Yu Niu

Detection of hepatitis B virus markers using a biosensor based on imaging ellipsometry

Summary.  A biosensor based on imaging ellipsometry (BIE) has been developed and validated in 169 patients for detecting five markers of hepatitis B virus (HBV) infection. The methodology has been established to pave the way for clinical diagnosis, including ligand screening, determination of the sensitivity, set‐up of cut‐off values (CoVs) and comparison with other clinical methods. A matrix assay method was established for ligand screening. The CoVs of HBV markers were derived with the help of receiver operating characteristic curves. Enzyme‐linked immunosorbent assay (ELISA) was the reference method. Ligands with high bioactivity were selected and sensitivities of 1 ng/mL and 1 IU/mL for hepatitis B surface antigen (HBsAg) and surface antibody (anti‐HBs) were obtained respectively. The CoVs of HBsAg, anti‐HBs, hepatitis B e antigen, hepatitis B e antibody and core antibody were as follows: 15%, 18%, 15%, 20% and 15%, respectively, which were the percentages over the values of corresponding ligand controls. BIE can simultaneously detect up to five markers within 1 h with results in acceptable agreement with ELISA, and thus shows a potential for diagnosing hepatitis B with high throughput.

Antibody oriented immobilization on gold using the reaction between carbon disulfide and amine groups and its application in immunosensing.

Carbon disulfide (CS(2)) can spontaneously react with amine groups to form dithiocarbamates on gold surface, providing the possibility to immobilize some compounds with primary or secondary amine groups in one step. Using this principle, an immunosensor interface prepared for immunoglobulin G (IgG) sensing surface toward anti-IgG has been fabricated for the first time by simply immersing gold slides into a mixed aqueous solution of CS(2) and protein A, followed by incubation in immunoglobulin G solution. The reaction between CS(2) and protein A has been followed by UV-vis spectroscopy, whereas cyclic voltammetry has been employed in the characterization of the modified gold surface with CS(2) and protein A, both methods indicating that protein A immobilization is implemented by CS(2). Conventional ellipsometry, atomic force microscopy (AFM), as well as surface plasmon resonance (SPR) have been used to evaluate the specific binding of protein A with IgG and IgG with anti-IgG, revealing that IgG is specifically captured to form the biosensing interface, maintaining its bioactivity. Compared to direct adsorption of IgG on the gold surface, the IgG sensing surface constructed of CS(2) and protein A is far more sensitive to capture anti-IgG as its target molecule. In addition, the modified surface is proven to have good capability to inhibit nonspecific adsorption, as supported by control experiments using lysozyme and BSA. To conclude, antibody immobilization using this one-step method has potential as a simple and convenient surface modification approach for immunosensor development.

Protein microarray biosensors based on imaging ellipsometry techniques and their applications

After years of development, biosensors based on imaging ellipsometry and biosensors based on total internal reflection imaging ellipsometry have been successfully implemented in various engineering systems. Their experimental setups, detection principles, and biological and clinical applications are briefly reviewed.

Analysis of the Interaction between Tropomyosin Allergens and Antibodies Using a Biosensor Based on Imaging Ellipsometry

A biosensor based on high spatial resolution imaging ellipsometry has been studied to examine its role in the rapid detection and analysis of the tropomyosin allergen existing in crustaceans. This methodology has been established for detection of the tropomyosin allergen and includes ligand screening, the determination of sensitivity, and a comparison with traditional detection methods. Three kinds of monoclonal antibodies (2F9, 4C7, and 2H6) known to have a high bioactivity against the tropomyosin allergen were screened and separately immobilized as ligands on a silicon wafer surface, thus allowing them to capture the tropomyosin allergen. Resulting changes on the wafer surface were visualized in gray scale variation on an ellipsometry image. Images showed that these antibodies are able to recognize the presence of the tropomyosin allergen in shrimp and crab with sensitivity of 1 mg L(-1), and at a detecting time of approximately 30 min for an extracted sample. This preliminary study has shown that the detection of the tropomyosin allergen is rapid and specific when using this type of assay on products containing shrimp and crab. When compared with the enzyme-linked immunosorbent assay, the biosensor based on imaging ellipsometry is able to perform a fast label-free analysis.

Detection of Cytomegalovirus Antibodies Using a Biosensor Based on Imaging Ellipsometry.

