Rapid microarray detection of DNA and proteins in microliter volumes with surface plasmon resonance imaging measurements

A four-chamber microfluidic biochip is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The 18 mm × 18 mm biochip consists of four 3 μL microfluidic chambers attached to an SF10 glass substrate, each of which contains three individually addressable SPRI gold thin film microarray elements. The 12-element (4 × 3) SPRI microarray consists of gold thin film spots (1 mm(2) area; 45 nm thickness), each in individually addressable 0.5 μL volume microchannels. Microarrays of single-stranded DNA and RNA (ssDNA and ssRNA, respectively) are fabricated by either chemical and/or enzymatic attachment reactions in these microchannels; the SPRI microarrays are then used to detect femtomole amounts (nanomolar concentrations) of DNA and proteins (ssDNA binding protein and thrombin via aptamer-protein bioaffinity interactions). Microarrays of ssRNA microarray elements are also used for the ultrasensitive detection of zeptomole amounts (femtomolar concentrations) of DNA via the technique of RNase H-amplified SPRI. Enzymatic removal of ssRNA from the surface due to the hybridization adsorption of target ssDNA is detected as a reflectivity decrease in the SPR imaging measurements. The observed reflectivity loss is proportional to the log of the target ssDNA concentration with a detection limit of 10 fM or 30 zeptomoles (18?000 molecules). This enzymatic amplified ssDNA detection method is not limited by diffusion of ssDNA to the interface, and thus is extremely fast, requiring only 200 s in the microliter volume format.     

Creating advanced multifunctional biosensors with surface enzymatic transformations

This paper summarizes our recent work on the coupling of surface enzyme chemistry and bioaffinity interactions on biopolymer microarrays for the creation of multiplexed biosensors with enhanced selectivity and sensitivity. The surface sensitive techniques of surface plasmon resonance imaging (SPRI) and surface plasmon fluorescence spectroscopy (SPFS) are used to detect the surface enzymatic transformations in real time. Three specific examples of novel coupled surface bioaffinity/surface enzymatic processes are demonstrated: (i) a surface enzymatic amplification method utilizing the enzyme ribonuclease H (RNase H) in conjunction with RNA microarrays that permits the ultrasensitive direct detection of genomic DNA at a concentration of 1 fM without labeling or PCR amplification, (ii) the use of RNA-DNA ligation chemistry to create renewable RNA microarrays from single stranded DNA microarrays, and (iii) the application of T7 RNA polymerase for the on-chip replication of RNA from double stranded DNA microarray elements. In addition, a simple yet powerful theoretical framework that includes the contributions of both enzyme adsorption and surface enzyme kinetics is used to quantitate surface enzyme reactivity. This model is successfully applied to SPRI and SPFS measurements of surface hydrolysis reactions of RNase H and Exonuclease III (Exo III) on oligonucleotide microarrays.     

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.     

Stabilizing structure-switching signaling RNA aptamers by entrapment in sol-gel derived materials for solid-phase assays

Structure-switching, fluorescence-signaling DNA and RNA aptamers have been reported as highly versatile molecular recognition elements for biosensor development. While structure-switching DNA aptamers have been utilized for solid-phase sensing, equivalent RNA aptamers have yet to be successfully utilized in solid-phase sensors due to their lack of chemical stability and susceptibility to nuclease attack. In this study, we examined entrapment into sol-gel derived organic-inorganic composite materials as a platform for immobilization of structure-switching fluorescence-signaling RNA aptamer reporters, using both the synthetic theophylline- and naturally occurring thiamine pyrophosphate-binding RNA aptamers as test cases. Structure-switching versions of both aptamers were entrapped into a series of sol-gel derived composites, ranging from highly polar silica to hydrophobic methylsilsesquioxane-based materials, and the target-binding and signaling capabilities of these immobilized aptamers were assessed relative to solution. Both immobilized aptamers demonstrated sensitivity and selectivity similar to that of free aptamers when entrapped in a composite material derived from 40% (v/v) methyltrimethoxysilane/tetramethoxysilane. Importantly, this material also conferred protection from nuclease degradation and imparted long-term chemical stability to the RNA reporter systems. Given the versatility of sol-gel entrapment for development of biosensors, microarrays, bioaffinity columns, and other devices, this entrapment method should provide a useful platform for numerous solid-phase RNA aptamer-based devices.     

