Development of surface chemistry for surface plasmon resonance based sensors for the detection of proteins and DNA molecules

The immobilisation of biological recognition elements onto a sensor chip surface is a crucial step for the construction of biosensors. While some of the optical biosensors utilise silicon dioxide as the sensor surface, most of the biosensor surfaces are coated with metals for transduction of the signal. Biological recognition elements such as proteins can be adsorbed spontaneously on metal or silicon dioxide substrates but this may denature the molecule and can result in either activity reduction or loss. Self assembled monolayers (SAMs) provide an effective method to protect the biological recognition elements from the sensor surface, thereby providing ligand immobilisation that enables the repeated binding and regeneration cycles to be performed without losing the immobilised ligand, as well as additionally helping to minimise non-specific adsorption. Therefore, in this study different surface chemistries were constructed on SPR sensor chips to investigate protein and DNA immobilisation on Au surfaces. A cysteamine surface and 1%, 10% and 100% mercaptoundecanoic acid (MUDA) coatings with or without dendrimer modification were utilised to construct the various sensor surfaces used in this investigation. A higher response was obtained for NeutrAvidin immobilisation on dendrimer modified surfaces compared to MUDA and cysteamine layers, however, protein or DNA capture responses on the immobilised NeutrAvidin did not show a similar higher response when dendrimer modified surfaces were used.     

Comparison of biosensor platforms for surface plasmon resonance based detection of paralytic shellfish toxins

Paralytic shellfish poisoning (PSP) toxins are produced by certain marine dinoflagellates and may accumulate in bivalve molluscs through filter feeding. The Mouse Bioassay (MBA) is the internationally recognised reference method of analysis, but it is prone to technical difficulties and regarded with increasing disapproval due to ethical reasons. As such, alternative methods are required. A rapid surface plasmon resonance (SPR) biosensor inhibition assay was developed to detect PSP toxins in shellfish by employing a saxitoxin polyclonal antibody (R895). Using an assay developed for and validated on the Biacore Q biosensor system, this project focused on transferring the assay to a high-throughput, Biacore T100 biosensor in another laboratory. This was achieved using a prototype PSP toxin kit and recommended assay parameters based on the Biacore Q method. A monoclonal antibody (GT13A) was also assessed. Even though these two instruments are based on SPR principles, they vary widely in their mode of operation including differences in the integrated μ-fluidic cartridges, autosampler system, and sensor chip compatibilities. Shellfish samples (n = 60), extracted using a simple, rapid procedure, were analysed using each platform, and results were compared to AOAC high performance liquid chromatography (HPLC) and MBA methods. The overall agreement, based on statistical 2 × 2 comparison tables, between each method ranged from 85% to 94.4% using R895 and 77.8% to 100% using GT13A. The results demonstrated that the antibody based assays with high sensitivity and broad specificity to PSP toxins can be applied to different biosensor platforms.     

Development of an optical biosensor assay for detection of β-lactam antibiotics in milk using the penicillin-binding protein 2x*

An assay was developed for the detection of residues of penicillins and cephalosporins in milk using a surface plasmon resonance (SPR) biosensor. The assay was based on the inhibition of the binding of digoxigenin-labelled ampicillin (DIG-AMPI) to a soluble penicillin-binding protein 2x derivative (PBP 2x*) of Streptococcus pneumoniae. Samples were incubated with PBP 2x* in a first step, whereby β-lactams in positive samples would bind to the PBP 2x*. Non-complexed PBP 2x* was then allowed to form a complex with DIG-AMPI in a second incubation step. The formed DIG-AMPI/PBP 2x*-complexes were detected in a SPR-based biospecific interaction assay (BIA) for digoxigenin with an antibody against digoxigenin immobilised on the sensor chip. Although binding of matrix components to the sensor chip (non-specific binding) occurred, benzylpenicillin, ampicillin, amoxicillin, cloxacillin, cephalexin and cefoperazone could be detected in defatted bulk raw milk samples at concentrations corresponding to the maximum residue limits (MRL) set by the European Union. The influence of matrix components on the performance of the assay was examined in more detail by analysing individual raw milk samples from 19 cows. Compared to bulk raw milk samples, individual samples showed a higher level and variation of matrix interferences. Non-specific binding could be reduced to a lower and more constant level by a heat-treatment step, a centrifugation step and the addition of carboxymethylated dextran to the samples. With this sample preparation, benzylpenicillin could be detected at MRL (4 μg kg⁻¹) in individual raw milk samples. Thus, the assay could be the basis for a screening test for routine use.     

