Surface Plasmon Resonance Imaging Signal Research Articles

Detection of morphine and data processing using surface plasmon resonance imaging sensor

Abstract Based on the surface plasmon resonance imaging (SPRi) instrument, we established a new method of analyzing morphine in urine by processing a calibration curve. According to an inhibition immunoassay, gradient concentration of morphine and morphine-BSA fixed on the chip competitively combine with morphine antibody on the chip. Given the three mathematical models, the data of SPRi signals generated from SPRi with morphine was processed to obtain the calibration curve. Ultrafiltration was used to pretreat blank urine samples with adding morphine, and then investigated the advantages and disadvantages of each model. With a limit detection of 6.57 ng·mL−1, the method and mathematical models can provide robust support for SPRi sensors used in further environmental detection, such as the epidemiological study of sewage.

Quantitatively sensitive and resolution-enhancing strategies in optical signal conversion of SPR images using extensive angle-scanning and image-reconstruction

Surface plasmon resonance imaging (SPRi) is a technology developed from SPR that has emerged in response to the growing need for high-throughput and for discovering diverse applications across multiple domains. Despite its potential, SPRi has encountered limitations in sensitivity when handling low-molecular-mass materials, which emphasizes the need for the quantitative processing of SPRi signals. In this study, an SPRi signal conversion process was developed to enhance the contrast of SPR images and convert them into quantitatively analyzed signals. At first, an optimum incident angle of light was determined as an angle that maximized the difference between sums of the brightness % of functionalized regions and the brightness % of background region in the SPR image, which enabled to obtain a distinct SPR image. In addition, the enhancement of contrast and reduction of noise in the SPR image was achieved using Contrast Limited Adaptive Histogram Equalization (CLAHE) method. Then various pixel domains of the SPR images were simultaneously quantified and visualized the multi-pixel processing technique. For validating the process, an SiO2/Au thin film was modified with four different functionalities and applied to the detection of gas molecules. The SPR images processed using the proposed process exhibited sensitive and quantitative information about the interactions of the functionalities with the target gas in the distinct multiple regions, thus, were applicable to the quantitative monitoring of various phenomena occurring on the plasmonic interface via the advantages of SPRi.

Unraveling the binding microprocess of individual Streptococcus mutans cells via sucrose-dependent adhesion based on surface plasmon resonance imaging

ABSTRACT The initial microprocess of a major cariogenic bacterium Streptococcus mutans forming biofilm at a single-cell level via sucrose-dependent adhesion has not been observed because the cells’ high moisture content caused measurement challenges. To develop a high-sensitivity biosensor chip and a real-time, label-free method to observe bioactive molecule interactions with single cells from oral biofilms. We made the chips of immobilized bacteria by micronano-processing. A surface plasmon resonance imaging (SPRI) system was used to detect and record the association and dissociation microprocess of S. mutans with sucrose/dextran solutions of various concentrations, and the calculus model was adopted to treat the data. At the location of S. mutans, a unique ‘comet-tail’ SPRI signal was observed. The binding patterns of S. mutans differed between individual cells exposed to the same solution as well as between sucrose and dextran. The different cells exhibited different affinities with dissociation constants for sucrose being 5.697 × 10−3 to 3.689 M and for dextran 1.235 × 10−3 to 1.282 M, indicating cell-to-cell heterogeneity. Our SPRI detection method is effective in investigating microbial binding, initial biofilm formation, and oral microecology. It offers new possibilities for studying oral microorganism characteristics and development of oral diseases.

Detection of carcinoembryonic antigens using a wavy gold-silver alloy nanoplate enhanced surface plasmon resonance imaging biosensor.

Carcinoembryonic antigen (CEA) is regarded as a promising broad spectrum tumor biomarker for clinical diagnosis, progression, and prognosis. Surface plasmon resonance imaging (SPRi) was considered as one of the powerful tools for immunoassay with advantages of label-free, real-time detection with high-throughput. Herein, wavy gold-silver alloy nanoplates functionalized with anti-CEA antibodies providing high protein loading capacity and high mass are used as signal enhancers for CEA detection through SPRi sandwich assay. The present method exhibits a dynamic range for CEA determination from 0.1 to 312.5 ng mL-1 and a detection limit of 0.55 ng mL-1, well below normal physiological levels. This biosensing approach demonstrates the advantages of wavy gold-silver alloy nanoplates compared to conventional gold nanoparticles as a signal amplifier to enhance the SPRi signal, which is expected to become a new prospect for detection of cancer markers in biomedical research and clinical diagnosis.

