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.     

Lable‐Free Electrochemical DNA Sensor Based on Gold Nanoparticles/Poly(neutral red) Modified Electrode

We present a new strategy for the label‐free electrochemical detection of DNA hybridization based on gold nanoparticles (AuNPs)/poly(neutral red) (PNR) modified electrode. Probe oligonucledotides with thiol groups at the 5‐end were covalently linked onto the surface of AuNPs/PNR modified electrode via S‐Au binding. The hybridization event was monitored by using differential pulse voltammetry (DPV) upon hybridization generates electrochemical changes at the PNR‐solution interface. A significant decrease in the peak current was observed upon hybridization of probe with complementary target ssDNA, whereas no obvious change was observed with noncomplementary target ssDNA. And the DNA sensor also showed a high selectivity for detecting one‐mismatched and three‐mismatched target ssDNA and a high sensitivity for detecting complementary target ssDNA, the detection limit is 4.2×10^?12 M for complementary target ssDNA. In addition, the DNA biosensor showed an excellent reproducibility and stability under the DNA‐hybridization conditions.     

Cyclically amplified fluorescent detection of theophylline and thiamine pyrophosphate by coupling self-cleaving RNA ribozyme with endonuclease

A structure-switching-based approach for the design of fluorescent biosensors from known RNA aptazymes were demonstrated for the detection of theophylline and thiamine pyrophosphate (TPP). Taking advantages of the ability of graphene oxide (GO) to protect ssDNA from nuclease cleavage and the cyclic amplification induced by deoxyribonuclease I (DNase I), the amplified assay showed high sensitivity. In the presence of target, the target-dependent hammerhead aptazyme cleaves off. The released Shine–Dalgarno (SD) sequence was introduced into the detection system, in which a FAM labeled probe ssDNA was noncovalently assembled on GO, and the fluorescence of the dye was completely quenched. In the presence of the released sequence, the binding between the dye-labeled DNA and the SD sequence alter the conformation of dye-labeled DNA, and disturb the interaction between the dye-labeled DNA and GO, liberating dye-labeled DNA from GO. The fluorescent intensity was increased, whereupon the DNase I can cleave the free DNA in the DNA/RNA complex, thereby liberating the fluorophore and ultimately releasing the SD RNA sequence. The released SD RNA sequence then binds another DNA probe, and the cycle starts anew, which leads to significant amplification of the fluorescent signal. The strategy showed good sensitivity and the dynamic ranges were of 0.1–10 μM and 0.5–100 μM for theophylline and TPP, respectively. The approach opens up a wide range of possibilities for sensing of other small molecules in biological entities.     

Insulin-binding aptamer-conjugated graphene oxide for insulin detection

This paper describes a simple and sensitive aptamer/graphene oxide (GO) based assay for insulin detection. GO can protect DNA from nuclease cleavage, but aptamers can be detached from the GO surface by specific target binding. This exposes the aptamers to enzymatic cleavage and releases the target for a new cycle. Cycling of targets leads to significant signal amplification and low LOD.     

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.     

Towards the optimization of an optical DNA sensor: control of selectivity coefficients and relative surface affinities

Short single-stranded DNA (ssDNA) oligonucleotides can be grown on the surface of fused silica by automated nucleic acid synthesis. The immobilized ssDNA can be deposited at a desired average density. The density of ssDNA provides a controlled parameter that in combination with temperature, ionic strength and pH, can be used to define the selectivity of hybridization. Furthermore, the density of ssDNA can be used to control the affinity of complementary DNA so that it associates with the nucleic acids on the surface rather than areas that are not coated with ssDNA. The characteristic melt temperature observed for immobilized double-stranded DNA (dsDNA) 20mer shifts by up to 10 °C when a single base pair mismatch is present in the center of a target oligonucleotide. Optimization of quantitative analysis of such single base pair mismatches requires use of select experimental conditions to maximize the formation of the fully matched target duplex while minimizing the formation of the mismatched duplex. Results based on fiber optic biosensors that are used to study binding of fluorescein-labeled complementary DNA demonstrate that it is possible to achieve a selectivity coefficient of fully matched to single base pair mismatch of approximately 85-1, while maintaining >55% of the maximum possible signal that can be obtained from the fully matched target duplex.     