Measurement uncertainty in analytical studies based on surface plasmon resonance

The sensitivity of surface plasmon resonance (SPR) transducers depends on the thickness and spatial organization of interfacial structures at their surfaces. This is because the response of the SPR sensor is determined by integrating the distance-dependent refractive index (spatial interfacial architectures), weighted by the square of the electromagnetic field, from zero to infinite distance. The effect of SPR transducer sensitivity variation on the accuracy of SPR analysis is considered. Our quantitative estimation (based on the results of refractometric studies) gave a value for sensitivity variation of about 3% for the formation of a self-assembled thiocyanate layer or a trypsin-soybean trypsin inhibitor surface complex. The estimated accuracy in measured variation (i.e., by 0.01) for the refractive index of the external medium was 3 × 10⁻⁴. This restriction, which follows immediately from the physical mechanism of the SPR phenomenon, should be taken into account when analyzing data obtained with the above technique.     

Development of an optical surface plasmon resonance biosensor assay for (fluoro)quinolones in egg, fish, and poultry meat

The aim of this study was to develop an optical biosensor inhibition immunoassay, based on the surface plasmon resonance (SPR) principle, for use as a screening test for 13 (fluoro)quinolones, including flumequine, used as veterinary drugs in food-producing animals. For this, we immobilised various quinolone derivatives on the sensor chip and tested binding of a range of different antibodies (polyclonal and one engineered antibody) in the presence and absence of free (fluoro)quinolones. The main challenge was to detect flumequine in an assay giving good results for the other compounds. One antigen–antibody combination proved satisfactory: polyclonal antibodies raised against a dual immunogen and, on the sensor chip, a fluoroquinolone derivative. It was the first time that this concept of the bi-active antibody was described in the literature.The assay, optimised for detection in three matrices (poultry muscle, fish, and egg), was tested on incurred samples prepared by liquid extraction followed by two washing steps. This rapid, simple method proved adequate for detecting at least 13 (fluoro)quinolones at concentrations below established maximum residue levels (MRLs). The reference molecule norfloxacin could be detected in the range of 0.1–10 μg kg⁻¹ in extracts of egg and poultry meat and in the range of 0.1–100 μg kg⁻¹ in extracts of fish. The determined midpoints of these calibration curves were about 1, 1.5 and 3 μg kg⁻¹ in poultry meat, egg and fish, respectively.     

Surface plasmon resonance biosensor for enrofloxacin based on deoxyribonucleic acid

A DNA-based surface plasmon resonance biosensor for enrofloxacin was developed. Heating denatured DNA immobilized on the gold-coated glass surface was exploited. The immobilization was performed by a layer-by-layer co-deposition with a cationic polymer. The sensor performance was tested with real biological probes. Direct and simple determination of enrofloxacin in milk samples was demonstrated. The sensor response obeys Langmuir binding isotherm being almost linear until about 20 μg mL⁻¹. The detection limit in milk samples was estimated to be 3 μg mL⁻¹.     