Rapid and Label-Free Immunosensing of Shiga Toxin Subtypes with Surface Plasmon Resonance Imaging.

Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world as well as in the United States. Current detection methods have limitation to implement for rapid field-deployable detection with high volume of samples that are needed for regulatory purposes. Surface plasmon resonance imaging (SPRi) has proved to achieve rapid and label-free screening of multiple pathogens simultaneously, so it was evaluated in this work for the detection of Shiga toxins (Stx1a and Stx2a toxoids were used as the less toxic alternatives to Stx1 and Stx2, respectively). Multiple antibodies (Stx1pAb, Stx1-1mAb, Stx1-2mAb, Stx1d-3mAb, Stx1e-4mAb, Stx2pAb, Stx2-1mAb, Stx2-2mAb, and Stx2-10mAb) were spotted one by one by programed microarrayer, on the same high-throughput biochip with 50-nm gold film through multiple crosslinking and blocking steps to improve the orientation of antibodies on the biochip surface. Shiga toxins were detected based on the SPRi signal difference (ΔR) between immobilized testing antibodies and immunoglobulin G (IgG) control. Among the antibodies tested, Stx1pAb showed the highest sensitivity for Stx1 toxoid, with the limit of detection (LOD) of 50 ng/mL and detection time of 20 min. Both Stx2-1mAb and Stx2-2mAb exhibited high sensitivity for Stx2 toxoid. Furthermore, gold nanoparticles (GNPs) were used to amplify the SPRi signals of monoclonal antibodies in a sandwich platform. The LOD reached the level of picogram (pg)/mL with the help of GNP-antibody conjugate. This result proved that SPRi biochip with selected antibodies has the potential for rapid, high-throughput and multiplex detection of Shiga toxins.

A simple and universal enzyme-free approach for the detection of multiple microRNAs using a single nanostructured enhancer of surface plasmon resonance imaging.

Here we describe a simple approach for the simultaneous detection of multiple microRNAs (miRNAs) using a single nanostructured reagent as surface plasmon resonance imaging (SPRi) enhancer and without using enzymatic reactions, sequence specific enhancers or multiple enhancing steps as normally reported in similar studies. The strategy involves the preparation and optimisation of neutravidin-coated gold nanospheres (nGNSs) functionalised with a previously biotinylated antibody (Ab) against DNA/RNA hybrids. The Ab guarantees the recognition of any miRNA sequence adsorbed on a surface properly functionalised with different DNA probes; at the same time, gold nanoparticles permit to detect this interaction, thus producing enough SPRi signal even at a low ligand concentration. After a careful optimisation of the nanoenhancer and after its characterisation, the final assay allowed the simultaneous detection of four miRNAs with a limit of detection (LOD) of up to 0.5pM (equal to 275attomoles in 500μL) by performing a single enhancing injection. The proposed strategy shows good signal specificity and permits to discriminate wild-type, single- and triple-mutated sequences much better than non-enhanced SPRi. Finally, the method works properlyin complex samples (total RNA extracted from blood) as demonstrated by the detection of four miRNAs potentially related to multiple sclerosis used as case study. This proof-of-concept study confirms that the approach provides the possibility to detect a theoretically unlimited number of miRNAs using a simple protocol and an easily prepared enhancing reagent, and may further facilitate the development of affordable multiplexing miRNA screening for clinical purposes.

Stability of Polydopamine Coatings on Gold Substrates Inspected by Surface Plasmon Resonance Imaging.

