Capillary electrophoretic separation of nuclei released from single cells

We report here the first capillary electrophoresis analysis of intact nuclei released on-column from single cells. Expression of the nuclear-targeted protein nuDsRed2 and the plasma membrane-bound farnesylated enhanced green fluorescent protein in cultured human DeltaH2-1 cells allowed fluorescent monitoring of the fate of these subcellular compartments upon injection of a single cell into the separation capillary. On-column treatment with digitonin allowed for the separation of the plasma membrane from the nucleus as indicated by their selective laser-induced fluorescence detection in two separate spectral regions. The data suggest that less than 0.1% of the plasma membrane remains bound to individually detected nuclei. In digitonin-treated cells, the electropherograms consisted of a prominent fluorescent peak attributed to nuDsRed2 localized to the nucleus and a collection of weakly fluorescent events (barely distinguishable from scattering) that seem to indicate additional localization of this protein to other subcellular regions. Taken together, this report points to the feasibility of studying intact organelles released from a single mammalian cell by capillary electrophoresis, which is a prerequisite to understanding the relevance of subcellular heterogeneity in biological systems.     

Pluronic F127 nanomicelles engineered with nuclear localized functionality for targeted drug delivery

PKKKRKV (Pro-Lys-Lys-Lys-Arg-Lys-Val, PV7), a seven amino acid peptide, has emerged as one of the primary nuclear localization signals that can be targeted into cell nucleus via the nuclear import machinery. Taking advantage of chemical diversity and biological activities of this short peptide sequence, in this study, Pluronic F127 nanomicelles engineered with nuclear localized functionality were successfully developed for intracellular drug delivery. These nanomicelles with the size ~ 100 nm were self-assembled from F127 polymer that was flanked with two PV7 sequences at its both terminal ends. Hydrophobic anticancer drug doxorubicin (DOX) with inherent fluorescence was chosen as the model drug, which was found to be efficiently encapsulated into nanomicelles with the encapsulation efficiency at 72.68%. In comparison with the non-functionalized namomicelles, the microscopic observation reveals that PV7 functionalized nanomicelles display a higher cellular uptake, especially into the nucleus of HepG2 cells, due to the nuclear localization signal effects. Both cytotoxicity and apoptosis studies show that the DOX-loaded nanomicelles were more potent than drug nanomicelles without nuclear targeting functionality. It was thus concluded that PV7 functionalized nanomicelles could be a potentially alternative vehicle for nuclear targeting drug delivery.     

Permeability of the nuclear envelope at isolated Xenopus oocyte nuclei studied by scanning electrochemical microscopy

In interphase eukaryotic cells, molecular transport between the cytoplasm and the nucleus is mediated by the nuclear pore complex (NPC), which perforates the double-membraned nuclear envelope (NE). Local permeability of the NE at large intact nuclei (approximately 400 microm in diameter) isolated from Xenopus laevis oocytes was studied by scanning electrochemical microscopy (SECM). Steady-state tip current versus tip-nucleus distance curves (approach curves) were measured with 10- and 2-microm-diameter Pt disk microelectrodes at the nuclei in isotonic buffer solutions containing redox-active molecules. The approach curves in the normalized form are independent of the tip diameter, indicating diffusion-limited membrane transport of the redox molecules. SECM chronoamperometry demonstrated that a decrease in the steady-state tip current at short tip-nucleus distances is due to smaller diffusion coefficients and concentrations of the redox molecules in the nucleus than those in the buffer solution. The experimental approach curves fit very well with theoretical ones for freely permeable membranes, yielding the NE permeability to the molecules that is at least 2 orders of magnitude larger than permeability of bilayer lipid membranes and cell membranes. This result indicates that passive transport of the redox molecules across the NE is facilitated by open NPC pores. The flux of the redox molecules sustainable by a single NPC channel (>9.8 x 10(6) molecules per NPC per second) and the diameter of the channel pore (>15 nm) were estimated from the SECM data by assuming the NE as an array of nanometer-sized NPC pores. The effects of the redox molecules on the nucleus and the NPC function were examined by studying signal-mediated nuclear import of rhodamine-labeled bovine serum albumin with and without nuclear localization signals by fluorescence microscopy.     

