Sensitive detection of proteins using assembled cascade fluorescent DNA nanotags based on rolling circle amplification

A novel cascade fluorescence signal amplification strategy based on the rolling circle amplification (RCA)-aided assembly of fluorescent DNA nanotags as fluorescent labels and multiplex binding of the biotin-streptavidin system was proposed for detection of protein target at ultralow concentration. In the strategy, fluorescent DNA nanotags are prepared relying on intercalating dye arrays assembled on nanostructured DNA templates by intercalation between base pairs. The RCA product containing tandem-repeat sequences could serve as an excellent template for periodic assembly of fluorescent DNA nanotags, which were presented per protein recognition event to numerous fluorescent DNA nanotags for assay readout. Both the RCA and the multiplex binding system showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. Using human IgG as a model protein, the designed strategy was successfully demonstrated for the ultrasensitive detection of protein target. The results revealed that the strategy exhibited a dynamic response to human IgG over a three-decade concentration range from 1.0 pM to 1.0 fM with a limit of detection as low as 0.9 fM. By comparison with the assay of multiple labeling antibodies with the dye/DNA conjugate, the limit of detection was improved by 4 orders. The designed signal amplification strategy would hold great promise as a powerful tool to be applied for the ultrasensitive detection of target protein in immunoassay.     

Fluorescence protection assay: a novel homogeneous assay platform toward development of aptamer sensors for protein detection

Development of novel aptamer sensor strategies for rapid and selective assays of protein biomarkers plays crucial roles in proteomics and clinical diagnostics. Herein, we have developed a novel aptamer sensor strategy for homogeneous detection of protein targets based on fluorescence protection assay. This strategy is based on our reasoning that interaction of aptamer with its protein target may dramatically increase steric hindrance, which protects the fluorophore, fluorescein isothiocyannate (FITC), labeled at the binding pocket from accessing and quenching by the FITC antibody. The aptamer sensor strategy is demonstrated using a model protein target of immunoglobulin E (IgE), a known biomarker associated with atopic allergic diseases. The results reveal that the aptamer sensor shows substantial (>6-fold) fluorescence enhancement in response to the protein target, thereby verifying the mechanism of fluorescence protection. Moreover, the aptamer sensor displays improved specificity to other co-existing proteins and a desirable dynamic range within the IgE concentration from 0.1 to 50 nM with a readily achieved detection limit of 0.1 nM. Because of great robustness, easy operation and scalability for parallel assays, the developed homogeneous fluorescence protection assay strategy might create a new methodology for developing aptamer sensors in sensitive, selective detection of proteins.     

Phage display screen for peptides that bind Bcl-2 protein

Bcl-2 family proteins are key regulators of apoptosis associated with human disease, including cancer. Bcl-2 protein has been found to be overexpressed in many cancer cells. Therefore, Bcl-2 protein is a potential diagnostic target for cancer detection. In the present study, the authors have identified several Bcl-2 binding peptides with high affinity (picomolar range) from a 5-round M13 phage display library screening. These peptides can be used to develop novel diagnostic probes or potent inhibitors with diverse polyvalencies.     

YB-1与肿瘤发生及治疗

Y-box结合蛋白(Y-box binding protein,YB)是一类广泛存在于从低等到高等多种生物中的蛋白质家族,在体内行使多种生物学功能.大量研究表明,YB-1作为该家族成员之一,与许多重要的生物大分子存在密切联系,并对细胞、组织和机体的生理机能产生重大影响.更为重要的是,YB-1在多种疾病,尤其是恶性肿瘤的发生和发展中也起到十分关键的作用,对癌细胞表型的维持及肿瘤多药耐药性(MDR)的产生具有全方位的影响.以YB-1为作用靶点的新型肿瘤治疗策略可望有效控制癌症患者病情恶化,改善耐药状况.现就对YB-1与肿瘤发生和发展之间关系的研究进展,以及针对YB-1治疗策略的制定作一评述和展望.     