Polydopamine (PDA)-based surface modification has been used in a variety of fields. However, a vague impression on the stability of PDA still exists due to a lack of systematic studies. To ascertain the issue and make better use of this surface modification method, a technique of surface plasmon resonance imaging (SPRi) was exploited to study the stability of PDA coated on gold surface. The results showed that PDA-coating stability was largely dependent on the pH of aqueous solutions, giving detachment ratios up to 66% and 80% at pH 1.0 and pH 14.0, respectively. However, increasing the ionic strength of aqueous solutions could reduce the detachment of PDA in strong acid and strong alkali conditions. Besides, organic solvents also made a difference on the PDA-coating stability. Among the tested 10 kinds of organic solvents, including n-hexane, toluene, ethyl ether, tetrahydrofuran, ethyl acetate, isopropanol, acetone, acetonitrile, dimethylformamide (DMF), and dimethyl sulfoxide (DMSO), DMSO caused the most serious detachment of PDA, up to 56%, followed by DMF with a detachment ratio of 31%. Ultrasonication caused less than 10% detachment of the coated PDA. It should be mentioned that the PDA coatings deposited on gold surface were not detached completely in all the test conditions, even at pH 14.0 (ca. 20% PDA retained). In alkaline conditions, detachment competes with further polymerization, which gave a slight increase of the SPRi signals at pH 9.0-11.0. Based on the obtained information about PDA-coating stability, thickness-controllable and alkali-resistant PDA coatings were prepared. Moreover, the alkali-resistant PDA coatings remained reactive to biomolecules, supporting further functionalization of PDA coatings.

Silver decahedral nanoparticles empowered SPR imaging-SELEX for high throughput screening of aptamers with real-time assessment

A highly efficient method for aptamer screening with real-time monitoring of the SELEX process was described by silver decahedra nanoparticles (Ag10-NPs) enhanced surface plasmon resonance imaging (SPRI). A microarray chip was developed by immobilization of target protein (Lactoferrin (Lac)) and control proteins (α-lactalbumin (α), β-lactoglobulin (β), casein, and bovine serum albumin (BSA)) on the biochip surface. Ag10-NPs were conjugated with an ssDNA library (lib) (Ag10-NPs-library) that consisted of a central 40 nt randomized sequence and a 20 nt fixed primer sequence. Introduction of the Ag10-NPs-library to the SPRI flow channels drastically increased the sensitivity of SPRI signal for real-time monitoring of SELEX. The work allows rapid screening of potential targets, and yields nine aptamers with high affinity (nanomolar range) for Lac after only six-rounds of selection. The aptamer Lac 13-26 was then further tested by SPRI, and the results demonstrated that the aptamer had the capacity to be ultra-sensitive for specific detection of Lac. The novel SPRI-SELEX method demonstrated here showed many advantages of real-time evaluation, high throughput, and high efficiency.

Surface Plasmon Resonance is an Analytically Sensitive Method for Antigen Profiling of Extracellular Vesicles.

Identification, enumeration, and characterization of extracellular vesicles (EVs) are hampered by the small size of EVs, a low refractive index, and low numbers of antigens on their surface. We investigated the potential of a 48-multiplex surface plasmon resonance imaging (SPRi) system to perform EV phenotyping. Antigen surface density of 11 antigens was measured on the human breast cancer cell lines HS578T, MCF7, and SKBR3 and their EVs by use of both SPRi and the widely used flow cytometry (FCM). For cells, the SPRi and FCM signals for antigen exposure correlated (RHS578T cells2 = 0.66, RMCF7 cells2 = 0.78, RSKBR3 cells2 = 0.60). With regard to EVs, SPRi detected 31 out of 33 tested antibody-EV pairs, whereas our flow cytometer detected 5 antibody-EV pairs because of high blank and isotype control signals. For HS578T-derived EVs, the SPRi and FCM signals correlated (R2HS578T EVs = 0.98). However, on MCF7- and SKBR3-derived EVs, insufficient antigens were detected by our flow cytometer. To confirm that the SPRi responses correlated with mean antigen density on EVs, the SPRi responses of EVs were correlated with antigen density on parental cells as measured by FCM (RHS578T2 = 0.77, RMCF72 = 0.49, RSKBR32 = 0.52). SPRi responses correlate with mean antigen density. Moreover, SPRi detects lower antigen-exposure levels than FCM because SPRi measures an ensemble of EVs binding to the sensor surface, whereas FCM detects antigens of single EV.