Visualization of nonengineered single mRNAs in living cells using genetically encoded fluorescent probes

Single mRNA imaging in live cells is a useful technique to elucidate its precise localization and dynamics. We developed a method for visualizing endogenous mRNAs in living cells with single molecule sensitivity using genetically encoded probes. An RNA-binding protein of human PUMILIO1 (PUM-HD) was used for recognizing base sequences of a target mRNA, β-actin mRNA. Two PUM-HDs were modified by amino acid mutations to bind specifically to tandem 8-base sequences of the target mRNA. Because each PUM-HD was connected with amino- and carboxyl-terminal fragments of enhanced green fluorescent protein (EGFP), the probes emit fluorescence by reconstitution of EGFP fragments upon binding to β-actin mRNAs. The EGFP reconstituted on the mRNAs was monitored with a total internal reflection fluorescence microscope. Results show that each fluorescent spot in live cells represented a single β-actin mRNA and that distinct spatial and temporal movement of the individual β-actin mRNAs was visualized. We also estimated the average velocity of the movement of the single mRNAs along microtubules in live cells. This method is widely applicable to tracking various mRNAs of interest in the native state of living cells with single-mRNA sensitivity.     

Localization and Dynamics of Glucocorticoid Receptor at the Plasma Membrane of Activated Mast Cells

In addition to their actions in the cell nucleus, glucocorticoids exhibit rapid non‐nuclear responses that are mechanistically not well understood. To explain these effects, the localization of a glucocorticoid receptor (GR) expressed in mast cells as a GFP fusion was analyzed after activation of the cells on allergenic lipid arrays. These arrays were produced on glass slides by dip‐pen nanolithography (DPN) and total internal reflection (TIRF) microscopy was used to visualize the GR. A rapid glucocorticoid‐independent and ‐dependent recruitment of the GR‐GFP to the plasma cell membrane was observed following contact of the cells with the allergenic array. In addition, the mobility of the GR at the membrane was monitored by fluorescence recovery after photobleaching (FRAP) and shown to follow binding kinetics demonstrating interactions of the receptor with membrane‐bound factors. Furthermore the recruitment of the GR to the cell membrane was shown to result in a glucocorticoid‐mediated increase in Erk phosphorylation. This is evidenced by findings that destruction of the membrane composition of the mast cells by cholesterol depletion impairs the membrane localization of the GR and subsequent glucocorticoid‐mediated enhancement of Erk phosphorylation. These results demonstrate a membrane localization and function of the GR in mast cell signaling.     

Measuring the doxorubicin content of single nuclei by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

Individual nuclei isolated from the human leukemia CCRF-CEM and CEM-C2 cells treated with doxorubicin (DOX) were in-column lysed with a sodium dodecyl sulfate (SDS) containing buffer, their contents were then separated by micellar electrokinetic capillary chromatography using the same lysing buffer, and the DOX content was detected by laser-induced fluorescence. Use of a microscope for the selection of one nucleus from the nuclear preparation decreases the possibility of introduction of other subcellular components that are commonly found as impurities in subcellular fractions. The presence of SDS in the running buffer made negligible the DNA's quenching effect on DOX fluorescence, which often compromises quantification of DOX by direct imaging, making it possible to carry out the first direct measurement of the doxorubicin content of isolated nuclei. On average, nuclei from CCRF-CEM and CEM/C2 cell lines contained 85 +/-64 (n = 6) and 91 +/- 51 (n =7) amol of DOX, respectively. These values correspond to 74 and 65% of the average total cellular content as determined by single-cell analysis of the corresponding cell types. It is envisioned that this approach could become an important bioanalytical tool to investigate the effect of treatments with fluorescent drugs targeting the nucleus.     

Intuitive, image-based cell sorting using optofluidic cell sorting

We present a microfluidic cell-sorting device which augments microscopy with the capability to perform facile image-based cell sorting. This combination enables intuitive, complex phenotype sorting based on spatio-temporal fluorescence or cell morphology. The microfluidic device contains a microwell array that can be passively loaded with mammalian cells via sedimentation and can be subsequently inspected with microscopy. After inspection, we use the scattering force from a focused infrared laser to levitate cells of interest from their wells into a flow field for collection. First, we demonstrate image-based sorting predicated on whole-cell fluorescence, which could enable sorting based on temporal whole-cell fluorescence behavior. Second, we demonstrate image-based sorting predicated on fluorescence localization (nuclear vs whole-cell fluorescence), highlighting the capability of our approach to sort based on imaged subcellular events, such as localized protein expression or translocation events. We achieve postsort purities up to 89% and up to 155-fold enrichment of target cells. Optical manipulation literature and a direct cell viability assay suggest that cells remain viable after using our technique. The architecture is highly scalable and supports over 10 000 individually addressable trap sites. Our approach enables sorting of significant populations based on subcellular spatio-temporal information, which is difficult or impossible with existing widespread sorting technologies.     

