Multi-wavelength immunoassays using surface plasmon-coupled emission

We describe a new method for multi-wavelength immunoassays using surface plasmon-coupled emission (SPCE). This phenomenon is coupling of excited fluorophores with a nearby thin metal film, in our case silver, resulting in strongly directional emission into the underlying glass substrate. The angle at which the radiation propagate through the prism depends on the surface plasmon angle for the relevant wavelength. These angles depend on emission wavelength, allowing measurement of multiple analytes using multiple emission wavelengths. We demonstrated this possibility using antibodies labeled with either Rhodamine Red-X or AlexaFluor 647. These antibodies were directed against an antigen protein bound to the silver surface. The emission from each labeled antibody occurred at a different angle on the glass prism, allowing independent measurement of surface binding of each antibody. This method of SPCE immunoassays can be readily extended to 4 or more wavelengths.     

Detection of hepatitis B virus surface antigen with the use of an optical biosensor

The detection of hepatitis B virus surface antigen (HBsAg) with the use of a model IAsys+ two-channel optical biosensor is based on the registration of interaction between anti-HBs monoclonal antibodies forming the surface layer of the biochip of the biosensor cuvette and blood serum HBsAg. For the first time a two-channel optical biosensor has been used for the detection of HBsAg in blood serum samples. The comparative analysis of the detection of HBsAg by two methods, viz. with the use of an optical biosensor and the enzyme immunoassay, has demonstrated lower sensitivity, but higher specificity of the detection of this antigen by means of a model IAsys+ biosensor with the biochip, prepared in the process of the work. The main advantages of the biosensor detection lie in the registration of interaction in real time without introducing special markers into the molecules under study.     

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g. by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-Pérot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a ''real time'' observation of bacteria attachment.The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 10⁴ cells/ml) in less than an hour.     

Grating-coupled surface plasmon resonance: a cell and protein microarray platform

Grating-coupled surface plasmon resonance (GCSPR) is a method for the accurate assessment of analyte in a multiplexed format using small amounts of sample. In GCSPR, the analyte is flowed across specific receptors (e.g. antibodies or other proteins) that have been immobilized on a sensor chip. The chip surface is illuminated with p-polarized light that couples to the gold surface's electrons to form a surface plasmon. At a specific angle of incidence, the GCSPR angle, the maximum amount of coupling occurs, thus reducing the intensity of reflected light. Shifts in the GCSPR angle can be correlated with refractive index increases following analyte capture by chip-bound receptors. Because regions of the chip can be independently analyzed, this system can assess 400 interactions between analyte and receptor on a single chip. We have used this label-free system to assess a number of molecules of immunological interest. GCSPR can simultaneously detect an array of cytokines and other proteins using the same chip. Moreover, GCSPR is also compatible with assessments of antigen expression by intact cells, detecting cellular apoptosis and identifying T cells and B cells. This technology represents a powerful new approach to the analysis of cells and molecular constituents of biological samples.     

Microarray of recombinant antibodies using a streptavidin sensor surface self-assembled onto a gold layer

We have developed a sensitive method for the detection of recombinant antibody-antigen interactions in a microarray format. The biochip sensor platform used in this study is based on an oriented streptavidin monolayer that provides a biological interface with well-defined surface architecture that dramatically reduces nonspecific binding interactions. All the antibody or antigen probes were biotinylated and coupled onto streptavidin-coated biochip surfaces (1 microL total volume). The detection limits for the immobilized probes on the microarray surface were 0.5 microgram/mL (200 fmol/spot) for the peptide antigen and 0.1 microgram/mL (3 fmol/spot) for the recombinant antibodies. Optimal concentrations for the detection of the Cy5-labeled protein target were in the range of 20 micrograms/mL. Protein microchips were used to measure antibody-antigen kinetics, to find optimal temperature conditions, and to establish the shelf life of recombinant antibodies immobilized on the streptavidin surface. For recombinant antibody fragments with a kDa of 10-100 nM, we have established an easy and direct immunoassay. In addition, we developed an indirect method for antibody detection with no need for expensive and time-consuming antibody purifications and modifications. Such a method was shown to be useful for large-scale screening of recombinant antibody fragments directly after their functional expression in bacteria. Our data demonstrate that recombinant antibody fragments are suitable components in the construction of antibody chips.     

