Detection of TP53 mutation using a portable surface plasmon resonance DNA-based biosensor

A DNA-based surface plasmon resonance (SPR) biosensor has been developed for the detection of TP53 mutation using the inexpensive and commercially available instrument, SPREETA SPR-EVM-BT, from Texas Instruments. A direct immobilisation procedure, based on the coupling of thiol-derivatised oligonucleotide probes (Probe-C6-SH) to bare gold sensor surfaces, was optimized using synthetic oligonucleotides. Hybridisation reactions between the immobilised probe and a short sequence (26 mer) complementary, non-complementary and one-point mutation DNA were then investigated. The main analytical parameters of the sensor system were studied in detail including selectivity, sensitivity, reproducibility and analysis time. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR)-amplified real samples, DNA extracted from both normal, wild-type, (Jurkat) and mutated (Molt 4), carrying the mutation at codon 248 of the TP53 cell lines. The results obtained demonstrate that the DNA-based SPR biosensor was able to distinguish sequences present in the various samples that differ only by one base; and hence, it appears to be a strong candidate technique for the detection of gene mutation.     

Quartz crystal microbalance (QCM) affinity biosensor for genetically modified organisms (GMOs) detection

A DNA piezoelectric sensor has been developed for the detection of genetically modified organisms (GMOs). Single stranded DNA (ssDNA) probes were immobilised on the sensor surface of a quartz crystal microbalance (QCM) device and the hybridisation between the immobilised probe and the target complementary sequence in solution was monitored. The probe sequences were internal to the sequence of the 35S promoter (P) and Nos terminator (T), which are inserted sequences in the genome of GMOs regulating the transgene expression. Two different probe immobilisation procedures were applied: (a) a thiol-dextran procedure and (b) a thiol-derivatised probe and blocking thiol procedure. The system has been optimised using synthetic oligonucleotides, which were then applied to samples of plasmidic and genomic DNA isolated from the pBI121 plasmid, certified reference materials (CRM), and real samples amplified by the polymerase chain reaction (PCR). The analytical parameters of the sensor have been investigated (sensitivity, reproducibility, lifetime etc.). The results obtained showed that both immobilisation procedures enabled sensitive and specific detection of GMOs, providing a useful tool for screening analysis in food samples.     

Oriented coupling of major histocompatibility complex (MHC) to sensor surfaces using light assisted immobilisation technology

Controlled and oriented immobilisation of proteins for biosensor purposes is of extreme interest since this provides more efficient sensors with a larger density of active binding sites per area compared to sensors produced by conventional immobilisation. In this paper oriented coupling of a major histocompatibility complex (MHC class I) to a sensor surface is presented. The coupling was performed using light assisted immobilisation--a novel immobilisation technology which allows specific opening of particular disulphide bridges in proteins which then is used for covalent bonding to thiol-derivatised surfaces via a new disulphide bond. Light assisted immobilisation specifically targets the disulphide bridge in the MHC-I molecule alpha(3)-domain which ensures oriented linking of the complex with the peptide binding site exposed away from the sensor surface. Structural analysis reveals that a similar procedure can be used for covalent immobilisation of MHC class II complexes. The results open for the development of efficient T cell sensors, sensors for recognition of peptides of pathogenic origin, as well as other applications that may benefit from oriented immobilisation of MHC proteins.     

DNA microdevice for electrochemical detection of Escherichia coli 0157:H7 molecular markers

An electrochemical DNA sensor based on the hybridization recognition of a single-stranded DNA (ssDNA) probe immobilized onto a gold electrode to its complementary ssDNA is presented. The DNA probe is bound on gold surface electrode by using self-assembled monolayer (SAM) technology. An optimized mixed SAM with a blocking molecule preventing the nonspecific adsorption on the electrode surface has been prepared. In this paper, a DNA biosensor is designed by means of the immobilization of a single stranded DNA probe on an electrochemical transducer surface to recognize specifically Escherichia coli (E. coli) 0157:H7 complementary target DNA sequence via cyclic voltammetry experiments. The 21 mer DNA probe including a C6 alkanethiol group at the 5' phosphate end has been synthesized to form the SAM onto the gold surface through the gold sulfur bond. The goal of this paper has been to design, characterise and optimise an electrochemical DNA sensor. In order to investigate the oligonucleotide probe immobilization and the hybridization detection, experiments with different concentration of DNA and mismatch sequences have been performed. This microdevice has demonstrated the suitability of oligonucleotide Self-assembled monolayers (SAMs) on gold as immobilization method. The DNA probes deposited on gold surface have been functional and able to detect changes in bases sequence in a 21-mer oligonucleotide.     