Label-free impedimetric immunosensor for ultrasensitive detection of cancer marker Murine double minute 2 in brain tissue

The detection of cancer biomarkers is as important tool for the diagnosis and prognosis of cancer such as brain cancer. Murine double minute 2 (MDM2) has been widely studied as prognostic marker for brain tumor. Here we describe development of a new sensitive label free impedimetric immunosensor for the detection of MDM2 based on cysteamine self assembled monolayers on a clean polycrystalline Au electrode surface. The amine-modified electrodes were further functionalized with antibody using homobifunctional 1,4-phenylene diisothiocyanate (PDITC) linker. The assembly processes of the immunosensor had been monitored with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques using Fe(CN)(6)(3-/4-) solution as redox probe. The impedance changes upon binding of MDM2 protein to the sensor surface was utilized for the detection of MDM2. The increase in relative electron-transfer resistance (ΔR/R(0)%) values was linearly proportional to the concentration of tumor marker MDM2 in the wide dynamic range of 1pg/ml-1μg/ml. The limit of detection was 0.29pg/ml in phosphate buffer saline (PBS) and 1.3pg/ml in mouse brain tissue homogenate, respectively. The immunosensor showed a good performance in comparison with ELISA for the analysis of the MDM2 in the cancerous mouse brain tissue homogenates. Moreover, the immunosensor had a good selectivity against epidermal growth factor receptor (EGFR) protein, long-storage stability and reproducibility. It might be become a promising assay for clinical diagnosis and early detection of tumors.     

Phosphatidylinositol 3,4,5-trisphosphate activity probes for the labeling and proteomic characterization of protein binding partners

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P?], regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P? that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins and a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by in-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P? headgroup analogue as well as through protein denaturation, indicating specific labeling. In addition, probes featuring linkers of different lengths between the PI(3,4,5)P? headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts; labeled proteins were observed by in-gel detection and characterized using postlabeling with biotin, affinity chromatography, and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P?-binding proteins.     

Highly sensitive detection of E2 activity in ubiquitination using an artificial RING finger

The ubiquitin‐conjugating (E2) enzymes of protein ubiquitination are associated with various diseases such as leukemia, lung cancer, and breast cancer. Rapid and accurate detection of E2 enzymatic activities remains poor. Here, we described the detection of E2 activity on a signal accumulation ISFET biosensor (AMIS sensor) using an artificial RING finger (ARF). The use of ARF enables the simplified detection of E2 activity without a substrate. The high‐sensitivity quantitative detection of E2 activities was demonstrated via real‐time monitoring over a response range of femtomolar to micromolar concentrations. Furthermore, the monitoring of E2 activities was successfully achieved using human acute promyelocytic leukemia cells following treatment with the anticancer drug bortezomib, which allowed the assessment of the pathological conditions. This strategy is extremely simple and convenient, and the present detection could be widely applied to specific E2s for various types of cancers. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization

The X-linked inhibitor of apoptosis protein (XIAP) is a potent cellular inhibitor of apoptosis. Designing small-molecule inhibitors that target the BIR3 domain of XIAP, where Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI) and caspase-9 bind, is a promising strategy for inhibiting the antiapoptotic activity of XIAP and for overcoming apoptosis resistance of cancer cells mediated by XIAP. Herein, we report the development of a homogeneous high-throughput assay based on fluorescence polarization for measuring the binding affinities of small-molecule inhibitors to the BIR3 domain of XIAP. Among four fluorescent probes tested, a mutated N-terminal Smac peptide (AbuRPFK-(5-Fam)-NH(2)) showed the highest affinity (Kd =17.92 nM) and a large dynamic range (deltamP = 231 +/- 0.9), and was selected as the most suitable probe for the binding assay. The binding conditions (DMSO tolerance and stability) have been investigated. Under optimized conditions, a Z' factor of 0.88 was achieved in a 96-well format for high-throughput screening. It was found that the popular Cheng-Prusoff equation is invalid for the calculation of the competitive inhibition constants (Ki values) for inhibitors in the FP-based competitive binding assay conditions, and accordingly, a new mathematical equation was developed, validated, and used to compute the Ki values. An associated Web-based computer program was also developed for this task. Several known Smac peptides with high and low affinities have been evaluated under the assay conditions and the results obtained indicated that the FP-based competitive binding assay performs correctly as designed: it can quantitatively and accurately determine the binding affinities of Smac-based peptide inhibitors with a wide range of affinities, and is suitable for high-throughput screening of inhibitors binding to the XIAP BIR3 domain.     

Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium

An immunosensor based on surface plasmon resonance (SPR) using protein G was developed for the detection of Salmonella typhimurium. A protein G layer was fabricated by binding chemically to self-assembly monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) on gold (Au) surface. The formation of protein G layer on Au surface modified with 11-MUA and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The effect of detergent such as Tween-20 on binding efficiency of antibody and antigen was investigated by SPR. The binding efficiency of antigen to the antibody immobilized on Au surface was improved up to about 85% and 100% by using protein G and Tween-20, respectively. The surface morphology analyses of 11-MUA monolayer on Au substrate, protein G layer on 11-MUA monolayer and antibody layer immobilized on protein G layer were performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. typhimurium using protein G was developed with a detection range of 10(2) to 10(9)CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. typhimurium could be applied to construct other immnosensors or protein chips.     

Copper sensing based on the far-red fluorescent protein, HcRed, from Heteractis crispa

In this article, we report for the first time on the copper (Cu(2+)) binding characteristics of the far-red fluorescent protein, HcRed, and its application in the development of a reagentless sensing system for copper. The far-red emission of HcRed (lambda(max) = 645 nm) where background cellular fluorescence is low should prove to be advantageous in the development of the sensing system. In the studies performed in our laboratory, we found that the fluorescence of HcRed is quenched in the presence of copper ions (Cu(2+)). The results obtained through UV-visible and circular dichroism spectra generated in the presence and absence of copper, as well as Stern-Volmer plots at different temperatures, indicate static quenching of HcRed fluorescence in the presence of copper, possibly through the formation of a copper-protein complex. On the basis of this observation, we developed a reagentless sensing system for the detection of copper(II) based on HcRed as the biosensing element. A detection limit for Cu(2+) in the nanomolar range was obtained. HcRed was found to bind copper ions selectively when compared with other divalent ions. A dissociation constant of 3.6muM was observed for copper binding. Histidine and cysteine residues are commonly involved in copper binding within proteins; therefore, to investigate the role of these amino acids present in HcRed, we chemically modified Cys and His residues using iodoacetamide and diethyl pyrocarbonate, respectively. The effect of copper addition on the fluorescence of the chemically modified HcRed was investigated. The His modification of HcRed substantially affected copper ion binding, pointing to histidine as the possible amino acid residue involved in the binding of copper ions in HcRed. A purification strategy for HcRed was also developed based on a copper immobilized affinity column without the addition of any affinity tag on the protein. The HcRed-based copper sensing system can potentially be employed to perform intracellular copper detection by genetically encoding the biosensing element or can be employed in environmental sensing.     

Three‐dimensional ring‐oven washing technique for a paper‐based immunodevice

Washing is a standard step for enzyme‐linked immunosorbent assays (ELISA) performed on a paper‐based chip, in which nonspecific‐binding antibodies and antigens should be removed completely from the paper surface. In this study, a novel three‐dimensional (3D) washing strategy using a heating ring‐oven was carried out on a paper‐based chip. Compared with a plane washing mode by a ring‐oven, this 3D washing strategy obtained a lower background, as gravity played an important role in the washing step. The paper‐based chip was placed on a 3D plastic holder and the waste area was connected to a heating ring. Use of a heating waste area meant that the nonspecific‐binding protein was continuously carried to the waste area through gravity and capillary action. The angle between the plastic holder and the ring plane was carefully selected. The effect of washing on different parts of the detection area was investigated by upconversion fluorescence and chemiluminescence (CL). This novel 3D washing strategy was performed for carcinoembryonic antigen detection through CL and a lower detection limit of 2 pg ml^?1 was obtained. This approach provides an effective washing strategy to remove nonspecific‐binding antibody from a paper‐based immunodevice.     