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml−1 and minimum levels of 0.03 ng ml−1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

Quantification of antibody production of individual hybridoma cells by surface plasmon resonance imaging

Surface plasmon resonance imaging (SPRi) is most frequently used for the label-free measurement of biomolecular interactions. Here we explore the potential of SPRi to measure antibody production of individual hybridoma cells. As a model system, cells from a hybridoma, producing monoclonal antibodies recognizing epithelial cell adhesion molecule (EpCAM), were used. Recombinant human EpCAM protein was immobilized on an SPR sensor and hybridoma cells were introduced into an IBIS MX96 SPR imager and the SPRi response was followed for 10h. SPRi responses were detected on the spots of the sensor only where ligands of the produced antibody were present. By measuring the SPRi signals on individual cells the antibody production of the individual cells was measured and production rates were calculated. For 53 single EpCAM hybridoma cells the production ranged from 0.16 to 11.95pg (mean 2.96pg per cell, SD 2.51) over a period of 10h. Antibody excretion per cell per hour ranged from 0.02 to 1.19pg (mean 0.30, SD 0.25). Here we demonstrate for the first time that antibody production of individual cells can be measured and quantified by SPRi, opening a new avenue for measuring excretion products of individual cells.

Applications of High Resolution Surface Plasmon Resonance Imaging to Adherent Cells: Single Mammalian Cells to Bacterial Biofilms

High resolution surface plasmon resonance imaging (SPRI) allows label-free imaging of subcellular features when performed using a high numerical aperture objective lens with a digital light projector to precisely position incident angle excitation. The SPRI signal is a result of the mass of material within the evanescent field, and can be interpreted as a density or a refractive index of the cellular material. The quantitative nature of SPR images and the direct relationship to refractive index changes at the surface sensor allow for visualization new insights into mechanisms of cell biology at an interface. When applied to mammalian cells, such as rat aortic smooth muscle cells, cellular components near the sensor surface such as the cell membrane, focal adhesions, and cell nucleus are visualized in the SPRI image. Focal adhesion sizes measured by SPRI are similar with those highlighted with fluorescent antibody stained vinculin. In addition, a positive correlation between focal adhesion size and protein density is observed by SPR imaging. When SPRI is applied to pathogenic biofilms of Streptococcus mutans, distinct components of the bacterial biofilm at the surface including individual bacteria, bacterial microcolonies, and extracellular polymeric substance (EPS) are observed. SPRI shows that the refractive index of bacteria in a biofilm increases over time compared to that of bacteria not in a biofilm, which remains constant. SPRI also indicates that the EPS material generated in the biofilm at early time points is thicker near the bacterial microcolony periphery. This suggests that the EPS matrix is generated at the colony edge and that SPRI can be used to monitor the dynamics of EPS production in biofilms.

Sensitive detection of multiple mycotoxins by SPRi with gold nanoparticles as signal amplification tags

Detection of multiple toxic mycotoxins is of importance in food quality control. Surface plasmon resonance imaging (SPRi) is an advanced tool for simultaneously multiple detections with accuracy; however, it suffers from limited sensitivity due to the instrumental constraint and small sizes of mycotoxins with only one epitope for an insensitive competitive immunoassay. In this work a gold nanoparticle (AuNP)-enhanced SPRi chip is designed to sensitively detect multiple mycotoxins using a competitive immunoassay format. The sensing surface is constructed by uniformly attaching dense mycotoxin antigens on poly[oligo(ethylene glycol) methacrylate-co-glycidyl methacrylate] (POEGMA-co-GMA) brush modified SPRi gold chip. After competitive binding in a sample solution containing respective monoclonal antibodies, secondary antibody-conjugated AuNPs are employed to bind with the captured monoclonal antibodies for further amplification of the SPRi signal. Highly specific and sensitive simultaneous detection is achieved for three typical mycotoxins including Aflatoxin B1 (AFB1), Ochratoxin A (OTA) and Zearalenone (ZEN) with low detection limits of 8, 30 and 15pgmL−1 and dynamic ranges covering three orders of magnitude.