Locating inositol 1,4,5-trisphosphate in the nucleus and neuronal dendrites with genetically encoded fluorescent indicators

Inositol 1,4,5-trisphosphate (InsP3) is a key second messenger in many cell types and also in distinct subcellular regions of single living cells; however, little is examined about the subcellular dynamics of InsP3 in a variety of cell types. We have developed fluorescent indicators to locate InsP3 dynamics in single living cells based on an intramolecular fluorescence resonance energy transfer. Our indicator has visualized InsP3 dynamics in the cytoplasm of cultured cells and even in single thin dendrites of hippocampal neurons, which has been unseen previously. We have further localized the present indicator in the nucleus and pinpointed nuclear InsP3 dynamics. The observation with our nuclear InsP3 indicator has solved a question on nuclear propagation of InsP3 from the cytoplasm and has drawn a conclusion that the nuclear InsP3 dynamics synchronously occurs with cytosolic InsP3 dynamics evoked by agonist stimulations. The present approach contributes to the understanding of when, where, and how InsP3 is generated and removed in a variety of living cells.     

Quantitation of DNA copy number in individual mitochondrial particles by capillary electrophoresis

Here, we present a direct method for determining mitochondrial DNA (mtDNA) copy numbers in individual mitochondrial particles, isolated from cultured cells, by means of capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. We demonstrate that this method can detect a single molecule of PicoGreen-stained mtDNA in intact DsRed2-labeled mitochondrial particles isolated from human osteosarcoma 143B cells. This ultimate limit of mtDNA detection made it possible to confirm that an individual mitochondrial nucleoid, the genetic unit of mitochondrial inheritance, can contain a single copy of mtDNA. The validation of this approach was achieved via monitoring chemically induced mtDNA depletion and comparing the CE-LIF results to those obtained by quantitative microscopy imaging and multiplex real-time PCR analysis. Owing to its sensitivity, the CE-LIF method may become a powerful tool for investigating the copy number and organization of mtDNA in mitochondrial disease and aging, and in molecular biology techniques requiring manipulation and quantitation of DNA molecules such as plasmids.     

Distribution of zeptomole-abundant doxorubicin metabolites in subcellular fractions by capillary electrophoresis with laser-induced fluorescence detection

Doxorubicin (DOX) treatment of NS-1 mouse hybridoma cells results in the formation of zeptomole amounts of metabolites per cell that are difficult to determine by confocal microscopy or HPLC. The native fluorescence of DOX and its metabolites together with laser-induced fluorescence detection (HF) has previously been used to detect a maximum of four components. In this study, we use capillary electrophoresis with postcolumn LIF (CE-LIF) to separate and detect 12 components attributed to DOX metabolism, resulting from treatment of NS-1 cells with 25 microM DOX for 8 h. The so-called metabolites 8 and 10 have been identified as doxorubicinone (DOXone) and 7-deoxydoxorubicinone (7-deoxyDOXone), respectively, by comigration with the corresponding synthetic standard. Due to comigration of DOX with doxorubicinol (DOXone), the presence of DOXone had to be determined separately by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The rest of the metabolites remain unidentified and are referred to by their number assignment. In comparison with the whole cell lysate, fractionation by differential centrifugation results in a better separation resolution of metabolites due to reduced amounts of metabolites in each fraction. This approach was chosen to compare the distribution of 13 metabolites in three subcellular fractions that form a pellet at < 1,400 g, 1,400-14,000 g, and > 14, 000 g and that generically are enriched in nuclei, organelles (mitochondria and lysosomes), and cytosolic components, respectively. The most abundant metabolite, DOXone, was estimated to be 90 +/- 15, 18 +/- 2, and 60 +/- 12 amol/cell (n = 5) in the nuclear-enriched, organelle-enriched, and cytosole-enriched fractions, respectively. In contrast, the total amount of other metabolites in a given fraction varied from 0 to 1,300 zmol. 7-DeoxyDOXone is the only metabolite that was present at similar levels in the three fractions. Other salient observations are metabolites 3, 7, and 11 are not detectable in the nuclear-enriched, organelle-enriched, and cytosole-enriched fractions, respectively; metabolite 9 and DOXone are more abundant in the nuclear-enriched fraction than in the other two fractions. The observations presented here suggest that subcellular fractionation followed by CE-LIF could be a powerful diagnostic for monitoring drug distribution, which is highly relevant to DOX cytoxicity studies.     