Protein A-based antibody immobilization onto polymeric microdevices for enhanced sensitivity of enzyme-linked immunosorbent assay

Highly efficient antibody immobilization is extremely crucial for the development of high-performance polymeric microdevices for enzyme-linked immunosorbent assay (ELISA). In this article, a site-selective tyrosinase (TR)-catalyzed protein A strategy for antibody immobilization was developed to enhance the sensitivity of ELISA in poly-(methyl methacrylate) (PMMA) microchannels for interferon-gamma (IFN-gamma) assay. To effectively immobilize the target antibodies, oxygen plasma was first used to activate the inert PMMA. This is followed by poly(ethyleneimine) (PEI) coating, an amine-containing functional polymer. For comparison, protein A was also immobilized through the commonly used amine-glutaraldehyde (GA) chemistry. Oxygen plasma treatment effectively increased the amount of PEI attachment and subsequent binding efficiency of the primary antibody. The antibody immobilized via TR-catalyzed protein A was able to provide much better specific antigen capture efficiency than GA chemistry due to the optimal spacing and orientation. Consequently, by using this new method, the detection signal and the signal-to-noise ratio of the ELISA immunoassay in microdevices were all significantly improved. In comparison to the standard assay carried out in the 96-well microtiter plate, the treated microchannels exhibited a broader detection range and a shorter detection time. And the detection limit was also decreased to 20 pg/mL, much lower than that obtained in other microdevices.     

Identification,Characterization and Application of a G-Quadruplex Structured DNA Aptamer against Cancer Biomarker Protein Anterior Gradient Homolog 2

Background

Anterior gradient homolog 2 (AGR2) is a functional protein with critical roles in a diverse range of biological systems, including vertebrate tissue development, inflammatory tissue injury responses, and cancer progression. Clinical studies have shown that the AGR2 protein is overexpressed in a wide range of human cancers, including carcinomas of the esophagus, pancreas, breast, prostate, and lung, making the protein as a potential cancer biomarker. However, the general biochemical functions of AGR2 in human cells remain undefined, and the signaling mechanisms that drive AGR2 to inhibit p53 are still not clearly illustrated. Therefore, it is of great interest to develop molecular probes specifically recognizing AGR2 for its detection and for the elucidation of AGR2-associated molecular mechanism.

Methodology/Principal Findings

Through a bead-based and flow cytometry monitored SELEX technology, we have identified a group of DNA aptamers that can specifically bind to AGR2 with K_d values in the nanomolar range after 14 rounds of selections. Aptamer C14B was chosen to further study, due to its high binding affinity and specificity. The optimized and shortened C14B1 has special G-rich characteristics, and the G-rich region of this binding motif was further characterized to reveal an intramolecular parallel G-quadruplex by CD spectroscopy and UV spectroscopy. Our experiments confirmed that the stability of the G-quadruplex structure was strongly dependent on the nature of the monovalent ions and the formation of G-quadruplex structure was also important for the binding capacity of C14B1 to the target. Furthermore, we have designed a kind of allosteric molecule beacon (aMB) probe for selective and sensitive detection of AGR2.

Conclusion/Significance

In this work, we have developed new aptamer probes for specific recognition of the AGR2. Structural study have identified that the binding motif of aptamer is an intramolecular parallel G-quadruplex structure and its structure and binding affinity are strongly dependent on the nature of the monovalent ion. Furthermore, with our design of AGR2-aMB, AGR2 could be sensitively and selectively detected. This aptamer probe has great potential to serve as a useful tool for early diagnosis and prognosis of cancer and for fundamental research to elucidate the biochemical functions of AGR2.     