Facile and rapid direct gold surface immobilization with controlled orientation for carbohydrates

Effective surface immobilization is a prerequisite for numerous carbohydrate-related studies including carbohydrate-biomolecule interactions. In the present work, we report a simple and rapid modification technique for diverse carbohydrate types in which direct oriented immobilization onto a gold surface is accomplished by coupling the amine group of a thiol group-bearing aminophenyl disulfide as a new coupling reagent with an aldehyde group of the terminal reducing sugar in the carbohydrate. To demonstrate the generality of this proposed reductive amination method, we examined its use for three types of carbohydrates: glucose (monosaccharide), lactose (disaccharide), and GM1 pentasaccharide. Through successful mass identifications of the modified carbohydrates, direct binding assays on gold surface using surface plasmon resonance and electrochemical methods, and a terminal galactose-binding lectin assay using atomic force microscopy, we confirmed several advantages including direct and rapid one-step immobilization onto a gold surface and exposure of functional carbohydrate moieties through oriented modification of the terminal reducing sugar. Therefore, this facile modification and immobilization method can be successfully used for diverse biomimetic studies of carbohydrates, including carbohydrate-biomolecule interactions and carbohydrate sensor or array development for diagnosis and screening.     

Surface plamon resonance imaging of DNA based biosensors for potential applications in food analysis

The adsorption processes of oligonucleotides immobilised onto suitable photolithographic patterned gold substrates have been investigated in aqueous buffer solution by using a home made surface plasmon resonance (SPR) imaging equipment. A rapid self-assembled method for the construction of DNA chips to be used in SPR imaging experiments have been followed. The immobilised DNA molecules (probes) adopted in our SPR experiments anchored to a gold surface via thiol group were 5'thiol-modified containing a (CH(2))(15) tail. The hybridisation processes taking place with its complementary sequence have been observed and characterized by monitoring phenomena by a SPR imaging system. The two analysed oligonucleotides (probes and target) are of interest in plant gene biotechnological application and differing for the presence at the 5'-end of a poly T16 spacer. Dynamic investigation of smallest changes in SPR imaging pictures performed in liquid phase in the presence of DNA complementary probes have been performed. Quantitative information in terms of threshold of sensitivity has been extracted by using a specific images treatment.     

Hybridisation of surface-immobilised single-stranded oligonucleotides and polymer monitored by surface plasmon resonance

We have investigated the hybridisation of thiol-modified single-stranded DNA embedded in a polyacrylamide layer through the technique of surface plasmon resonance (SPR). Kinetic studies were carried out by two different immobilisation methods: (a) SH-ssDNA was firstly attached on gold and the remaining free space was filled with polymer and (b) SH-ssDNA and the polymer was attached onto the surface from the same solution. The immobilisation methods were compared for various concentrations of SH-ssDNA. Hybridisation was dependent on both the immobilisation method and the concentration of the components. The highest hybridisation was obtained when SH-ssDNA and the polymer was immobilised from the same solution at low SH-ssDNA concentration or when high concentrations of oligos were spread onto the surface and the surface was post-treated with polymer. The target response corresponded to a surface coverage of 100+/-15 ng/cm2. The same surface coverage on hybridisation was also obtained when low concentration of SH-ssDNA and polymer was attached onto the surface from the same solution. The non-specific binding of sample DNA was very low at optimal concentrations due to the polymer and the hybridisation was linearly dependent on target concentration.     

Improved procedures for immobilisation of oligonucleotides on gold-coated piezoelectric quartz crystals

The high sensitivity and specificity of DNA hybridisation techniques makes them powerful tools for environmental or clinical analysis. This work describes the development of a DNA piezoelectric biosensor for the detection of the hybridisation reaction. Attention was focused on the choice of the coating chemistry that could be used for the immobilisation of oligonucleotides onto the gold surface of the quartz crystal. Four immobilisation procedures were tested and compared considering the amount of immobilised probe, the extent of the hybridisation reaction, the possibility of regeneration and the absence of non-specific adsorption. All the experiments were performed with oligonucleotides of 25 bases (probe, target and non-complementary oligonucleotide). The four coating methods were all based on the use of self-assembled monolayers (SAM). Three of them employed the interaction between streptavidin and biotin for the immobilisation of a biotinylated probe. Results indicated that immobilisation of a biotinylated probe on streptavidin linked to a layer of carboxylated dextran provides higher sensitivity for the detection of the hybridisation reaction, absence of non-specific adsorption and a higher stability with respect to the regeneration step.     