Polydopamine-Functionalization of Graphene Oxide to Enable Dual Signal Amplification for Sensitive Surface Plasmon Resonance Imaging Detection of Biomarker

Surface plasmon resonance imaging (SPRi) is one of the powerful tools for immunoassays with advantages of label-free, real-time, and high-throughput; however, it often suffers from limited sensitivity. Herein we report a dual signal amplification strategy utilizing polydopamine (PDA) functionalization of reduced graphene oxide (PDA-rGO) nanosheets for sensitive SPRi immunoassay in serum. The PDA-rGO nanosheet is synthesized by oxidative polymerization of dopamine in a gentle alkaline solution in the presence of graphene oxide (GO) sheets and then is antibody-conjugated via a spontaneous reaction between the protein and the PDA component. In the dual amplification mode, the first signal comes from capture of the antibody-conjugated PDA-rGO to form sandwiched immunocomplexes on the SPRi chip, followed by a PDA-induced spontaneous gold reductive deposition on PDA-rGO to further enhance the SPRi signal. The detection limit as low as 500 pg mL(-1) is achieved on a nonfouling SPRi chip with high specificity and a wide dynamic range for a model biomarker, carcinoembryonic antigen (CEA) in 10% human serum.

Dual signal amplification of surface plasmon resonance imaging for sensitive immunoassay of tumor marker

Surface plasmon resonance imaging (SPRi) is an intriguing technique for immunoassay with the inherent advantages of being high throughput, real time, and label free, but its sensitivity needs essential improvement for practical applications. Here, we report a dual signal amplification strategy using functional gold nanoparticles (AuNPs) followed by on-chip atom transfer radical polymerization (ATRP) for sensitive SPRi immunoassay of tumor biomarker in human serum. The AuNPs are grafted with an initiator of ATRP as well as a recognition antibody, where the antibody directs the specific binding of functional AuNPs onto the SPRi sensing surface to form immunocomplexes for first signal amplification and the initiator allows for on-chip ATRP of 2-hydroxyethyl methacrylate (HEMA) from the AuNPs to further enhance the SPRi signal. High sensitivity and broad dynamic range are achieved with this dual signal amplification strategy for detection of a model tumor marker, α-fetoprotein (AFP), in 10% human serum.

High Resolution Surface Plasmon Resonance Imaging of Focal Adhesions in Single Cells

Surface plasmon resonance imaging (SPRI) is a powerful label-free technique that has been only recently applied to the field of cell biology. High resolution label-free imaging of subcellular features can be performed using a high numerical aperture objective lens with a digital light projector to precisely position incident angle excitation. The SPRI signal is a result of the mass of material within the evanescent field, the refractive index of the material, and the distance between the material and the substrate. Cellular components near the sensor surface such as the cell membrane, focal adhesions, cell nucleus, and extracellular material deposited at cell periphery are visualized in the SPRI image. The optical contrast in the SPR images is sufficient to define the edge of the cell with relatively high signal-to-noise corresponding to the cell membrane. Within this cell membrane region, SPR images show punctate regions of high reflectivity that are putatively the cellular focal adhesions. Focal adhesions are known to be regions of high protein density that reside at the cell-substratum interface. Comparing SPR analyzed images with fluorescent antibody stain for Vinculin in rat aortic smooth muscle cells reveals a similar distribution of focal adhesion size and normalized intensity. Subsequently, comparing SPR images for several cell types reveals a distinct difference in focal adhesion intensity levels corresponding to a difference in protein density. In general, a positive correlation between focal adhesion size and intensity level is revealed by SPR imaging. Also, visualized around the cell edge is extracellular deposited material that corresponds to an approximate monolayer coating that extends beyond the cell, up to 40 μm around the cell periphery, indicating the ability to visualize cell secreted modification of the substrate for specific cell types.

Improving surface plasmon resonance imaging of DNA by creating new gold and silver based surface nanostructures

The use of nanoparticles (NPs) can substantially improve the analytical performance of surface plasmon resonance imaging (SPRi) in general, and in DNA sensing in particular. In this work, we report on the modification of the gold surface of commercial biochips with gold nanospheres, silica-coated gold nanoshells, and silver nanoprisms, respectively. The NPs were tethered onto the surface of the chip and functionalized with a DNA probe. The effects of tethering conditions and varying nanostructures on the SPRi signals were evaluated via hybridization assays. The results showed that coupling between planar surface plasmons and electric fields, generated by localized surface plasmons of the NPs, is mandatory for signal enhancement. Silver nanoprisms gave the best results in improving the signal change at a target DNA concentration of <50 nM by +50 % (compared to a conventional SPRi chip). The limit of detection for the target DNA was 0.5 nM which is 5 times less than in conventional SPRi.