Ultrasensitive electrical detection of protein using nanogap electrodes and nanoparticle-based DNA amplification

The present study describes an ultrasensitive protein biochip that employs nanogap electrodes and self-assembled nanoparticles to electrically detect protein. A bio-barcode DNA technique amplifies the concentration of target antigen at least 100-fold. This technique requires the establishment of conjugate magnetic nanoparticles (MNPs) and gold nanoparticles (AuNPs) through binding between monoclonal antibodies (2B2), the target antigen, and polyclonal antibodies (GP). Both GP and capture ssDNA (single-strand DNA) bonds to bio-barcode ssDNA are immobilized on the surface of AuNPs. A denature process releases the bio-barcode ssDNAs into the solution, and a hybridization process establishes multilayer AuNPs over the gap surface between electrodes. Electric current through double-layer self-assembled AuNPs is much greater than that through self-assembled monolayer AuNPs. This significant increase in electric current provides evidence that the solution contains the target antigen. Results show that the protein biochip attains a sensitivity of up to 1 pg/μL.     

Labeless AC impedimetric antibody-based sensors with pgml(-1) sensitivities for point-of-care biomedical applications

This paper describes the development and characterisation of labeless immunosensors for (a) the cardiac drug digoxin and (b) bovine serum albumin (BSA). Commercial screen-printed carbon electrodes were used as the basis for the sensors. Two methods were used to immobilise antibodies at the electrode surface. Aniline was electropolymerised onto these electrodes to form a thin planar film of conductive polyaniline; the polyaniline film was then utilised as a substrate to immobilise biotinylated anti-digoxin using a classical avidin-biotin affinity approach. As an alternative approach, poly(1,2-diaminobenzene) was electrodeposited onto the carbon electrodes and this modified surface was then sonochemically ablated to form an array of micropores. A second electropolymerisation step was then used to co-deposit conductive polyaniline along with antibodies for BSA within these pores to produce a microarray of polyaniline protrusions with diameters of several mum, containing entrapped anti-BSA. The resulting antibody grafted electrodes were interrogated using an AC impedance protocol before and following exposure to digoxin or BSA solutions, along with control samples containing a non-specific IgG antibody. The impedance characteristics of both types of electrode were changed by increasing concentrations of antigen up to a saturation level. Calibration curves were obtained by subtraction of the non-specific response from the specific response, thereby eliminating the effects of non-specific adsorption of antigen. Both the use of microelectrode arrays and affinity binding protocols showed large enhancements in sensitivity over planar electrodes containing entrapped antibodies and gave similar sensitivities to our other published work using affinity-based planar electrodes. Detection limits were in the order of 0.1ngml(-1) for digoxin and 1.5ngml(-1) for BSA.     

Use of surface plasmon-coupled emission to measure DNA hybridization

The authors describe a new approach to measuring DNA hybridization based on surface plasmon-coupled emission (SPCE). SPCE is the resonance coupling of excited fluorophores with electron motions in thin metal films, resulting in efficient transfer of energy through the film and radiation into the glass substrate. The authors evaluated the use of SPCE for detection of DNA hybridization. An unlabeled capture biotinylated oligonucleotide was attached near the surface of a thin (50 nm) silver film using streptavidin. The authors then measured the emission intensity of single-stranded Cy5-labeled DNA upon binding to a complementary oligomer attached to a silver film. Hybridization could be detected by an increase in SPCE, which appeared as light radiated into the substrate at a sharply defined angle near 73 degrees from the normal. The largest signals were observed when the excitation angle of incidence equaled the surface plasmon wavelength, but directional emission was also observed without excitation by the surface plasmon evanescent field. The increased intensity is due to proximity to the metal surface, so that hybridization can be detected without a change in the quantum yield of the fluorophore. These results indicate that SPCE can provide highly sensitive real-time measurement of DNA hybridization.     