Comparison between different strategies of covalent attachment of DNA to glass surfaces to build DNA microarrays

DNA microarray is a powerful tool allowing simultaneous detection of many different target molecules present in a sample. The efficiency of the array depends mainly on the sequence of the capture probes and the way they are attached to the support. The coupling procedure must be quick, covalent, and reproducible in order to be compatible with automatic spotting devices dispensing tiny drops of liquids on the surface. We compared several coupling strategies currently used to covalently graft DNA onto a glass surface. The results indicate that fixation of aminated DNA to an aldehyde-modified surface is a choice method to build DNA microarrays. Both the coupling procedure and the hybridization efficiency have been optimized. The detection limit of human cytomegalovirus target DNA amplicons on such DNA microarrays has been estimated to be 0.01 nM by fluorescent detection.     

Investigation into the effect that probe immobilisation method type has on the analytical signal of an EIS DNA biosensor

The analytical performance of an enhanced surface area electrolyte insulator semiconductor (EIS) device was investigated for DNA sensor development. The work endeavored to advance EIS performance by monitoring the effect of DNA probe layers have on the impedimetric signal during target hybridisation detection. Two universally employed covalent chemistries, direct and spacer-mediated attachment of amino modified probe molecules to amino-functionalised surfaces were investigated. Relative areal densities of immobilised probe were measured on planar and enhanced surface area substrates using epi-fluorescence microscopy. The reproducibility of the each immobilisation method was seen to have a direct effect on the reproducibility of the impedimetric signal. The sensitivity and selectivity was seen to be dependent on the type of immobilisation method. Real time, impedimetric detection of target DNA hybridisation concentrations as low as 25 and 1 nM were possible. The impact that probe concentration had on the impedimetric signal for selective and non-selective interactions was also investigated.     

Label-free genosensor based on immobilized DNA hairpins on gold surface

In this report, we demonstrate a label-free genosensor based on DNA hairpins coupled to gold coated sensor surfaces. The hairpin probes were labeled with a thiolated moiety for immobilization at the 5' end and with a fluorophore for signal transduction at the 3' end. In the absence of the complement, the fluorophore is quenched by energy transfer to the gold surface. Addition of the target sequence leads to the hairpin unfolding, and releases the fluorescent signal. This built-in property, using a gold film as both the immobilizing substrate and quenching agent, has the advantage of simplicity in design and ease of further integration. Our results showed that lengths of both the stem and the loop structures have significant effects on the sensor performance. Hybridization kinetics was investigated for various probe/target lengths and concentrations. An optimized hairpin probe gave a fluorescent signal increase of 39 folds after hybridization, which is much higher than the earlier reported results. A limit of detection (LOD) down to 0.3 nM for the complementary target DNA detection has been achieved. The developed sensor was further successfully applied for the detection of single-base mismatch targets, as well as for the direct detection of PCR products.     

A disposable polymer sensor chip combined with micro-fluidics and surface plasmon read-out

This paper describes a new type of disposable polymeric sensor chip based on the grating coupling of surface plasmon modes combined with a micro-fluidic channel system. A specifically designed silicon stamp with nano-structure (grating) on the micro-structures (micro-channel) was fabricated by combining a holographic method and photolithography. By using such a stamp the micro-channels, the grating coupler and the gold which was first thermally evaporated onto the stamp were transferred to the polymeric substrate successfully in one step. It is demonstrated that the grating profile in the micro-channels allowed a very efficient coupling of the laser light to the surface plasmons propagating at the bottom of the micro-channels. The transferred gold exhibits properties of a freshly cleaned surface, and the self-assembly of a functional thiol derivative (mercapto-PEG) onto the sensor chip can be monitored by surface plasmon spectroscopy. The results obtained in this sensor chip show no difference from those obtained on a regular grating-coupled SPR sensor chip.     