Preparation and characterization of a polyclonal antibody from rabbit for detection of trinitrotoluene by a surface plasmon resonance biosensor

A polyclonal antibody against trinitrophenyl (TNP) derivatives was raised in rabbit, and the antibody was applied to detection of trinitrotoluene (TNT) using a surface plasmon resonance (SPR) biosensor. TNP-keyhole limpet hemocyanine (TNP-KLH) conjugate was injected into a rabbit, and a polyclonal anti-TNP antibody was realized after purification of the sera using protein G. Aspects of the anti-TNP antibody against various nitroaromatic compounds, such as cross-reactivities and affinities, were characterized. The temperature dependence of the affinity between the anti-TNP antibody and TNT was also evaluated. The quantification of TNT was based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-β-alanine-ovalbumin (TNP-β-ala-OVA) and anti-TNP antibody was inhibited in the presence of free TNT in solution. TNP-β-ala-OVA was immobilized to the dextran matrix on the Au surface by amine coupling. The addition of a mixture of free TNT to the anti-TNP antibody was found to decrease the incidence angle shift due to the inhibitory effect of TNT. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range of 3 × 10⁻¹¹ to 3 × 10⁻⁷ g/ml. To increase the sensitivity of the sensor, anti-rabbit IgG antibody was used. After flowing the mixture of free TNT and anti-TNP antibody, anti-rabbit IgG antibody was injected, and the incidence angle shift was measured. Amplification of the signal was observed and the detection limit was improved to 1 × 10⁻¹¹ g/ml.     

Development of an ultra-low volume flow cell for surface plasmon resonance detection in a miniaturized capillary electrophoresis system

A miniaturized capillary electrophoresis system coupled to a surface plasmon resonance (SPR) sensor on a microfluidic platform fabricated from PDMS is detailed. A previously described split-flow injection technique is first utilized to manipulate sample into the microfluidic chip, followed by separation within the fused-silica capillary and final off-capillary detection of analytes via SPR. Instead of using commercial SPR flow cells requiring relatively large detection volumes, samples of less than 1 nL volume are utilized. The interface between the CE system and SPR sensor made it possible to detect minute volumes of sample with minimal dispersion. The flow cell has the potential to be applicable to miniaturized flow-injection (FI) systems where submicroliter volumes of sample are frequently only available for analysis. The components present in solution, but not bound to the sensor surface, were also investigated. The sensitivity of the CE-SPR system was similar to that found in UV-spectrometric instruments and nonchromophoric components could also be measured.     

Surface plasmon resonance biosensor for highly sensitive detection of microRNA based on DNA super-sandwich assemblies and streptavidin signal amplification

MicroRNAs (miRNAs) play an important regulatory role in cells and dysregulation of miRNA has been associated with a variety of diseases, making them a promising biomarker. In this work, a novel biosensing strategy has been developed for label-free detection of miRNA using surface plasmon resonance (SPR) coupled with DNA super-sandwich assemblies and biotin–strepavidin based amplification. The target miRNA is selectively captured by surface-bound DNA probes. After hybridization, streptavidin is employed for signal amplification via binding with biotin on the long DNA super-sandwich assemblies, resulting in a large increase of the SPR signal. The method shows very high sensitivity, capable of detecting miRNA at the concentration down to 9 pM with a wide dynamic range of 6 orders of magnitude (from 1 × 10⁻¹¹ M to 1 × 10⁻⁶ M) in 30 min, and excellent specificity with discriminating a single base mismatched miRNA sequence. This biosensor exhibits good reproducibility and precision, and has been successfully applied to the detection of miRNA in total RNA samples extracted from human breast adenocarcinoma MCF-7 cells. It, therefore, offers a highly effective alternative approach for miRNA detection in biomedical research and clinical diagnosis.     

An optical immunosensor based on surface plasmon resonance for determination of bFGF

An optical immunosensor based on surface plasmon resonance (SPR) has been developed for immunosensing. The sensor is designed on the basis of fixing incident angle of light and measuring the reflected intensities in the wavelength range of 400-800 nm simultaneously. The SPR spectrum was shown in terms of reflected light intensities versus wavelengths of incident light. The intensity of the reflected light reaches the minimum at the resonant wavelength. Molecular self-assembling in solution is used to form the sensing membrane on gold substrate. The kinetic processes of sensing monolayer formation were studied. The basic fibroblast growth factor, a kind of basic polypeptide, was determined in the concentration range of 0.24-9.6 μg/ml. Under optimum experimental conditions, the sensor has a good repeatability, reversibility and selectivity.     