Directional surface plasmon-coupled emission: application for an immunoassay in whole blood

We present a new approach for performing fluorescence immunoassay in whole blood using fluorescently labeled anti-rabbit immunoglobulin G (IgG) on a silver surface. This approach, which is based on surface plasmon-coupled emission (SPCE), provides increased sensitivity and substantial background reduction due to exclusive selection of the signal from the fluorophores located near a bioaffinity surface. This article describes the effect of an optically dense sample matrix, namely human whole blood and serum, on the intensity of the SPCE. An antigen (rabbit IgG) was adsorbed to a slide covered with a thin silver metal layer, and the SPCE signal from the fluorophore-labeled anti-rabbit antibody, binding to the immobilized antigen, was detected. The effect of the sample matrix (buffer, human serum, or human whole blood) on the end-point immunoassay SPCE signal was studied. It was demonstrated that the kinetics of binding could be monitored directly in whole blood or serum. The results showed that human serum and human whole blood attenuate the SPCE end-point signal and the immunoassay kinetic signal only approximately two- and threefold, respectively, as compared with buffer, resulting in signals that are easily detectable even in whole blood. The high optical absorption of the hemoglobin can be tolerated because only fluorophores within a couple of hundred nanometers from the metallic film contribute to SPCE. Excited fluorophores outside the 200-nm layer do not contribute to SPCE, and their free space emission is not transmitted through the opaque metallic film into the glass substrate. We believe that SPCE has the potential of becoming a powerful approach for performing immunoassays based on surface-bound analytes or antibodies for many biomarkers directly in dense samples such as whole blood with no need for washing steps.     

Photoengraving of coverslips and slides to facilitate monitoring of micromanipulated cells or chromosome spreads

A simple photolithographic technique has been developed which can be used to produce microscopic grid patterns on glass coverslips. The grid pattern is first photo-reduced onto film, and the resulting photographic negative is then used as a mask. A glass slide or coverslip, coated with a layer of photoresist, is then exposed to tungsten light through the mask. After developing and etching, the grid pattern is transferred permanently onto glass. This simple and rapid procedure allows one to mass-produce very small, high resolution grids which are useful for monitoring individual microinjected cells or chromosomal spreads under the microscope.     

Immuno-capture of Cryptosporidium parvum using micro-well array

A glass slide and micro-well array chip on which anti-Cryptosporidium parvum antibody was immobilized were used for the rapid capture and detection of C. parvum. Biotinylated anti-C. parvum antibodies were spotted onto the streptavidin-coated glass slides. C. parvum oocysts were captured specifically on the spot when more than 73 ng of anti-C. parvum antibody was applied onto the glass slide. However, C. parvum oocysts captured on the glass slide were detached by repeating washing steps. To improve the capture efficiency of oocysts, capture was performed in a micro-well format consisting of 1024 wells/2.5 mm2 (32 x 32 wells) fabricated as a chip by photolithography. Instead of a flat surface on a glass slide, each well was 30 microm in diameter and 10 microm in depth. Streptavidin was also immobilized onto the micro-well array. The biotinylated anti-C. parvum antibodies were immobilized efficiently onto the chip using a buffer containing 20% methanol. Using this technique C. parvum oocysts were stably captured onto the chip after repeated washing procedures. These data show that the newly designed micro-well array technique described here is useful for antibody-mediated C. parvum capture.     

Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology.

We report here the development and application of a biosensor-based technology that employs surface plasmon resonance for label-free studies of molecular interactions in real time. The sensor chip interface, comprising a thin layer of gold deposited on a glass support, is derivatized with a flexible hydrophilic polymer to facilitate the attachment of specific ligands to the surface and to increase the dynamic range for surface concentration measurements. The sensor can be used to measure surface concentrations down to 10 pg/mm2. Typical coefficients of variation are from two to five percent. We anticipate that the ability to monitor multi-molecular complexes as they form will greatly contribute to the understanding of biorecognition and the structural basis of molecular function.     