Immobilisation of DNA to polymerised SU-8 photoresist

SU-8 is an epoxy-based photosensitive resist, which is currently used for a large variety of MEMS and lab-on-a-chip applications. Here, we demonstrate a one-step process to functionalize SU-8 with DNA probes. The immobilisation procedure relies on direct coupling of DNA to SU-8 and resulted in surfaces with functional capture probe densities of approximately 10 fmol/mm(2) as determined by hybridisation assays with fluorescent labelled target molecules. A comparable density of functional capture probes was measured on commercial aldehyde coated glass. DNA probes did not decrease in hybridisation performance after 10 min incubation in water at 98 degrees C prior to hybridisation, indicating a covalent bond between DNA and SU-8. Finally, DNA microarrays of high quality were obtained on SU-8 by contact printing of probe solution directly on SU-8 demonstrating a simple method for the implementation of microarrays in microsystems.     

Independently-addressable micron-sized biosensor elements

With the continuing development of micro-total analysis systems and sensitive biosensing technologies, it is often desirable to immobilize biomolecules onto a surface in a small well-defined area. A novel method was developed to electrochemically attach DNA probes to micron-sized regions of a gold surface using biotin-LC-hydrazide (BH). Previously, we have found that the radical produced during the oxidation of BH will attach to a wide variety of electroactive surfaces. An array of micron-sized gold band electrodes (75 microm wide) was fabricated onto glass microscope slides and BH was deposited onto each electrode through the application of an oxidizing potential. Subsequent attachment of avidin to the biotinylated surface created the 'molecular sandwich' architecture necessary for further immobilization of biotinylated biomolecules to the surface. In this work, we utilized biotinylated DNA probes of varying sequence to illustrate the specificity of the attachment scheme. The immobilization of avidin, DNA probe, and hybridization of DNA target is visualized with fluorescence tags and the spatially selective attachment and hybridization of unique DNA sequences is demonstrated.     

Surface Plasmon Resonance Studies of the Hybridization Behavior of DNA-Modified Gold Nanoparticles with Surface-Attached DNA Probes

Colloidal gold nanoparticles (AuNPs) have been extensively investigated as amplification tags to improve the sensitivity of surface plasmon resonance (SPR) biosensors. When using the so-called AuNP-enhanced SPR technique for DNA detection, the density of single-stranded DNA (ssDNA) on both the AuNPs and planar gold substrates is of crucial importance. Thus, in this work, we carried out a systematical study about the influence of surface ssDNA density onto the hybridization behavior of various DNA-modified AuNPs (DNA-AuNPs) with surface-attached DNA probes by using surface plasmon resonance spectroscopy. The lateral densities of the ssDNA on both the AuNPs and planar gold substrates were controlled by using different lengths of oligo-adenine sequence (OAS) as anchoring group. Besides SPR measurements, the amount of the captured DNA-AuNPs after the hybridization was further identified via atomic force microscope (AFM). SPR and AFM results clearly indicated that a higher ssDNA density on either the AuNPs or the gold substrates would give rise to better hybridization efficiency. Moreover, SPR data showed that the captured DNA-AuNPs could not be removed from SPR sensor surfaces using various dehybridization solutions regardless of surface ssDNA density. Consequently, it is apparent that the hybridization behavior of DNA-AuNPs was different from that of solution-phase ssDNA. Based on these data, we hypothesized that both multiple recognitions and limited accessibility might account for the hybridization of DNA-AuNPs with surface-attached ssDNA probes.

     

Directed immobilization of nucleic acids at ultramicroelectrodes using a novel electro-deposited polymer

A two-step method for the directed immobilization of nucleic acids at ultramicroelectrodes with micron-size dimensions is described. The approach is based on the immobilization of streptavidin at the surface of carbon or noble metal electrodes within a novel electro-deposited polymer, formed by electropolymerization of the natural compound scopoletin (7-hydroxy-6-methoxy-coumarin) at potentials between 0.4 and 0.7 V vs. Ag/AgCl. Biotin-tagged nucleic acids or proteins are immobilized on top of the modified electrodes in a second step. The new method has some advantages compared to classical electropolymerization approaches (e.g. polypyrrole, polyphenol), because the growing polymer is highly hydrophilic, resulting in efficient incorporation of streptavidin and a high biotin binding capacity of 6 pmol cm(-2). The polymer film seems to be non-conductive but shows good swelling properties in aqueous solutions. The feasibility of the method for the electro-directed biochemical modification of individual microelectrodes has been demonstrated by sequential immobilization of two different single strand oligonucleotides onto interdigitated ultramicroelectrodes. The resulting miniature DNA probe was used for single base mutation detection with two synthetic targets (fluorescence-labeled 17-mer oligomers) by evaluating the fluorescence patterns after hybridisation with the immobilised DNA probes. The new method is useful for the production of microelectrode based DNA chips and for the electro-directed immobilisation of biomolecules at microelectrode structures with high spatial resolution and yield.     