Use of a small reporter molecule to determine cell-surface proteins by capillary electrophoresis and laser-induced fluorescence: use of 5-SAENTA-x8f for quantitation of the human equilibrative nucleoside transporter 1 protein

The human equilibrative nucleoside transporter 1 protein (hENT1) is a major mediator of cellular entry of nucleosides and anticancer nucleoside drugs; its assay is important in understanding and diagnosing chemotherapy resistance. Here we present a novel assay for quantifying hENT1 using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). A cellular population is treated with 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine-x8-fluorescein (5-SAENTA-x8f), which binds with high affinity and specificity to the hENT1 protein. The cells are washed to remove excess reagent, lysed, and centrifuged, and the supernatant is analyzed by CE-LIF with the use of an internal standard. Accuracy was evaluated by comparing the capillary electrophoresis results with those obtained by flow cytometry; the results were highly correlated, r = 0.96. The relative standard deviation of the hENT1 assay was 10%, determined from nine independent assays of the same cell line, which is 3 times superior to results obtained in a flow cytometry assay. The detection limit for 5-SAENTA-x8f was 4300 molecules injected into the capillary.     

Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein-protein interactions in bacteria: improved sensitivity and reduced screening time.

In this research, an improved detection system is described that allows an easy in vivo screening and selection of functional interactions between two interacting proteins in bacteria. We earlier reported a new concept for detecting protein-protein interactions based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing (Ozawa, T.; et al. Anal. Chem. 2000, 72, 5151-5157.): Two putative interacting proteins are genetically fused to the split VDE inteins, which are linked directly to the N- and C-terminal halves of the split EGFP. Association of the interacting proteins results in functional complementation of VDE and protein-splicing reaction that leads to formation of an EGFP fluorophore. This technique simplified detection of protein interactions, but because of the low splicing efficiency of VDE intein, its sensitivity and screening time were not enough for detecting the protein interactions directly in living cells. In this paper, we have explored the use of the DnaE split intein from Synechocystis sp. PCC6803 for intracellular reconstitution of the split EGFP. We examined efficiency of the fluorophore formation by preparing four different split-EGFP types, among which EGFP dissected at the position between 157 and 158 was found to show the strongest fluorescence intensity upon protein interactions. A time required for the formation of EGFP after protein interactions was only 4 h, as compared to 3 days with the VDE intein. The protein interactions were thereby detected by an in vivo selection and screening assay in Escherichia coli on Luria broth agar plates. This improvement permits versatile designs of screening procedures either for ligands that bind to particular proteins or for molecules or mutations that block particular interactions between two proteins of interest.     

CdTe nanoparticles display tropism to core histones and histone-rich cell organelles

The disclosure of the mechanisms of nanoparticle interaction with specific intracellular targets represents one of the key tasks in nanobiology. Unmodified luminescent semiconductor nanoparticles, or quantum dots (QDs), are capable of a strikingly rapid accumulation in the nuclei and nucleoli of living human cells, driven by processes of yet unknown nature. Here, it is hypothesized that such a strong tropism of QDs could be mediated by charge-related properties of the macromolecules presented in the nuclear compartments. As the complex microenvironment encountered by the QDs in the nuclei and nucleoli of live cells is primarily presented by proteins and other biopolymers, such as DNA and RNA, the model of human phagocytic cell line THP1, nuclear lysates, purified protein, and nucleic acid solutions is utilized to investigate the interactions of the QDs with these most abundant classes of intranuclear macromolecules. Using a combination of advanced technological approaches, including live cell confocal microscopy, fluorescent lifetime imaging (FLIM), spectroscopic methods, and zeta potential measurements, it is demonstrated that unmodified CdTe QDs preferentially bind to the positively charged core histone proteins as opposed to the DNA or RNA, resulting in a dramatic shift off the absorption band, and a red shift and decrease in the pholuminescence (PL) intensity of the QDs. FLIM imaging of the QDs demonstrates an increased formation of QD/protein aggregates in the presence of core histones, with a resulting significant reduction in the PL lifetime. FLIM technology for the first time reveals that the localization of negatively charged QDs to their ultimate nuclear and nucleolar destinations dramatically affects the QDs' photoluminescence lifetimes, and offers thereby a sensitive readout for physical interactions between QDs and their intracellular macromolecular targets. These findings strongly suggest that charge-mediated QD/histone interactions could provide the basis for QD nuclear localization downstream of intracellular transport mechanisms.     