Probing high affinity sequences of DNA aptamer against VEGF165

Vascular endothelial growth factor (VEGF(165)) is a potent angiogenic mitogen commonly overexpressed in cancerous cells. It contains two main binding domains, the receptor-binding domain (RBD) and the heparin-binding domain (HBD). This study attempted to identify the specific sequences of the VEa5 DNA aptamer that exhibit high binding affinity towards the VEGF(165) protein by truncating the original VEa5 aptamer into different segments. Using surface plasmon resonance (SPR) spectroscopy for binding affinity analysis, one of the truncated aptamers showed a >200-fold increase in the binding affinity for HBD. This truncated aptamer also exhibited high specificity to HBD with negligible binding affinity for VEGF(121), an isoform of VEGF lacking HBD. Exposing colorectal cancer cells to the truncated aptamer sequence further confirmed the binding affinity and specificity of the aptamer to the target VEGF(165) protein. Hence, our approach of aptamer truncation can potentially be useful in identifying high affinity aptamer sequences for the biological molecules and targeting them as antagonist for cancer cell detection.     

Concise machinery for monitoring ubiquitination activities using novel artificial RING fingers

Protein ubiquitination is involved in many cellular processes, such as protein degradation, DNA repair, and signal transduction pathways. Ubiquitin‐conjugating (E2) enzymes of the ubiquitination pathway are associated with various cancers, such as leukemia, lung cancer, and gastric cancer. However, to date, detection of E2 activities is not practicable for capturing the pathological conditions of cancers due to complications related to the enzymatic cascade reaction. To overcome this hurdle, we have recently investigated a novel strategy for measuring E2 activities. Artificial RING fingers (ARFs) were developed to conveniently detect E2 activities during the ubiquitination reaction. ARFs were created by grafting the active sites of ubiquitin‐ligating (E3) enzymes onto amino acid sequences with 38 residues. The grafting design downsized E3s to small molecules (ARFs). Such an ARF is a multifunctional molecule that possesses specific E2‐binding capabilities and ubiquitinates itself without a substrate. In this review, we discuss the major findings from recent investigations on a new molecular design for ARFs and their simplified detection system for E2 activities. The use of the ARF allowed us to monitor E2 activities using acute promyelocytic leukemia (APL)‐derived cells following treatment with the anticancer drug bortezomib. The molecular design of ARFs is extremely simple and convenient, and thus, may be a powerful tool for protein engineering. The ARF methodology may reveal a new screening method of E2s that will contribute to diagnostic techniques for cancers.     

Surface plasmon resonance immunosensor using self-assembled protein G for the detection of Salmonella paratyphi

A surface plasmon resonance (SPR) based immunosensor using self-assembled protein G was developed for the detection of Salmonella paratyphi. In order to endow a solid substrate binding affinity to protein G, the free amine (-NH2) of protein G was substituted into thiol (-SH) using 2-iminothiolane. Thus, self-assembled protein G was fabricated on gold (Au) substrate. The formation of protein G layer on Au surface, and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analysis of the protein G layer on Au surface was performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. paratyphi using self-assembled protein G was developed with a detection range of 10(2)-10(7) CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. paratyphi could be applied to construct other immnosensors or protein chips.     

A two-step antibody strategy for surface plasmon resonance spectroscopy detection of protein-DNA interactions in nuclear extracts

Surface plasmon resonance (SPR) spectroscopy has emerged as a powerful alternative to conventional biochemistry methods for studying protein-DNA interactions that involve recombinant proteins of known identity. There are, however, limited demonstrations of SPR detection of protein-DNA bindings in crude samples, e.g., cell extracts, where the challenge is to detect and identify specific DNA binding protein(s) among other protein components in a physiological setting. We have developed a two-step antibody approach for an SPR study of estrogen receptor α (ERα)-DNA interactions, in which nuclear extracts prepared from MCF-7 breast cancer cells were used as the source of ERα protein. Following the binding of nuclear extracts to surface-immobilized estrogen response elements, rabbit anti-ERα antibody followed by a secondary antibody (goat anti-rabbit IgG) were applied to recognize the bound ERα and amplify the signals, respectively. Through a series of experiments, we have demonstrated that the magnitude of the binding signals from the secondary antibody reflects the affinity by which ERα binds to different DNA sequences. The detection sensitivity is determined by the amount of nuclear extracts and the concentration of primary antibody used. The sequence specificity of the nuclear ERα measured using the two-step antibody approach is in agreement with that measured for recombinant ERα protein (using receptor binding signals).     