Finding the Optimal Surface Density of Aptamer Monolayers by SPR Imaging Detection‐based Aptamer Microarrays

Surface plasmon resonance imaging (SPRi) by enabling label‐free, real time assessment of biomolecular interactions in multiplexed manner is one of the methods of choice for high throughput characterization of large pools of DNA aptamer candidates following in vitro selection. Moreover, with major advances in in situ amplification methods SPRi became also a viable detection platform for aptamer microarrays. In case of aptamer microarrays, commonly prepared by microspotting, the direct assessment of the surface density of aptamer probes, which is essential for both kinetic and sensing measurements is not possible. Therefore, here we introduce a methodology for simple, one‐step determination of surface densities of thiol labelled aptamer monolayers microspotted on gold SPRi chips. Based on this methodology we investigated in detail the effect of the surface density of aptamers on target binding through two aptamer‐target systems, i. e. human immunoglobulin E (hIgE) and six histidine tag 6xHis‐tag. We found that the surface density of the aptamers is indeed critical and shows a sharp maximum in terms of target binding efficiency, which is largely determined by the size of the target. The optimal aptamer surface densities determined, the immobilization chemistry (shared by many detection platforms, e. g., electrochemical, surface acoustic) and the trends identified may be used for rapid rational optimization of aptamer‐target assays.     

Microfluidic networks for surface plasmon resonance imaging real-time kinetics experiments

The coupling of microfluidic devices with surface plasmon resonance imaging (SPRI) has emerged in recent years as a novel approach for the simultaneous monitoring of interactions of biomolecules arrayed onto gold substrates. In order to minimize a variety of effects which affect the final determination of kinetic parameters (non-specific interactions above all), difficult choices of appropriate references are often encountered in carrying out SPRI investigations. A common solution to these problems consists of laborious experimental setup involving the use of specially designed microchannels and tedious manipulation of the gold substrate that often produces surface degradation.In this work, a discussion about appropriate choice of references in SPRI measurements is opened and the use of alternative microfluidic patterns coupled to the SPRI system is proposed as a solution to the above mentioned problems. Specifically, a Y-shaped SPRI flow cell has been constructed from masters in polyvinyl chloride and it has been identified as one of the most suitable experimental approach for obtaining appropriate referencing during SPRI experiments. The experimental set up has been tested in a real time study of the interaction between the Datura Stramonium Agglutinin and the asialofetuin and the obtained results demonstrate the suitability of such microfluidic network in SPRI investigations.     

Structure-switching signaling aptamers: transducing molecular recognition into fluorescence signaling

The development of aptamer technology considerably broadens the utility of nucleic acids as molecular recognition elements, because it allows the creation of DNA or RNA molecules for binding a wide variety of analytes (targets) with high affinity and specificity. Several recent studies have focused on developing rational design strategies for transducing aptamer-target recognition events into easily detectable signals, so that aptamers can be widely exploited for detection directed applications. We have devised a generalizable strategy for designing nonfluorescent aptamers that can be turned into fluorescence-signaling reporters. The resultant signaling probes are denoted "structure-switching signaling aptamers" as they report target binding by switching structures from DNA/DNA duplex to DNA/target complex. The duplex is formed between a fluorophore-labeled DNA aptamer and an antisense DNA oligonucleotide modified with a quencher (denoted QDNA). In the absence of the target, the aptamer hybridizes with QDNA, bringing the fluorophore into close proximity of the quencher for efficient fluorescence quenching. When this system is exposed to the target, the aptamer switches its binding partner from QDNA to the target. This structure-switching event is coupled to the generation of a fluorescent signal through the departure of QDNA, permitting the real-time monitoring of the aptamer-target recognition. In this article, we discuss the conceptual framework of the structure-switching approach, the essential features of structure-switching signaling aptamers as well as remaining challenges and possible solutions associated with this new methodology.     

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.     

Direct immobilization of DNA probes on non-modified plastics by UV irradiation and integration in microfluidic devices for rapid bioassay

DNA microarrays have become one of the most powerful tools in the field of genomics and medical diagnosis. Recently, there has been increased interest in combining microfluidics with microarrays since this approach offers advantages in terms of portability, reduced analysis time, low consumption of reagents, and increased system integration. Polymers are widely used for microfluidic systems, but fabrication of microarrays on such materials often requires complicated chemical surface modifications, which hinders the integration of microarrays into microfluidic systems. In this paper, we demonstrate that simple UV irradiation can be used to directly immobilize poly(T)poly(C)-tagged DNA oligonucleotide probes on many different types of plastics without any surface modification. On average, five- and fourfold improvement in immobilization and hybridization efficiency have been achieved compared to surface-modified slides with aminated DNA probes. Moreover, the TC tag only costs 30% of the commonly used amino group modifications. Using this microarray fabrication technique, a portable cyclic olefin copolymer biochip containing eight individually addressable microfluidic channels was developed and used for rapid and parallel identification of Avian Influenza Virus by DNA hybridization. The one-step, cost-effective DNA-linking method on non-modified polymers significantly simplifies microarray fabrication procedures and permits great flexibility to plastic material selection, thus making it convenient to integrate microarrays into plastic microfluidic systems.     