Background Cytomegalovirus (CMV) is the most common infectious cause of mental disability in newborns in developed countries. There is an urgent need to establish an early detection and high-throughput screening method for CMV infection using portable detection devices. Methods An antibody analysis method is reported for the detection and identification of CMV antibodies in serum using a biosensor based on high spatial resolution imaging ellipsometry (BIE). CMV antigen (CMV-3A) was immobilized on silicon wafers and used to capture CMV antibodies in serum. An antibody against human immunoglobulin G (anti-IgG) was used to confirm the IgG antibody against CMV captured by the CMV-3A. Results Our results show that this assay is rapid and specific for the identification of IgG antibody against CMV. Further, patient serum was quantitatively assessed using the standard curve method, and the quantitative results were in agreement with the enzyme-linked immunosorbent assay. The CMV antibody detection sensitivity of BIE reached 0.01 IU/mL. Conclusions This novel biosensor may be a valuable diagnostic tool for analysis of IgG antibody against CMV during CMV infection screening.

Joint detection of tumor markers with imaging ellipsometry biosensor

Abstract Tumor marker detection contributes to the early diagnosis of cancers. However, due to the lack of detection specificity, its results cannot act as a direct evidence to confirm cancer occurrence in clinic. Joint detection of tumor markers may improve the detection specificity. As a trial for clinical diagnosis of hepatocellular carcinoma, α-fetoprotein, α-l-fucosidase and ferritin have been combined and detected with a label-free, phase sensitive and high throughput imaging ellipsometry biosensor (IEB). Eighty-two sera have been quantitatively detected with IEB and the results are in agreement with the clinical standard approaches. Evaluated by receiver operating characteristic analysis, the specificity of joint detection improves remarkably with IEB for hepatocellular carcinoma. It can be foreseen that the joint detection of tumor markers with IEB has a potential for clinical cancer diagnosis.

Phospholipid/cholesterol/decanethiol mixtures for direct assembly of immunosensing interfaces

In this work, a simple yet robust method to prepare lipid-based biosensing interfaces on gold using common lipids (a phospholipid and cholesterol) and an alkanethiol is reported. The lipids were carefully chosen to tailor the biophysical properties of the bilayer. The simplicity of the method relies on the incorporation of a small percentage of decanethiol in the lipid vesicles for a direct formation of a thiol-linked supported lipid bilayer, which is advantageous in several respects. It prevents the use of specially synthesized thiolipids and preserves the natural fluidity and dynamics of the lipids. As a consequence the whole arrangement is extremely stable regarding ionic strength changes and solution flow during surface plasmon resonance experiments. Moreover, we show that this interface is very effective on suppressing the nonspecific adsorption of proteins on the surface, and enables the covalent attachment of the recognition antibody. The subsequent detection of specific interaction toward antigen was monitored in real-time by SPR and confirmed by ellipsometric measurements. This lipid-based biosensing platform is versatile and can be adapted to the biorecognition reaction of interest.

CD146 detection with real-time total internal reflection imaging ellipsometry

Biosensor with the total internal reflection imaging ellipsometry (TIRIE) uses an evanescent wave as optical probe to monitor bio-molecular interaction with a high sensitivity due to its property of phase sensitive. Here, the biosensor is applied for a quantitative detection of CD146 with concentrations of 0.1 to 100 ng/mL in order to realize a high sensitive quantitative detection. Moreover, the regression curve between the signal of biosensor (y) and CD146 concentration (x.. lnC+2.4) is deduced as a linear y=1.0544x+0.7839.

Kinetic analysis of the weak affinity interaction between tris and lysozyme.

The biosensor based on total internal reflection imaging ellipsometry (TIRIE), regarded as an automotive real-time research approach for biomolecular interaction, is introduced to analyze the kinetic process of the weak interaction between tris and lysozyme. The experiment is performed by delivering lysozyme solution diluted to different concentrations to the biosensor substrate interface immobilized with tris. By applying pseudo-first-order interaction kinetics model, we are able to obtain the kinetic parameters from fitting experimental data. The calculated association rate constant and dissociation rate constant of tris and lysozyme interaction are in 10(-2) mol(-1) s(-1) and 10(3)s(-1) magnitude, respectively. To further improve TIRIEs ability for kinetically characterizing biomolecular interaction, a theoretical method to deduce associate rate constant before experiment is proposed.

Approach to Quantitative Detection of CD146 with the Label-free Protein Biosensor Based on Imaging Ellipsometry

CD146 glycoprotein belonging to cell adhesion molecules is considered to be a novel target on endothelial cell involved in tumor angiogenesis. The biosensor based on imaging ellipsometry (BIE) which is performed in null and off-null mode is used for CD146 detection as a trial by the following steps. Firstly, anti-CD146 antibody as ligand is immobilized on Protein G modified silicon substrate. Then, CD146 test is carried out and its calibration curve is established for the requirement of quantitative detection. Finally, 18 serum samples are detected quantitatively and their results are validated by ELISAs. The sensitivity for CD146 detection achieves the order of ng/ml and the relationship between BIE signal y (grayscale value) and CD146 concentration x (ng/ml) is y=3.3ln(x) +91.3. Compared with ELISAs, the majority of results are in agreement, and the results of two approaches have significant statistic relevance.