Electrochemical DNA Biosensor Based on Partially Reduced Graphene Oxide Modified Carbon Ionic Liquid Electrode for the Detection of Transgenic Soybean A2704‐12 Gene Sequence

In this work a partially reduced graphene oxide (p‐RGO) modified carbon ionic liquid electrode (CILE) was prepared as the platform to fabricate an electrochemical DNA sensor, which was used for the sensitive detection of target ssDNA sequence related to transgenic soybean A2704‐12 sequence. The CILE was fabricated by using 1‐butylpyridinium hexafluorophosphate as the binder and then p‐RGO was deposited on the surface of CILE by controlling the electroreduction conditions. NH₂ modified ssDNA probe sequences were immobilized on the electrode surface via covalent bonds between the unreduced oxygen groups on the p‐RGO surface and the amine group at the 5′‐end of ssDNA, which was denoted as ssDNA/p‐RGO/CILE and further used to hybridize with the target ssDNA sequence. Methylene blue (MB) was used as electrochemical indicator to monitor the DNA hybridization. The reduction peak current of MB after hybridization was proportional to the concentration of target A2704‐12 ssDNA sequences in the range from 1.0×10^?12 to 1.0×10^?6 mol/L with a detection limit of 2.9×10^?13 mol/L (3σ). The electrochemical DNA biosensor was further used for the detection of PCR products of transgenic soybean with satisfactory results.     

A graphene-based real-time fluorescent assay of deoxyribonuclease I activity and inhibition

Using the remarkable difference in the affinity of graphene oxide (GO) with double strand DNA (dsDNA) and short DNA fragments, we report for the first time a GO-based nonrestriction nuclease responsive system. Our system was composed of GO and a fluorescent dye fluorescein amidite (FAM)-labeled dsDNA substrate (F-dsDNA). At first, the fluorescence of this F-dsDNA substrate was quenched upon addition of GO. When nuclease was added to the mixture of dsDNA and GO, hydrolysis of dsDNA was initiated and small DNA fragments were produced. As a result, the short FAM-linked DNA fragments were released from GO due to the weak affinity of GO with short DNA fragments, and the fluorescence got a restoration. At present, many sensing systems are based on the fact that GO prefers to bind long single strand DNA (ssDNA) over dsDNA or short ssDNA. As for our system, GO has a prior binding with dsDNA over short DNA fragments. Compared with previous methods, this assay platform has some advantages. First, since GO can be prepared in large quantities from graphite available at very low cost, this method shows advantages of simplicity and cost efficiency. Besides, the proposed GO-based nuclease assay provides high sensitivity due to the super quenching capacity of GO. Using deoxyribonuclease I (DNase I) as a model system, DNase I activity can be quantitatively analyzed by the velocity of the enzymatic reaction, and 1.75 U mL⁻¹ DNase I can be significantly detected. Moreover, the fluorescent intensity with various concentrations of nuclease becomes highly discriminating after 3–8 min. Thus, it is possible to detect nuclease activity within 3–8 min, which demonstrates another advantage of quick response of the present system. Finally, use of dsDNA as substrate, our method can achieve real-time nuclease activity/inhibition assay, which is time-saving and effortless.     

MALDI‐MS detection of noncovalent interactions of single stranded DNA with Escherichia coli single‐stranded DNA‐binding protein

The Escherichia coli single‐stranded DNA binding protein (SSB) selectively binds single‐stranded (ss) DNA and participates in the process of DNA replication, recombination and repair. Different binding modes have previously been observed in SSB?ssDNA complexes, due to the four potential binding sites of SSB. Here, chemical cross‐linking, combined with high‐mass matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS), is used to determine the stoichiometry of the SSB?ssDNA complex. SSB forms a stable homotetramer in solution, but only the monomeric species (m/z 19 100) can be detected with standard MALDI‐MS. With chemical cross‐linking, the quaternary structure of SSB is conserved, and the tetramer (m/z 79 500) was observed. We found that ssDNA also functions as a stabilizer to conserve the quaternary structure of SSB, as evidenced by the detection of a SSB?ssDNA complex at m/z 94 200 even in the absence of chemical cross‐linking. The stability of the SSB?ssDNA complex with MALDI strongly depends on the length and strand of oligonucleotides and the stoichiometry of the SSB?ssDNA complex, which could be attributed to electrostatic interactions that are enhanced in the gas phase. The key factor affecting the stoichiometry of the SSB?ssDNA complex is how ssDNA binds to SSB, rather than the protein‐to‐DNA ratio. This further suggests that detection of the complex by MALDI is a result of specific binding, and not due to non‐specific aggregation in the MALDI plume. Copyright © 2012 John Wiley & Sons, Ltd.     

Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid‐State NMR Spectroscopy

DnaB helicases are bacterial, ATP‐driven enzymes that unwind double‐stranded DNA during DNA replication. Herein, we study the sequential binding of the “non‐hydrolysable” ATP analogue AMP‐PNP and of single‐stranded (ss) DNA to the dodecameric DnaB helicase from Helicobacter pylori using solid‐state NMR. Phosphorus cross‐polarization experiments monitor the binding of AMP‐PNP and DNA to the helicase. ¹³C chemical‐shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The helicase switches upon AMP‐PNP addition into a conformation apt for ssDNA binding, and AMP‐PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP‐PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid‐state NMR can provide for the characterization of protein–DNA interactions and the interplay with ATP or its analogues.     

基于石墨烯和量子点自组装膜的能量转移构筑的高灵敏度DNA界面荧光传感

以QDs作为荧光探针, HIV1病毒序列DNA为研究对象, 设计了Quartz/PDDA/PSS/PDDA/CdTe/ssDNA自组装膜, 利用氧化石墨烯(GO)猝灭自组装膜上CdTe QDs的荧光, 而靶DNA(tDNA)与自组装膜表面ssDNA的互补配对作用使GO与CdTe QDs的距离增加, 导致自组装膜上量子点的荧光恢复, 由此建立了一种基于GO与QDs自组装膜之间荧光共振能量转移的快速灵敏检测DNA的界面分析方法, 检出限为7.26×10^-14 mol/L. 本方法制备的DNA探针操作简单, 自组装膜表面修饰的ssDNA提高了方法的选择性, GO的猝灭作用降低了检测背景, 极大地提高了荧光分析方法的灵敏度.     

Efficient Preparation of Large‐Sized Rings of Single‐Stranded DNA through One‐Pot Ligation of Multiple Fragments

Circular single‐stranded DNA (c‐ssDNA) has significant applications in DNA detection, the development of nucleic acid medicine, and DNA nanotechnology because it shows highly unique features in mobility, dynamics, and topology. However, in most cases, the efficiency of c‐ssDNA preparation is very low because polymeric byproducts are easily formed due to intermolecular reaction. Herein, we report a one‐pot ligation method to efficiently prepare large c‐ssDNA. By ligating several short fragments of linear single‐stranded DNA (l‐ssDNA) in one‐pot by using T4 DNA ligase, longer l‐ssDNAs intermediates are formed and then rapidly consumed by the cyclization. Since the intramolecular cyclization reaction is much faster than intermolecular polymerization, the formation of polymeric products is suppressed and the dominance of intramolecular cyclization is promoted. With this simple approach, large‐sized single‐stranded c‐ssDNAs (e.g., 200‐nt) were successfully synthesized in high selectivity and yield.     

Label‐Free Detection of MicroRNA: Two‐Step Signal Enhancement with a Hairpin‐Probe‐Based Graphene Fluorescence Switch and Isothermal Amplification

A label‐free approach with multiple enhancement of the signal for microRNA detection has been introduced. The key idea of this strategy is achieved by taking advantage of a novel graphene oxide (GO)/intercalating dye based fluorescent hairpin probe (HP) and an isothermal polymerization reaction. In this paper, we used microRNA‐21 (mir‐21) as the target to examine the desirable properties of this assay. When the target, as a “trigger”, was hybridized with the HP and caused a conformation change, an efficient isothermal polymerization reaction was activated to achieve the first step of the “signal” amplification. After incubation with the platform of GO/intercalating dye, the formed complex of DNA interacted with the high‐affinity dye and then detached from the surface of the GO, a process that was accompanied by distinguishable fluorescence recovery. Further signal enhancement has been accomplished by a mass of intercalating dye inserting into the minor groove of the long duplex replication product. Due to the efficient and multiple amplification steps, this approach exerted a substantial enhancement in sensitivity and could be used for rapid and selective detection of Mir‐21 at attomole levels. Proof‐of‐concept evidence has been provided for the proposed cost‐effective strategy; thus, this strategy could expand the application of GO‐material‐based bioanalysis for nucleic acid studies.     