Analysis of gamma-carboxyglutamic acid content of protein, urine, and plasma by capillary electrophoresis and laser-induced fluorescence

When the properties of an analyte are known, the separation system can be designed to make the analyte of interest migrate at either a much faster or a much slower velocity compared to other molecules in the sample matrix. A simple and sensitive method to analyze the gamma-carboxyglutamic acid (Gla) content of protein, urine, and plasma was developed using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The separation method is designed according to the specific properties of three amino acids of interest. The number of Gla residues from three vitamin K-dependent proteins were estimated by quantifying the amount of fluorescein thiocarbamyl derivative of Gla after alkaline hydrolysis and fluorescein isothiocyanate labeling. Human prothrombin, blood coagulation factor X, and bovine osteocalcin were calculated to have 10.0 +/- 0.7, 11.0 +/- 0.6, and 2.1 +/- 0.1 Gla residues per mole of protein, respectively, which agreed well with amino acid sequencing data. The analysis of free Gla content in urine and plasma was also demonstrated by this method. It was demonstrated that submicrograms of protein can be characterized by CE-LIF.     

Individual acidic organelle pH measurements by capillary electrophoresis

This report describes the pH measurement of individual acidic organelles isolated from the human leukemia CCRF-CEM and CEM/C2 cells. These cells were allowed to endocytose fluorescein tetramethylrhodamine dextran (FRD), a ratiometric probe that has fluorescein as a pH-dependent fluorophore and tetramethylrhodamine as a pH-independent fluorophore. Isolated organelle fractions from these cells were then subjected to capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) analysis. The detection of individual organelle fluorescence at two different wavelengths, selected on the basis of the emission range of the FRD probe, gives a fluorescence intensity ratio used to calculate the pH from a calibration curve. This curve was constructed from CE-LIF measurements of individual liposomes loaded with several pH buffer standards. The respective median pH values are 5.1 +/- 0.2 in CEM/C2 cells and 6.1 +/- 0.4 in CCRF-CEM cells. These measurements compare well with pixel-based epifluorescence microscopy measurements of whole cells where the corresponding average pH values are 5.0 +/- 0.6 (n = 15) and 6.2 +/- 0.7 (n = 15). A pH comparison between the two cell types suggests that the lower pH in the CEM/C2 cells may be relevant to the protonation and sequestration of weak base anticancer drugs such as doxorubicin. The determination of the pH of individual vesicles, liposomes, and acidic organelles is a new resource for measuring and investigating the role of the acid-base properties of subcellular-size compartments.     

Fold and flexibility: what can proteins' mechanical properties tell us about their folding nucleus?

The determination of a protein''s folding nucleus, i.e. a set of native contacts playing an important role during its folding process, remains an elusive yet essential problem in biochemistry. In this work, we investigate the mechanical properties of 70 protein structures belonging to 14 protein families presenting various folds using coarse-grain Brownian dynamics simulations. The resulting rigidity profiles combined with multiple sequence alignments show that a limited set of rigid residues, which we call the consensus nucleus, occupy conserved positions along the protein sequence. These residues'' side chains form a tight interaction network within the protein''s core, thus making our consensus nuclei potential folding nuclei. A review of experimental and theoretical literature shows that most (above 80%) of these residues were indeed identified as folding nucleus member in earlier studies.     