Label-free colorimetric and quantitative detection of cancer marker protein using noncrosslinking aggregation of Au/Ag nanoparticles induced by target-specific peptide probe

Assays for non-enzyme protein based on peptide-protein interaction are few due to the fact that most of peptide-protein bindings do not produce easily measurable output signals. Here we report a homogenous assay for colorimetric and quantitative detection of a cancer marker and promising antitumor target, cyclin A(2), using noncrosslinking aggregation of unmodified AuNPs/AgNPs by utilizing the difference of coagulating ability of a cationic peptide probe (P1) and its binding form toward naked AuNPs/AgNPs. In the absence of cyclin A(2), P1 coagulates particles immediately, whereas cyclin A(2) binding prevents the interaction of P1 with metal particles surface, significantly reducing the magnitude of aggregation. The extent of aggregation is dependent on the concentration of the target protein cyclin A(2) and the difference in color can readily be distinguished by spectrometer and naked eyes. The assay is sensitive and selective. Cyclin A(2) assay using AuNPs as colorimetric indicator is more easily monitored by naked eyes owing to the distinct color change, and 40 nM cyclin A(2) can be detected without the aid of any instruments. Using inexpensive desktop spectrometer, cyclin A(2) assay using AgNPs as colorimetric indicator can detect as low as 30 nM cyclin A(2), which is 20 fold lower than that of cyclin A(2) assay using terbium-chelating peptide as the probe reported recently (Pazos et al., 2008, 130, 9652-9653). This strategy will shed light on developing of unlabeled peptide-based protein biosensors.     

In vitro selection of DNA aptamers binding pesticide fluoroacetamide

Fluoroacetamide (Mw = 77.06) is a lethal rodenticide to humans and animals which is still frequently abused in food storage somewhere in China. The production of antibodies for fluoroacetamide is difficult due to its high toxicity to animals, which limits the application of immunoassay method in poison detection. In this work, aptamers targeting N-fluoroacetyl glycine as an analog of fluoroacetamide were selected by a specific systematic evolution of ligands by exponential enrichment (SELEX) strategy. The binding ability of the selected aptamers to fluoroacetamide was identified using surface plasmon resonance (SPR)-based assay. The estimated K_D values in the low micromolar range showed a good affinity of these aptamers to the target. Our work verified that the SELEX strategy has the potential for developing aptamers targeted to small molecular toxicants and aptamers can be employed as new recognition elements instead of antibodies for poison detection.     

Amplification of antigen-antibody interactions based on biotin labeled protein-streptavidin network complex using impedance spectroscopy.

Antibody was covalently immobilized by amine coupling method to gold surfaces modified with a self-assembled monolayer of thioctic acid. The electrochemical measurements of cyclic voltammetry and impedance spectroscopy showed that the hexacyanoferrate redox reactions on the gold surface were blocked due to the procedures of self-assembly of thioctic acid and antibody immobilization. The binding of a specific antigen to antibody recognition layer could be detected by measurements of the impedance change. A new amplification strategy was introduced for improving the sensitivity of impedance measurements using biotin labeled protein-streptavidin network complex. This amplification strategy is based on the construction of a molecular complex between streptavidin and biotin labeled protein. This complex can be formed in a cross-linking network of molecules so that the amplification of response signal will be realized due to the big molecular size of complex. The results show that this amplification strategy causes dramatic improvement of the detection sensitivity of hIgG and has good correlation for detection of hIgG in the range of 2-10 microg/ml.