Adsorptive Stripping Voltammetric Detection of Single‐Stranded DNA at Electrochemically Modified Glassy Carbon Electrode

Electrochemically modified glassy carbon electrode (GCE) was used to study the electrochemical oxidation and detection of denatured single‐stranded (ss) DNA by means of adsorptive stripping voltammetry. The modification of GCE, by electrochemical oxidation at +1.75 V (vs.SCE) for 10 min and cyclic sweep between +0.3 V and ?1.3 V for 20 cycles in pH 5.0 phosphate buffer, results in 100‐fold improvement in sensitivity for ssDNA detection. We speculated that the modified GCE has a high affinity to single‐stranded DNA through hydrogen bond (specific static adsorption). Single‐stranded DNA can accumulate at the GCE surface at open circuit and produce a well‐defined oxidation peak corresponding to the guanine residues at about +0.80 V in pH 5.0 phosphate buffer, while the native DNA gives no signal under the same condition. The peak currents are proportional to the ssDNA concentration in the range of 0–18.0 μg mL^?1. The detection limit of denatured ssDNA is ca. 0.2 μg mL^?1 when the accumulation time is 8 min at open circuit. The accumulation mechanism of ssDNA on the modified GCE was discussed.     

Simple and label-free electrochemical assay for signal-on DNA hybridization directly at undecorated graphene oxide

Exploring graphene oxide (GO), DNA hybridization detection usually relies on either GO decoration or DNA sequences labeling. The former endows GO with desired chemical, optical, and biological properties. The latter adopts labeled molecules to indicate hybridization. In the present work, we propose a simple, label-free DNA assay using undecorated GO directly as the sensing platform. GO is anchored on diazonium functionalized electrode through electrostatic attraction, hydrogen bonding or epoxy ring-opening. The π–π stacking interaction between hexagonal cells of GO and DNA base rings facilitates DNA immobilization. The adsorbed DNA sequence is specially designed with two parts, including immobilization sequence and probe sequence. In the absence of target, the two sequences lie nearly flat on GO platform. In the presence of target, probe hybridizes with it to form double helix DNA, which ‘stands’ on GO. While the immobilization sequence part remains ‘lying’ on GO surface. Hence, DNA hybridization induces GO interfacial property changes, including negative charge and conformational transition from ‘lying’ ssDNA to ‘standing’ dsDNA. These changes are monitored by electrochemical impedance spectroscopy and adopted as the analytical signal. This strategy eliminates the requirement for GO decoration or DNA labeling, representing a comparatively simple and effective way. Finally, the principle is applied to the detection of conserved sequence of the human immunodeficiency virus 1 pol gene fragment. The dynamic detection range is from 1.0 × 10⁻¹² to 1.0 × 10⁻⁶ M with detection limit of 1.1 × 10⁻¹³ M with 3σ. And the sequences with double- or four-base mismatched are readily distinguishable. In addition, this strategy may hold great promise for potential applications from DNA biosensing to nanostructure framework construction based on the versatile DNA self-assembly.     

Label‐Free and Label Based Electrochemical Detection of Hybridization by Using Methylene Blue and Peptide Nucleic Acid Probes at Chitosan Modified Carbon Paste Electrodes

A chitosan modified carbon paste electrode (ChiCPE) based DNA biosensor for the recognition of calf thymus double stranded DNA (dsDNA), single stranded DNA (ssDNA) and hybridization detection between complementary DNA oligonucleotides is presented. DNA and oligonucleotides were electrostatically attached by using chitosan onto CPE. The amino groups of chitosan formed a strong complex with the phosphate backbone of DNA. The immobilized probe could selectively hybridize with the target DNA to form hybrid on the CPE surface. The detection of hybridization was observed by using the label‐free and label based protocols. The oxidation signals of guanine and adenine greatly decreased when a hybrid was formed on the ChiCPE surface. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with target. The signals of MB were investigated at dsDNA modified ChiCPE and ssDNA modified ChiCPE and the increased peak currents were observed, in respect to the order of electrodes. The hybridization of peptide nucleic acid (PNA) probes with the DNA target sequences at ChiCPE was also investigated. Performance characteristics of the sensor were described, along with future prospects.     