Surface plasmon resonance sensor for domoic acid based on grafted imprinted polymer

A molecularly imprinted polymer (MIP) film for domoic acid (DA) was synthesised by direct photo-grafting onto a gold chip suitable for a surface plasmon resonance (SPR) based bioanalytical instrument system, the BIAcore 3000. The gold surface was first functionalised with a self-assembled monolayer of 2-mercaptoethylamine and subsequent carbodiimide chemistry was performed for covalent attachment of the photoinitiator, 4,4'-azobis(cyanovaleric acid). This ensured that the formation of the MIP thin film, comprising 2-(diethylamino) ethyl methacrylate as functional monomer and ethylene glycol dimethacrylate as cross-linker, occurred only at the surface level. Optimisation and control over the grafting procedure were achieved using contact angle measurements and atomic force microscope (AFM) imaging. The surface grafting resulted in the formation of thin and homogeneous MIP film with thickness of 40 nm. A competitive binding assay was performed with free DA and its conjugate with horseradish peroxidase, which was used as a refractive label. The sensor was evaluated for its sensitivity, cross-reactivity, and robustness by using a BIAcore 3000. Likewise, monoclonal antibodies acting as natural receptors for the toxin were studied with the same BIAcore system. Results of a comparison between the artificial and natural receptors are reported. In contrast to monoclonal antibodies, the regeneration of MIP chip did not affect its recognition properties and continuous measurement was possible over a period of at least 2 months.     

Sensitivity enhancement of surface plasmon resonance biosensor with colloidal gold

We enhanced the sensitivity of surface plasmon resonance biosensor by the conversion of the real-time direct binding immunoassay into the sandwich immunoassay, in which colloidal gold particles coated with anti-mouse IgG was used. By the immobilization of anti-mouse IgG onto the carboxymethyl dextran surface of thin gold film, the direct binding of analyte (mouse IgG) onto the sensor chip, and the injection of colloidal gold particles coated with antimouse IgG, about 100 times of sensitivity enhancement was obtained. This result suggests that nanoparticles, which has a high refractive index, homogeneous ultrafine structure and capability of size control, would be applicable for the detection of very small quantity of biomaterial.     

Electroaddressed immobilization of recombinant HIV-1 P24 capsid protein onto screen-printed arrays for serological testing

A serological chemiluminescent biochip was designed based on screen-printed electrode arrays composed of nine 1-mm(2) electrodes. Arrays were shown to be produced with good batch-to-batch reproducibility (standard deviations of 4.4 and 12.0% for ferricyanide oxidation potential and current, respectively) and very good reproducibility within a particular array (2.0 and 7.5% standard deviations for the same controls). Electrode arrays were used to electroaddress various bioconjugate structures comprising a recombinant HIV-1 P24 capsid protein (RH24K) in polypyrrole film. Entrapment of RH24K preimmobilized onto maleic anhydride-alt-methyl vinyl ether copolymer was shown to be the more efficient immobilization procedure. This addressed sensing layer enabled the detection of anti-P24 antibodies at a concentration of 3.5 ng/ml through peroxidase-labeled anti-human immunoglobulin G reaction. The biochip was used to perform an HIV-1 serological test in human sera. HIV-1 seropositive and seronegative sera were easily discriminated using serum dilutions greater than 1/10,000.     

蛋白质微阵列检测抗原-抗体相互作用

为了制备蛋白质微阵列和研究芯片表面抗原-抗体的相互作用,研究了如何在玻片表面固化蛋白质和用荧光染料（Cy3,Cy5）对蛋白质进行标记.结果表明,在醛基修饰的玻璃表面,通过共价偶联的方法将抗原或抗体固定到芯片表面,能使二者保持其特异性结合能力.同时,荧光标记后的抗原或抗体仍然具有特异性结合能力.蛋白质微阵列是通过机械手在玻片表面排阵制作的.芯片上的荧光信号获取采用了激光共焦荧光扫描系统.用不同浓度的抗原探针阵列,对其相应的抗体靶分子的特异性结合进行了分析和研究.此外,还通过在玻片表面固定兔IgG和固定鼠IgG,对羊抗兔和羊抗鼠抗体与其相应抗原的特异性相互作用进行了检测.     