Assay for glucosamine 6-phosphate using a ligand-activated ribozyme with fluorescence resonance energy transfer or CE-laser-induced fluorescence detection

A naturally occurring aptazyme, the glmS ribozyme, is adapted to an assay for glucosamine 6-phosphate, an effector molecule for the aptazyme. In the assay, binding of analyte allosterically activates aptazyme to cleave a fluorescently labeled oligonucleotide substrate. The extent of reaction, and hence analyte concentration, is detected by either fluorescence resonance energy transfer (FRET) or capillary electrophoresis with laser-induced fluorescence (CE-LIF). With FRET, assay signal is the rate of increase in FRET in presence of analyte. With CE-LIF, the assay signal is the peak height of cleavage product formed after a fixed incubation time. The assay has a linear response up to 100 (CE-LIF) or 500 microM (FRET) and detection limit of approximately 500 nM for glucosamine 6-phosphate under single-turnover conditions. When substrate is present in excess of the aptazyme, it is possible to amplify the signal by multiple turnovers to achieve a 13-fold improvement in sensitivity and detection limit of 50 nM. Successful signal amplification requires a temperature cycle to alternately dissociate cleaved substrate and allow fresh substrate to bind aptazyme. The results show that aptazymes have potential utility as analytical reagents for quantification of effector molecules.     

Cellular uptake of gold nanoparticles passivated with BSA-SV40 large T antigen conjugates

Internalization and subcellular localization in HeLa cells of gold nanoparticles modified with the SV40 large T antigen were quantified using inductively coupled plasma optical emission spectroscopy (ICP-OES). Internalization was monitored as a function of incubation time, temperature, nanoparticle diameter, and large T surface coverage. Increasing the amount of large T peptides per gold nanoparticle complex, by either increasing the coverage at constant nanoparticle diameter or by increasing the nanoparticle diameter at constant large T coverage, resulted in more cellular internalization. In addition, nuclear fractionation was performed to quantify nuclear localization of these complexes as a function of large T coverage. In contrast to our prior qualitative investigations of nuclear localization by video-enhanced color differential interference contrast microscopy (VEC-DIC), ICP-OES was able to detect nanoparticles inside fractionated cell nuclei. Although increasing the large T coverage was found to afford higher cell internalization and nuclear targeting, quantitative evaluation of cytotoxicity revealed that higher large T coverages also resulted in greater cytotoxicity. The ICP-OES and nuclear fractionation techniques reported here are valuable tools that can add important quantitative information to optical and electron imaging methods such as VEC-DIC and transmission electron microscopy regarding the fate of nanoparticles in cells.     

茭白黑粉菌核相变化及ISSR分子标记筛选的研究

通过显微观察和Hoechst 33258荧光染色方法,系统观察了茭白灰茭中黑粉菌厚垣孢子不同萌发时期的核相变化.研究表明,厚垣孢子萌发过程中黑粉菌细胞核的数目不断变化,核相在萌发过程中是不规则的.茭白灰茭厚垣孢子中没有观察到细胞核,菌丝体细胞核的数目在1~4个,大多数小孢子仅有一个细胞核或没有细胞核.同时获得了18个可用于茭白黑粉菌遗传变异分析的ISSR分子标记,作为深入探讨茭白黑粉菌的生物学特性的参考.     

Protein nanopatterns and biosensors using gold binding polypeptide as a fusion partner

An efficient strategy for immobilizing proteins on a gold surface was developed by employing the gold binding polypeptide (GBP) as a fusion partner. Using the enhanced green fluorescent protein (EGFP), severe acute respiratory syndrome coronavirus (SARS-CoV) envelope protein (SCVme), and core streptavidin (cSA) of Streptomyces avidinii as model proteins, specific immobilization of the GBP-fusion proteins onto the gold nanoparticles and generation of protein nanopatterns on the bare gold surface were demonstrated. The GBP-fused SCVme bound to gold nanoparticles successfully interacted with its antibody and showed changes in absorbance and color, allowing efficient diagnosis of SARS-CoV. The fusion proteins could be successfully immobilized on the gold surface by nanopatterning and microcontact printing as examined by atomic force microscopy and surface plasmon resonance analysis. The poly(dimethylsiloxane) microfluidic channels were created on the gold surface and were used for antigen-antibody and DNA-DNA interaction studies. Specific immobilization of GBP-EGFP fusion protein and its interaction with the antibody in the microchannels could be demonstrated. By immobilizing the DNA probe through the use of GBP-fused cSA, specific hybridization of the target DNA prepared from Salmonella could also be achieved. The GBP-fusion method allows immobilization of proteins onto the gold surface without surface modification and in bioactive forms suitable for studying protein-protein, DNA-DNA, and other biomolecular interaction studies. Furthermore, these studies can be carried out in a microfluidic system, which allows high-throughput analysis of biomolecular interactions.