New Anthraquinone Derivatives as Electrochemical Redox Indicators for the Visualization of the DNA Hybridization Process

Interactions of dsDNA and ssDNA, at the surface of gold and silver electrodes, with three novel anthraquinone derivatives: 3‐(9′,10′‐dioxo‐9′,10′‐dihydro‐anthracen‐1‐yl)‐7,11‐di(carboxymethyl)‐3,7,11‐triazatridecanedioic acid, (AQ‐1); 1‐(9′,10′‐dioxo‐9′,10′‐dihydro‐anthracen‐1yl)‐9‐carboxymethyl‐5‐methyl‐1,5,9‐triazaundecanoicacid, (AQ‐2); and N‐(2‐(9,10‐dioxo‐9,10‐dihydro‐anthracen‐1‐ylamino)ethyl)‐2‐(1,4,10,13‐tetraoxa‐7,16‐diazacyclooctadecan‐7‐yl)acetamide, (AQ‐3) are studied. These derivatives are well soluble in water and phosphate buffer solutions. The square wave voltammetric behavior of these redox indicators is described and the parameters of interactions with DNA are reported. It is also pointed out that these compounds can be employed as the hybridization indicators. The difference in the binding ability of the particular redox indicator to single and double stranded DNA can be used for the detection of the complementary nucleic acids.     

Design, synthesis, conformational analysis and nucleic acid hybridisation properties of thymidyl pyrrolidine-amide oligonucleotide mimics (POM)

Pyrrolidine-amide oligonucleotide mimics (POM) 1 were designed to be stereochemically and conformationally similar to natural nucleic acids, but with an oppositely charged, cationic backbone. Molecular modelling reveals that the lowest energy conformation of a thymidyl-POM monomer is similar to the conformation adopted by ribonucleosides. An efficient solution phase synthesis of the thymidyl POM oligomers has been developed, using both N-alkylation and acylation coupling strategies. 1H NMR spectroscopy confirmed that the highly water soluble thymidyl-dimer, T2-POM, preferentially adopts both a configuration about the pyrrolidine N-atom and an overall conformation in D2O that are very similar to a typical C3'-endo nucleotide in RNA. In addition the nucleic acid hybridisation properties of a thymidyl-pentamer, T5-POM, with an N-terminal phthalimide group were evaluated using both UV spectroscopy and surface plasmon resonance (SPR). It was found that T5-POM exhibits very high affinity for complementary ssDNA and RNA, similar to that of a T5-PNA oligomer. SPR experiments also showed that T5-POM binds with high sequence fidelity to ssDNA under near physiological conditions. In addition, it was found possible to attenuate the binding affinity of T5-POM to ssDNA and RNA by varying both the ionic strength and pH. However, the most striking feature exhibited by T5-POM is an unprecedented kinetic binding selectivity for ssRNA over DNA.     

Amplified electrochemical detection of nucleic acid hybridization via selective preconcentration of unmodified gold nanoparticles

The common drawback of optical methods for rapid detection of nucleic acid by exploiting the differential affinity of single-/double-stranded nucleic acids for unmodified gold nanoparticles (AuNPs) is its relatively low sensitivity. In this article, on the basis of selective preconcentration of AuNPs unprotected by single-stranded DNA (ssDNA) binding, a novel electrochemical strategy for nucleic acid sequence identification assay has been developed. Through detecting the redox signal mediated by AuNPs on 1, 6-hexanedithiol blocked gold electrode, the proposed method is able to ensure substantial signal amplification and a low background current. This strategy is demonstrated for quantitative analysis of the target microRNA (let-7a) in human breast adenocarcinoma cells, and a detection limit of 16 fM is readily achieved with desirable specificity and sensitivity. These results indicate that the selective preconcentration of AuNPs for electrochemical signal readout can offer a promising platform for the detection of specific nucleic acid sequence.