations. Based on these results, we apply this selective fragmentation pathway for the development of CIT reagents. For demonstration purposes, duplex CIT reagent is prepared using a single isotope-coded precursor, allyl-d(5)-bromide, with reporter ions appearing at m/z 164 and 169. Isotope-coded allyl azides for the construction of the reporter ion group can be prepared from halogenated alkyl groups which are also employed for the mass balance group via N-alkylation, reducing the cost and effort for synthesis of isobaric pairs. Owing to their modular designs, an unlimited number of isobaric combinations of CIT reagents are, in principle, possible. The reporter ion mass can be easily tuned to avoid overlapping with common peptide MS/MS fragments as well as the low mass cutoff problems inherent in ion trap mass spectrometers. The applicability of the CIT reagent is tested with several model systems involving protein mixtures and cellular systems.     

Noncovalent fluorous interactions for the synthesis of carbohydrate microarrays

A surface patterning method based on noncovalent immobilization of fluorous-tagged sugars on fluorous-derivatized glass slides allows the facile fabrication of carbohydrate microarrays. To expand the scope of these arrays, the first syntheses are reported of arabinose, rhamnose, lactose, maltose, and glucosamine tagged with a single C₈F₁₇-tail for ease of purification as well as array formation. Screening of these carbohydrate microarrays against lectins from Triticum vulgaris (WGA) and Arachis hypogaea (PNA) demonstrate that the noncovalent fluorous–fluorous interaction is sufficient to retain not only mono- but also disaccharides under the biological assay conditions.     

A modular system for the synthesis of multiplexed magnetic resonance probes

We have developed a modular architecture for preparing high-relaxivity multiplexed probes utilizing click chemistry. Our system incorporates azide bearing Gd(III) chelates and a trialkyne scaffold with a functional group for subsequent modification. In optimizing the relaxivity of this new complex, we undertook a study of the linker length between a chelate and the scaffold to determine its effect on relaxivity. The results show a strong dependence on flexibility between the individual chelates and the scaffold with decreasing linker length leading to significant increases in relaxivity. Nuclear magnetic resonance dispersion (NMRD) spectra were obtained to confirm a 10-fold increase in the rotational correlation time from 0.049 to 0.60 ns at 310 K. We have additionally obtained a crystal structure demonstrating that modification with an azide does not impact the coordination of the lanthanide. The resulting multinuclear center has a 500% increase in per Gd (or ionic) relaxivity at 1.41 T versus small molecule contrast agents and a 170% increase in relaxivity at 9.4 T.     

QconCATs: design and expression of concatenated protein standards for multiplexed protein quantification

Systems biology requires knowledge of the absolute amounts of proteins in order to model biological processes and simulate the effects of changes in specific model parameters. Quantification concatamers (QconCATs) are established as a method to provide multiplexed absolute peptide standards for a set of target proteins in isotope dilution standard experiments. Two or more quantotypic peptides representing each of the target proteins are concatenated into a designer gene that is metabolically labelled with stable isotopes in Escherichia coli or other cellular or cell-free systems. Co-digestion of a known amount of QconCAT with the target proteins generates a set of labelled reference peptide standards for the unlabelled analyte counterparts, and by using an appropriate mass spectrometry platform, comparison of the intensities of the peptide ratios delivers absolute quantification of the encoded peptides and in turn the target proteins for which they are surrogates. In this review, we discuss the criteria and difficulties associated with surrogate peptide selection and provide examples in the design of QconCATs for quantification of the proteins of the nuclear factor κB pathway.     

Chemical synthesis of picornaviral protein primers of RNA replication

Naturally occurring nucleopeptidic replication primers (VPg-pUpU) of poliovirus and coxsackie virus were chemically synthesized. The synthesis was accomplished via block-coupling of two minimally protected fragments of the target structures: a short RNA-nucleopeptide and a longer peptide segment containing diverse side-chain functionalities. The synthetic VPg-pUpU of coxsackie virus was characterized by NMR spectroscopy.