Surface Plasmon Resonance Imaging for Cell Biology: Direct Measurement of the Evanescent Wave Penetration Depth

Surface plasmon resonance imaging (SPRI) is a powerful label-free technique that has been known to provide sensitive biochemical surface measurements, but has only recently been applied to the field of cell biology. High resolution label-free imaging of cell-substrate contacts can be performed using a high numerical aperture objective lens. The SPRI signal is a result of the mass of material within the evanescent field, the refractive index of the material, and the distance between the material and the substrate. Cell focal contacts and cellular organelles can contribute to the SPRI signal. Unambiguous interpretation of the SPRI data is therefore complicated by uncertainties regarding the penetration depth of the plasmon wave. To gain a better theoretical understanding of the signal present in the image we used micron scale beads of polymers with a refractive index similar to cells as reference materials to calibrate SPRI reflectivity as a function of distance-to-substrate. Multi-wavelength measurements of these beads show that it is possible to tailor the effective depth of penetration of the evanescent wave into the cellular environment. The application of multiple wavelengths will assist the interpretation of the SPRI image.

Design of a universal biointerface for sensitive, selective, and multiplex detection of biomarkers using surface plasmon resonance imaging

This paper reports on the sensitive, selective, and simultaneous detection of four protein biomarkers involved in metastasis of various cancers, namely Fas, angiopoietin-2 (Ang-2), human epidermal growth factor receptor 2 (HER2), and matrix metallopeptidase-9 (MMP-9) using an antibody-conjugated quantum dot (QD) chip and surface plasmon resonance imaging (SPRi) biosensors. Initially, a self-assembled monolayer film of l-cysteine, using glutaraldehyde as a linker and QDs as signal enhancement moieties, was employed to immobilize anti-Fas for the detection of Fas as a model protein in buffer. The biointerface was characterized using confocal microscopy, atomic force microscopy, and scanning electron microscopy to provide evidence of uniform surface coverage by the QDs. The SPRi signal was enhanced 100-fold to achieve a detection limit of 25 pg mL(-1) after applying biotinylated detection antibody-conjugate streptavidin-modified QDs. Secondly, this signal amplification strategy was applied to sequentially detect Fas, HER2, MMP-9, and Ang-2 at low concentrations on a protein-microprinted/gold-coated SPRi chip. The results showed the absence of cross-reactivity among these proteins and the feasibility of the approach for multiplex detection of biomarkers as required for the accurate diagnosis of various diseases.

Poly(acrylic acid) brushes pattern as a 3D functional biosensor surface for microchips

Poly(acrylic acid) (PAA) brushes, a novel three dimensional (3D) precursor layer of biosensor or protein microarrays, possess high protein loading level and low non-specific protein adsorption. In this article, we describe a simple and convenient way to fabricate 3D PAA brushes pattern by microcontact printing (μCP) and characterize it with FT-IR and optical microscopy. The carboxyl groups of PAA brushes can be applied to covalently immobilize protein for immunoassay. Thriving 3D space made by patterning PAA brushes thin film is available to enhance protein immobilization, which is confirmed by measuring model protein interaction between human immunoglobulin G (H-IgG) and goat anti-H-IgG (G-H-IgG) with fluorescence microscopy and surface plasmon resonance imaging (SPRi). As expected, the SPRi signals of H-IgG coating on 3D PAA brushes pattern and further measuring specific binding with G-H-IgG are all larger than that of 3D PAA brushes without pattern and 2D bare gold surface. We further revealed that this surface can be used for high-throughput screening and clinical diagnosis by label-free assaying of Hepatitis-B-Virus surface antibody (HBsAb) with Hepatitis-B-Virus surface antigen (HBsAg) concentration array chip. The linearity range for HBsAb assay is wider than that of conventional ELISA method.