Multiplexed protein detection using an affinity aptamer amplification assay

Affinity probe capillary electrophoresis (APCE) is potentially one of the most versatile technologies for protein diagnostics, offering an excellent balance between robustness, analysis speed and sensitivity. Combining the immunosensing and separating strength of capillary electrophoresis with the signal enhancement power of nucleic acid amplification, aptamers can further push the analytical limits of APCE to offer ultrasensitive, multiplexed detection of protein biomarkers, even when differences in electrophoretic mobility between the different aptamer-target complexes are limited. It is demonstrated how, through careful selection of experimental parameters, simultaneous detection of picomolar levels of three target proteins can be achieved even with aptamers that were initially selected under very different conditions and further taking into account that the aptamers need to be modified to allow successful PCR amplification. Aptamer-enhanced APCE offers limits of detection that are orders of magnitude lower than those that can be achieved through traditional capillary electrophoresis-based immunosensing. With recent developments in aptamer selection that for the first time realise the promise of aptamers as easily accessible, high affinity recognition molecules, it can therefore be envisioned that aptamer-enhanced APCE on parallel microfluidic platforms can be the basis for a truly high-throughput multiplexed proteomics platform, rivalling genetic screening for the first time.     

Nonequilibrium capillary electrophoresis of equilibrium mixtures: a universal tool for development of aptamers

Aptamers are DNA (or RNA) ligands selected from large libraries of random DNA sequences and capable of binding different classes of targets with high affinity and selectivity. Both the chances for the aptamer to be selected and the quality of the selected aptamer are largely dependent on the method of selection. Here we introduce selection of aptamers by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). The new method has a number of advantages over conventional approaches. First, NECEEM-based selection has exceptionally high efficiency, which allows aptamer development with fewer rounds of selection. Second, NECEEM can be equally used for selecting aptamers and finding their binding parameters. Finally, due to its comprehensive kinetic capabilities, the new method can potentially facilitate selection of aptamers with predefined K(d), k(off), and k(on) of the aptamer-target interaction. In this proof-of-principle work, we describe the theoretical bases of the method and demonstrate its application to a one-step selection of DNA aptamers with nanomolar affinity for protein farnesyltransferase.     

Surface plasmon resonance spectroscopy study of interfacial binding of thrombin to antithrombin DNA aptamers

We have applied surface plasmon resonance (SPR) spectroscopy, in combination with one-step direct binding, competition, and sandwiched assay schemes, to study thrombin binding to its DNA aptamers, with the aim to further the understanding of their interfacial binding characteristics. Using a 15-mer aptamer that binds thrombin primarily at the fibrinogen-recognition exosite as a model, we have demonstrated that introducing a DNA spacer in the aptamer enhances thrombin-binding capacity and stability, as similarly reported for hydrocarbon linkers. The bindings are aptamer surface coverage and salt concentration dependent. When free aptamers or DNA sequences complementary to the immobilized aptamer are applied after the formation of thrombin/aptamer complexes, bound thrombin is displaced to a certain extent, depending on the stability of the complexes formed under different conditions. When the 29-mer aptamer (specific to thrombin's heparin-binding exosite) is immobilized on the surface, its affinity to thrombin appears to be lower than the immobilized 15-mer aptamer, although the 29-mer aptamer is known to have a higher affinity in the solution phase. These findings underline the importance of aptamers' ability to fold into intermolecular structures and their accessibility for target capture. Using a sandwiched assay scheme followed by an additional signaling step involving biotin-streptavidin chemistry, we have observed the simultaneous binding of the 15- and 29-mer aptamers to thrombin protein at different exosites and have found that one aptamer depletes thrombin's affinity to the other when they bind together. We believe that these findings are invaluable for developing DNA aptamer-based biochips and biosensors.     

Selection of DNA Aptamers with Affinity for Pro-gastrin-Releasing Peptide (proGRP), a Tumor Marker for Small Cell Lung Cancer

Aptamers are single-strand oligonucleotides that are generated by the systemic evolution of ligands by exponential enrichment (SELEX) technique and that can bind to target molecules specifically. However, only a few aptamers have been developed to date against tumor markers. To utilize aptamers for tumor diagnosis, a variety of aptamers are required. Here, a single-stranded DNA aptamer specific for pro-gastrin-releasing peptide (proGRP), a marker for small cell lung cancer, was selected using SELEX. After selection, identical sequences were found in the DNA library. This sequence was selected and its binding affinity to proGRP was evaluated using surface plasmon resonance.     