Protein patterning on silicon-based surface using background hydrophobic thin film

A new and convenient protein patterning method on silicon-based surface was developed for protein array by spin coating of hydrophobic thin film (CYTOP). Photolithographic lift-off process was used to display two-dimensional patterns of spatially hydrophilic region. The background hydrophobic thin film was used to suppress nonspecific protein binding, and the hydrophilic target protein binding region was chemically modified to introduce aldehyde group after removal of the photoresist layer. The difference in surface energy between the hydrophilic pattern and background hydrophobic film would induce easier covalent binding of proteins onto defined hydrophilic areas having physical and chemical constraints. Below 1 microg/ml of total protein concentration, the CYTOP hydrophobic film effectively suppressed nonspecific binding of the protein. During the process of protein patterning, inherent property of the hydrophobic thin film was not changed judging from static and dynamic contact angle survey. Quantitative analysis of the protein binding was demonstrated by streptavidin-biotin system.     

Photochemical attachment of biomolecules onto fibre-optics for construction of a chemiluminescent immunosensor.

We report herein a simple and effective way to photochemically immobilize biomolecules onto a fibre-optic silica surface. The system is based on a photoreactive benzophenone derivative that is bound to SiO2 surfaces of the optical fibre via a silane anchor. The benzophenone derivative was 4-allyloxybenzophenone, synthesized by standard procedures that were later used to synthesize the 4-(3'-chlorodimethylsilyl) propyloxybenzophenone and 4-(3'-dichloromethylsilyl) propyloxybenzophenone by regular hydrosilation procedures. After silanization with the benzophenone derivatives, the fibres were immersed in a cholera toxin B subunit solution and illuminated with UV light (wavelength > 345 nm). As a result of the photochemical reaction, a thin layer of the antigen was covalently bound to the benzophenone-modified surface. The photochemically modified fibre-optics were then tested as immunosensors in the detection of cholera anti-toxin antibody and revealed through chemiluminescence measurements. A secondary antibody labelled with horseradish peroxidase acted as the marker for the cholera toxin antibody. A photo-electronic set-up was designed specifically to monitor the signal. The immunosensor system was shown to be both specific and sensitive. The lowest rabbit serum titre detected was 1:1 700,000.     

Immunological biochips for studies of human erythrocytes

Immunological microarrays (biochips) for detecting erythrocyte surface antigens, viz., blood group antigens (A, B, 0) and Rhesus system antigens (D, E, e, C, and c), are described. The biochips represent transparent plastic supports onto which 1.5-mm spots of specific immobilized antibodies (IgM) are coated in different dilutions. The volume of tested blood samples is rather small (1–2 μl). Binding of erythrocytes to antibodies immobilized on the biochips is specific and allows further morphological analysis of bound cells. Analysis of the dynamics of cell detachment from biochip spots using a microfluidic chamber at different flow rates of the washing solution showed that combination of a biochip with a microfluidic chamber is a promising approach to concentration of cells of various immunotypes even if their content in the mixture is very low.     

A surface plasmon resonance immunosensor based on the streptavidin-biotin complex.

It is shown that a streptavidin monolayer immobilized onto an evaporated gold film with biotin forms the basis of a highly specific sensing element. As an example, we show that by immobilizing the biotinylated antibody sex hormone binding globulin (alpha-SHBG) to the bound streptavidin monolayer a specific sensor for the antigen SHBG is readily fabricated. The interaction between immobilized antibody and corresponding antigen is monitored by surface plasmon resonance spectroscopy and is shown to follow a classic Langmuir isotherm. Detection of SHBG at nanomolar concentrations is demonstrated.