Surface Plasmon Resonance‐based Inhibitive Immunoassay Coupled with Dummy Template Molecularly Imprinted Polymer Solid Phase Extraction for On‐line Analysis of Trace Clenbuterol

A new indirect inhibitive immunoassay using surface plasmon resonance (SPR) coupled with molecularly imprinted polymer (MIP) was developed and applied for the analysis of trace clenbuterol (CL). A MIP coating using phenylephrine as the dummy template was synthesized in a flexible quartz capillary (30 cm×0.25 mm i.d.) by in situ polymerization technique, which then was used as the online solid phase extraction (SPE) tube before SPR detection. The thickness of the coating was 198 nm on average, illustrated by scanning electron microscope (SEM) image. The mechanism for adsorption of CL on dummy template MIP was found to be a Freundlich isotherm and pseudo‐second‐order model. The immunoassay was conducted on BIAcore 3000 biosensor automatically without pre‐treatment. The calibration curve was generated by linear fit in the range of 0.1–10 ng L^‐1 and 10–100 ng L^‐1. The detection limit was 0.1 ng L^–1, which is super sensitive. This method was directly applied for the analysis of real‐world samples without pretreatment.     

A novel detection of single-stranded DNA binding protein based on ss-DNA modified chip using surface plasmon resonance microscopy

An ss-DNA gold chip was prepared based on self-assembly of the thiol-derivatized oligonucleotide,and used for thedetermination of single-stranded binding protein(SSB)by surface plasmon resonance microscopy(SPR).The experiment resultsshowed that SSB binds ss-DNA with high specificity,and relative signal of SPR response is proportional to the concentration of SSBin the range of 0.1-100 ng/mL with a detection limit(S/N=3)of 0.07 ng/mL.     

基于表面等离子体子共振成像技术研究苏拉明和顺铂对肝癌细胞生长的抑制作用

基于表面等离子体子共振成像(SPRi)技术提出了一种实时、 非标记的新型抗癌药物药效评估方法. 以聚二甲基硅氧烷(PDMS)为材料, 制作了包含微柱结构的微流控芯片作为流通反应池, 配合自行设计组装的SPRi生物传感器完成肿瘤细胞的特异性捕获及检测, 研究了苏拉明和顺铂对肝癌细胞HepG2的生长抑制作用. 同时引入辅助验证实验, 即采用常规八肽胆囊收缩素(简称CCK-8)法测定上述药物对肝癌细胞增殖的抑制作用. SPRi检测结果表明, 苏拉明和顺铂能抑制肿瘤细胞HepG2增殖并呈现剂量、 时间依赖关系.     

Ag Nanowire‐Ag Nanoparticle Hybrids for the Highly Enhanced Fluorescene Detection of Protoporphyrin IX Based on Surface Plasmon‐Enhanced Fluorescence

In this study, we developed an approach to fabricate novel 1D Ag NWs‐Ag NPs hybrid substrate for enhanced fluorescene detection of protoporphyrin IX (PpIX) based on surface plasmon‐enhanced fluorescence. The Ag NWs‐Ag NPs hybrid was synthesized by combining the hydrothermal method and self‐assembly method with the asisstance of polyvinylpyrrolidone (PVP). When the Ag NWs‐Ag NPs hybrid was deposited on the glass substrate and employed as active substrate to detect PpIX, the fluorescence intensity of PpIX was enhanced greatly due to the coupling effect of localized surface plasmon‐localized surface plasmon (LSP‐LSP) and localized surface plasmon‐surface plasmon propagation (LSP‐SPP) which induced great enhancement of the electromagnetic field. Furthermore, the enhancement effect was approximately linear when the concentration of PpIX was ranged from 1×10^?7 mol/L to 2×10^?5 mol/L, and the photobleaching phenomenon of PpIX was reduced greatly, indicating that the fabricated Ag NWs‐Ag NPs hybrid substrate had well performance for PpIX imaging. This work provides an effective approach to prepare highly sensitive and stable fluorescence enhancement substrate, and has great potential application in fluorescence imaging.     