On-chip chemiluminescent signal enhancement using nanostructured gold-modified carbon microarrays

An original method for the enhancement of chemiluminescent (CL) on-chip detection of protein and oligonucleotides is presented. This enhancement is based on the electrodeposition of a gold nanostructured layer onto a screen-printed (SP) carbon microarray prior to the immobilization of biomolecules through a well-established diazonium adduct electrodeposition. Morphological studies of the Au layer (optical and atomic force microscopy) show that the metal film is composed of nanostructured 800 nm diameter particles covering the entire graphite surface and yielding a high surface area. Using these modified SP microarrays, enhancement factors of 229 and 126 were obtained for prostate-specific antigen (PSA) and p53 oligonucleotide detection, respectively. These enhancements were associated with three different phenomena: an enhancement of the catalyzed chemiluminescent reaction by the gold surface, an increase of the specific surface area for immobilization of the probe biomolecules, and an opposite quenching effect due to the overlapping of the gold absorption and CL emission peaks. For free PSA and target oligonucleotide detection, enhanced performances were obtained, giving detection limits of 5 ng/mL and 0.1 nM, respectively.     

Recent Progress in Aptamer‐Based Functional Probes for Bioanalysis and Biomedicine

Nucleic acid aptamers are short synthetic DNA or RNA sequences that can bind to a wide range of targets with high affinity and specificity. In recent years, aptamers have attracted increasing research interest due to their unique features of high binding affinity and specificity, small size, excellent chemical stability, easy chemical synthesis, facile modification, and minimal immunogenicity. These properties make aptamers ideal recognition ligands for bioanalysis, disease diagnosis, and cancer therapy. This review highlights the recent progress in aptamer selection and the latest applications of aptamer‐based functional probes in the fields of bioanalysis and biomedicine.     

Polymer microfluidic chip for online monitoring of microarray hybridizations

A disposable single use polymer microfluidics chip has been developed and manufactured by micro injection molding. The chip has the same outer dimensions as a standard microscope slide (25 x 76 x 1.1 mm) and is designed to be compatible with existing microscope slide handling equipment like microarray scanners. The chip contains an inlet, a 10 microL hybridization chamber capable of holding a 1000 spot array, a waste chamber and a vent to allow air to escape when sample is injected. The hybridization chamber ensures highly homogeneous hybridization conditions across the microarray. We describe the use of this chip in a flexible setup with fluorescence based detection, temperature control and liquid handling by computer controlled syringe pumps. The chip and the setup presented in this article provide a powerful tool for highly parallel studies of kinetics and thermodynamics of duplex formation in DNA microarrays. The experimental setup presented in this article enables the on-chip microarray to be hybridized and monitored at several different stringency conditions during a single assay. The performance of the chip and the setup is demonstrated by on-line measurements of a hybridization of a DNA target solution to a microarray. A presented numerical model indicates that the hybridization process in microfluidic hybridization assays is diffusion limited, due to the low values of the diffusion coefficients D of the DNA and RNA molecules involved.     

DNA and RNA aptamers: from tools for basic research towards therapeutic applications

The systematic evolution of ligands by exponential enrichment (SELEX) is a combinatorial oligonucleotide library-based in vitro selection approach in which DNA or RNA molecules are selected by their ability to bind their targets with high affinity and specificity, comparable to those of antibodies. Nucleic acids with high affinity for their targets have been selected against a wide variety of compounds, from small molecules, such as ATP, to membrane proteins and even whole organisms. Recently, the use of the SELEX technique was extended to isolate oligonucleotide ligands, also known as aptamers, for a wide range of proteins of importance for therapy and diagnostics, such as growth factors and cell surface antigens. The number of aptamers generated as inhibitors of various target proteins has increased following automatization of the SELEX process. Their diagnostic and therapeutic efficacy can be enhanced by introducing chemical modifications into the oligonucleotides to provide resistance against enzymatic degradation in body fluids. Several aptamers are currently being tested in preclinical and clinical trials, and aptamers are in the process of becoming a new class of therapeutic agents. Recently, the anti-VEGF aptamer pegaptanib received FDA approval for treatment of human ocular vascular disease.     

信号核酸识体用于药物托普霉素的高灵敏度检测

提出了一种利用RNA核酸识体和光开关化合物[Ru(phen)2dppz\]2+ (RU)高灵敏检测药物分子的荧光分析新方法. 以托普霉素(TOB)为靶分子, 检测限可达到10 nmol/L, 同时该方法有较好的选择性, 能在溶液中实现无需分离的实时检测. 这种无标记检测方法有利于不稳定RNA核酸识体在分析检测中的应用, 简化实验操作, 为其它药物分子的检测提供了新思路.