Liquid phase SPR imaging experiments for biosensors applications

Surface plasmon resonance (SPR) has recently gained attention as a label-free method for the detection of biological molecules binding onto functionalised surfaces. It is one of the most sensitive detection method for monitor variations in the thickness and refractive index in ultra-thin films. Here, the adsorption processes of oligonucleotides onto gold substrates have been investigated in aqueous buffer solution using SPR imaging measurements. The hybridization of a thiol-modified, single stranded oligonucleotide anchored to a gold surface via thiol group, with its complementary sequence has been observed and characterised monitoring the hybridization process by SPR equipment. In situ investigation of smallest changes in SPR imaging measurements dynamically performed in liquid phase in the presence of DNA complementary probes was performed. Infrared spectroscopy and scanning electron microscopy characterisation of the functionalised gold surfaces of the biosensor were compared with the images obtained by SPR experimental apparatus.     

A Love wave immunosensor for whole E. coli bacteria detection using an innovative two-step immobilisation approach

The efficiency of a monomolecular film of (3-glycidoxypropyl) trimethoxysilane (GPTS) on a shear horizontal guided (Love) acoustic wave immunosensor to detect whole Escherichia coli (E. coli) bacteria is demonstrated. Direct anti-E. coli antibodies grafting onto the sensor surface did not lead to a significant bacteria immobilisation, partially attributed to the SiO2 sensor surface roughness. An innovative method has been set up to get around this difficulty and to detect whole bacteria. It consists in grafting goat anti-mouse antibodies (GAM) onto the sensor surface in a first step and introducing E. coli bacteria mixed with anti-E. coli antibodies onto the sensor in a second step. We describe the characteristics of such a technique like sample preparation time (lower than 30 min) and temperature improvements. A 37 degrees C experimental temperature led to the fastest bacteria binding kinetic, reducing the total analysis time. This method enables to keep the specificity of the antibody/antigen interaction and provides significant results in less than 1h. This leads to a detection threshold of 10(6) bacteria/ml in a 500 microl chamber.     

Detection of proteins using a colorimetric bio-barcode assay

The colorimetric bio-barcode assay is a red-to-blue color change-based protein detection method with ultrahigh sensitivity. This assay is based on both the bio-barcode amplification method that allows for detecting miniscule amount of targets with attomolar sensitivity and gold nanoparticle-based colorimetric DNA detection method that allows for a simple and straightforward detection of biomolecules of interest (here we detect interleukin-2, an important biomarker (cytokine) for many immunodeficiency-related diseases and cancers). The protocol is composed of the following steps: (i) conjugation of target capture molecules and barcode DNA strands onto silica microparticles, (ii) target capture with probes, (iii) separation and release of barcode DNA strands from the separated probes, (iv) detection of released barcode DNA using DNA-modified gold nanoparticle probes and (v) red-to-blue color change analysis with a graphic software. Actual target detection and quantification steps with premade probes take approximately 3 h (whole protocol including probe preparations takes approximately 3 days).     

Immobilization of immunoglobulins using lectins with an immune sensor method based on surface plasmon resonance

The effect of different lectins upon the response of immune sensor based on surface plasmon resonance (SPR) was investigated. Lectins affinity to carbohydrates of the IgG can be used to increase the density and orientation of IgG molecules at their immobilisation on the sensor surface. Conditions were elaborated for enhancement of immune sensor response in comparison with that one for bare or preliminary treated with dodecanthiol thin gold sensor surface. It was shown that human IgG revealed maximal affinity to wheat germ lectin and mouse monoclonal antibodies and rabbit IgG--to helix pomatia lectin. Pig antibodies, similar to human IgG, showed the greatest affinity to wheat germ lectin.