Single-crystal sapphire-fiber optic sensors based on surface plasmon resonance spectroscopy for in situ monitoring

Single-crystal sapphire-fiber optic sensors based on surface plasmon resonance (SPR) for refractive index (RI) measurements of aqueous and hydrothermal water solutions are described. Accurate measurement of RIs is essential to efficient operation and control of broad range of engineering processes. Some of these processes are carried out with harsh environments, such as high-temperature, high pressure, and chemical corrosion. These extreme physical conditions are proving a limiting factor in application of the conventional silica-based optical sensors. Single-crystal sapphire is an ideal material for sensor applications, where reliable performance is required in the extreme environment conditions. With regard to the liquid species detection, most applications of SPR sensors are designed to function near the refractive index of water (1.3330 RI). The RI changes of aqueous solution can be easily monitored by silica-fiber (RI, 1.4601 at 550 nm) based SPR sensor. However, the sapphire waveguide has a prohibitively high RI (1.7708 at 546 nm) for unmodified monitoring of the RI changes of aqueous solutions. For that purpose, a practical SPR probe geometry has been applied to the ability to tune the SPR coupling wavelength/angle pair with sapphire-fiber based SPR probe.     

Fabrication of a novel immunosensor using functionalized self-assembled monolayer for trace level detection of TNT by surface plasmon resonance

We have developed a new immunosensor based on self-assembly chemistry for highly sensitive and label-free detection of 2,4,6-trinitrotoluene (TNT) using surface plasmon resonance (SPR). A monolayer of amine terminated poly(ethylene glycol) hydrazinehydrochloride (PEG-NH₂) thiolate was constructed on an activated gold surface and immobilized with trinitrophenyl-β-alanine (TNPh-β-alanine) by amide coupling method. The binding interaction of a monoclonal anti-TNT Ab (M-TNT Ab) with TNPh-β-alanine immobilized thiolate monolayer surface was monitored and evaluated for detection of TNT based on the principle of indirect competitive immunoreaction. Here, the competition between the self-assembled TNT derivative and the TNT in solution for binding with antibody yields in the response signal that is inversely proportional to the concentration of TNT in the linear detection range. With the present immunoassay format, TNT could be detected in the concentration range from 0.008 ng/ml (8 ppt) to 30 ng/ml (30 ppb). The response time for an immunoreaction was 2 min and one immunocycle could be done with in 4 min including surface regeneration. Bound antibodies could be easily eluted from the self-assembled immunosurface at high recoveries (more than 100 cycles) using pepsin solution without any damage to the TNT derivatives immobilized on the surface. The compact self-assembled monolayer was highly stable and prevented the non-specific adsorption of proteins on the surface favoring error free measurement.     

A microfluidic chip‐based fluorescent biosensor for the sensitive and specific detection of label‐free single‐base mismatch via magnetic beads‐based “sandwich” hybridization strategy

A novel microfluidic chip‐based fluorescent DNA biosensor, which utilized the electrophoretic driving mode and magnetic beads‐based “sandwich” hybridization strategy, was developed for the sensitive and ultra‐specific detection of single‐base mismatch DNA in this study. In comparison with previous biosensors, the proposed DNA biosensor has much more robust resistibility to the complex matrix of real saliva and serum samples, shorter analysis time, and much higher discrimination ability for the detection of single‐base mismatch. These features, as well as its easiness of fabrication, operation convenience, stability, better reusability, and low cost, make it a promising alternative to the SNPs genotyping/detection in clinical diagnosis. By using the biosensor, we have successfully determined oral cancer‐related DNA in saliva and serum samples without sample labeling and any preseparation or dilution with a detection limit of 5.6 × 10^?11 M, a RSD (n = 5) < 5% and a discrimination factor of 3.58–4.54 for one‐base mismatch.     

Comparison of an antibody and its recombinant derivative for the detection of the small molecule explosive 2,4,6-trinitrotoluene

Antibodies are commonly used as recognition elements in immunoassays because of their high specificity and affinity, and have seen extensive use in competitive assays for the detection of small molecules. However, these complex molecules require production either in animals or by mammalian cell cultures, and are not easily tailored through genetic manipulation. Single chain antibodies (scFv), recombinantly expressed molecules consisting of only the antibody's binding region joined via a linking peptide, can provide an alternative to intact antibodies. We describe the characterization of a new monoclonal antibody (mAb), 2G5B5, able to detect the small molecule explosive 2,4,6-trinitrotoluene (TNT) and the scFv derived from its variable regions. The mAb and scFv were tested by surface plasmon resonance to determine their affinity for an immobilized TNT surrogate; dissociation constants were determined to be 1.5 × 10⁻¹³ M and 4.8 × 10⁻¹⁰ M respectively. Circular dichroism was used to determine their melting temperatures. The mAb is more stable melting at ∼75 °C while the scFv melts at ∼65 °C. The recognition elements were incorporated into a competitive assay format using a bead-based multiplexing platform to examine their sensitivity and specificity. The scFv was able to detect TNT ∼10-fold more sensitively than the mAb in this assay format, allowing detection of TNT concentrations down to at least 1 μg L⁻¹. The 2G5B gave similar detection limits to a commercial anti-TNT mAb, but was less specific, recognizing 1,3,5-trinitrobenzene (TNB) equally well as TNT.     

Development of a surface plasmon resonance-based assay for the detection of Corynebacterium pseudotuberculosis infection in sheep

Caseous lymphadenitis (CLA), a disease affecting sheep and goats, is caused by Corynebacterium pseudotuberculosis and is difficult to detect, especially at early stages in its development. A surface plasmon resonance-based biosensor assay for the detection of antibodies to the phospholipase D (PLD) exotoxin of C. pseudotuberculosis in sheep serum was successfully generated. It employed a recombinant form of PLD, which was immobilised, and all aspects of the assay including minimisation of non-specific binding, and the regeneration of the chip, were optimised. The applicability of the assay was initially demonstrated using sera collected from experimentally infected sheep and from sheep with no prior history of infection. The assay was then evaluated on a panel of clinical samples and the results obtained compared very favourably to those obtained by a double sandwich ELISA (over 90% similarity) and clearly verified its analytical value.     

Immobilization of antibody fragment for immunosensor application based on surface plasmon resonance

Biosurface fabrication using the Fab′ fragment of immunoglobulin (IgG) was carried out by self-assembly (SA) technique. The pepsin-digested monoclonal antibody (Mab) against bovine insulin containing the F(ab′)₂ fragment and residual proteins was separated using affinity chromatography and dialysis. To prevent the nonspecific binding of F(ab′)₂ onto gold (Au) substrate, the native disulfide bridge was reduced using dithiothreitol (DTT) to convert F(ab′)₂ into Fab′, which made the immobilization to be carried out via the native thiol (–SH) group. The fabricated biosurface using SA technique showed the formation of stable thin film through AFM topography. Through the concentration change of DTT and Fab′, the absorption characteristics against the Au surface were investigated using surface plasmon resonance (SPR) with the flow cell. The amount of immobilized antibody fragment and the antigen binding capacity were regulated with respect to the reduction state and concentration of F(ab′)₂. Based on the biosurface of the fabricated Fab′, the insulin-detection was carried out by the measurement of SPR. The proposed antibody surface could successfully detect the bovine insulin at the concentration from 100 ng/mL to 10 μg/mL.